Shrimp Scampi Foil Packets (Oven or Grill)

By electricdiet / July 16, 2020


These shrimp scampi foil packets are healthy, flavorful, easy to cook, and a breeze to clean-up! Make them in the oven or on the grill for a quick and tasty meal.

Cooked Shrimp Scampi in an open foil packet topped with bread crumbs and fresh parsley on a plate that's on top of a cutting board

Are you always on the look-out for meals that feature easy clean-up? No one wants to finish a delicious dinner only to face a mountain of dishes in the kitchen.

That’s why I love these shrimp scampi foil packets with artichoke hearts! The topping comes together in one pan, and everything else cooks right in the foil packets. How easy is that?

And you can cook the shrimp packets either in the oven or on the grill. Choose whatever is most convenient for you!

Did I mention that this shrimp dish is unbelievably delicious? It’s a little rich, but with just the right amount of herbs, lemon, and spices to balance it all out. And it’s low-carb, too!

So the next time you’re craving shrimp or just want a meal that’s easy to cook AND clean-up, give this quick and tasty shrimp scampi recipe a try!

How to make shrimp scampi foil packets

This dish is so easy to make either in the oven or on the grill. Everything cooks right in the foil packets!

Step 1: Heat a small skillet over medium heat.

Step 2: Add 1 teaspoon of olive oil and the panko breadcrumbs, then cook, stirring frequently, until the panko is golden brown, about 3 minutes.

Step 3: Transfer the mixture to a small bowl. Stir in the lemon zest, Parmesan, and garlic, then set aside.

Step 4: Tear off 4 large sheets of heavy-duty foil, about 18 inches long each.

Step 5: Place 1/4 of the artichoke hearts in the middle of each sheet of foil. Season with salt to taste, then top each with a thyme sprig, 3 to 4 lemon slices, and 1 teaspoon of butter.

Step 6: In a large bowl, toss the shrimp with the crushed red pepper and garlic.

Step 7: Add 1/4 of the shrimp mixture on top of the artichoke mixture on each sheet of foil. Drizzle each with 1 tablespoon of white wine and a 1/2 tablespoon of olive oil.

Uncooked shrimp scampi in the foil packet ready to be wrapped and cooked

Step 8: Bring the opposite sides of foil together and fold at least twice to seal, leaving a little headroom for the air to circulate. Then, fold the open sides over at least twice.

Step 9: Cook the packets either directly on an oven rack for 15 to 20 minutes at 400°F or on a hot grill for about 10 minutes. The shrimp should be opaque when done.

Step 10: Open the packets carefully and top with the toasted panko breadcrumbs and fresh parsley.

Now all you have to do to clean up is toss the foil packets… after you enjoy your shrimp scampi, of course!

What is shrimp scampi?

In Italian, scampi refers to langoustines, which are tiny lobsters. They are traditionally cooked in a sauce that contains olive oil, white wine, and garlic.

Since langoustines aren’t as common in the U.S., chefs started replacing them with shrimp in this recipe. And that’s how “shrimp scampi” came to be.

There are so many flavor variations for this dish. It can be rich and buttery or light and herby. It might be served over pasta (or not). It may be lemony, spicy, or both. The possibilities are endless!

The beauty of this version is that it’s a little bit rich, a little bit herby, a little bit spicy, and a little bit lemony. If you ask me, it’s really the best of all worlds!

What to serve with this dish

I like to choose sides that are light but with vibrant and fresh flavors to compliment this recipe.

One of my favorites is a salad made with my homemade citrus vinaigrette. It adds a wonderful tanginess! And you can easily whip up the dressing while the shrimp are cooking.

If you can afford the carbs, a breadstick or whole-grain roll are a nice indulgence. And if you’re feeling really ambitious, try my garlic knots (just be sure to plan ahead).

Storage

Once cooked, shrimp can be stored covered in the refrigerator for about 3 or 4 days.

Just keep in mind that the crispy bread-crumb topping might get a bit soggy in the refrigerator. For that reason, I prefer to serve this dish right away.

Other healthy shrimp recipes

Shrimp is a great option for a lean, healthy protein that can be made with so many different flavors! Here are a few more healthy and delicious shrimp recipes that I know you’ll enjoy:

When you’ve tried this dish, please don’t forget to let me know how you liked it and rate the recipe in the comments below!

Recipe Card

Shrimp Scampi in foil packets

Shrimp Scampi Foil Packets (Oven or Grill)

These shrimp scampi foil packets with artichoke hearts are healthy, flavorful, easy to cook, and a breeze to clean-up! Make them in the oven or on the grill for a quick and tasty meal.

Prep Time:25 minutes

Cook Time:15 minutes

Total Time:40 minutes

Author:Shelby Kinnaird

Servings:4

Instructions

  • Heat a small skillet over medium heat.

  • Add 1 teaspoon of olive oil and the panko breadcrumbs, then cook, stirring frequently, until the panko is golden brown, about 3 minutes.

  • Transfer the mixture to a small bowl. Stir in the lemon zest, Parmesan, and garlic, then set aside.

  • Tear off 4 large sheets of heavy-duty foil, about 18 inches long each.

  • Place 1/4 of the artichoke hearts in the middle of each sheet of foil. Season with salt to taste, then top each with a thyme sprig, 3 to 4 lemon slices, and 1 teaspoon of butter.

  • In a large bowl, toss the shrimp with the crushed red pepper and garlic.

  • Add 1/4 of the shrimp mixture on top of the artichoke mixture on each sheet of foil. Drizzle each with 1 tablespoon of white wine and a 1/2 tablespoon of olive oil.

  • Bring the opposite sides of foil together and fold at least twice to seal, leaving a little headroom for the air to circulate. Then, fold the open sides over at least twice.

  • Cook the packets either directly on an oven rack for 15 to 20 minutes at 400°F or on a hot grill for about 10 minutes. The shrimp should be opaque when done.

  • Open the packets carefully and top with the toasted panko breadcrumbs and fresh parsley.

Recipe Notes

This recipe is for 4 servings. Each serving is the contents of 1 foil packet. Leftovers can be stored covered in the refrigerator for 3-4 days. However, the breadcrumbs may become soggy, so I would recommend serving this dish right away.

Nutrition Info Per Serving

Nutrition Facts

Shrimp Scampi Foil Packets (Oven or Grill)

Amount Per Serving (1 packet)

Calories 304 Calories from Fat 128

% Daily Value*

Fat 14.2g22%

Saturated Fat 3.9g24%

Trans Fat 0g

Polyunsaturated Fat 4.2g

Monounsaturated Fat 3.1g

Cholesterol 14.6mg5%

Sodium 867.6mg38%

Potassium 51.8mg1%

Carbohydrates 13.6g5%

Fiber 2g8%

Sugar 3.1g3%

Protein 29.7g59%

Vitamin A 550IU11%

Vitamin C 22.3mg27%

Calcium 80mg8%

Iron 8.1mg45%

Net carbs 11.6g

* Percent Daily Values are based on a 2000 calorie diet.

Course: Fish & Seafood

Cuisine: Mediterranean

Diet: Diabetic

Keyword: shrimp foil packets, shrimp scampi, shrimp scampi foil packets



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BBQ Turkey Burger – My Bizzy Kitchen

By electricdiet / July 14, 2020


My late husband and I loved to go out to eat, and on the weekends we would eat out at least twice if not three times.  The weekends were a slippery slope for me.  

I would do great Monday through Friday and then come Friday night it’s as if I lost my mind and ate whatever I wanted, knowing that I “did good” the previous five days.  Well, that doesn’t work my friends.  Unless you want to gain and lose the same five pounds.

I hit the reset button on June 13 when I weighed in at 190.2.  That’s the highest adult weight I’ve been in a very long time.  Not my all time high of 211 but I needed to focus and get my shit together.

Guess what?  Just following the WW plan.  However, I do it a bit differently.  I am on Team Purple so I get 300 “free” foods to chose from and 16 points a week, plus an additional 35 weekly points, or a total of 147 points for the week.  Getting “blue dots” on the WW app means that you stayed within your point range for the day.

Me?  I switch it up!  My points this past week from Saturday through Friday: 25, 17, 22, 18, 25, 26, 23 for a total of 156 points.  I lost 3 pounds this week.  I think it’s important to keep your body guessing about what it’s going to get, instead of eating exactly 5 points for breakfast, 5 points for lunch and 6 points for dinner.

Hopefully that makes sense?!  My mantra this millionth time getting “back on track” is consistency, not perfection.  That’s it.  Oh, and it helped that I am nearly half way through my #dryjuly challenge!

But let’s talk about turkey burgers!  I remember going out to Applebees with my late husband (he loved their 32 ounce $3 miller lite beers!) and I was bound and determined to eat healthy even as I was eyeing the three appetizer for dinner choice (um, fried zucchini, mozzarellas sticks and coconut shrimp – yes please!).

I chose the turkey burger with sweet potato fries.  Go me!  It was delicious and I ate every bite. Until I got home and tracked the points and it was 33 points and 1250 calories and 40 grams of fat – what the what?!

As my late husband used to say if something made him mad “turkey burgers were dead to me” and I didn’t make one for years.

But now that ground turkey breast is zero points on my WW plan, I knew I had to give them another try.  My secret to a juicy turkey breast burger?  I Can’t Believe It’s Not Butter.

It makes all the difference.  It seriously is the key to a juicy burger.  Another tip:  don’t flip the burger too soon.  Whether on the grill or in a cast iron skillet, let the burger cook to get that browned caramelized goodness.

Print

BBQ Turkey Burger

The juiciest turkey breast burger you’ll ever make!  I am posting the recipe for one serving, so scale as needed.

  • Author: Biz
  • Prep Time: 5 minutes
  • Cook Time: 15 minutes
  • Total Time: 20
  • Yield: 1 1x

Scale

Ingredients

  • 4 ounces ground turkey breast
  • 1/4 teaspoon salt
  • 1/4 teaspoon pepper
  • 1 tablespoon favorite BBQ sauce (I used G. Hughes sugar free)
  • 2 tablespoons real bacon bits
  • 1 teaspoon I Can’t Believe It’s Not Butter Light
  • 14 grams favorite cheese
  • baby spinach or lettuce
  • 1 brioche bun (mine was 5 points and 160 calories)

Instructions

Mix all the ingredients from turkey breast to bacon bits. Divide burger into two patties. On top of one patty, spread the butter, top with the other half of the burger and, using your hands, squeeze the burger together, kind of pinching the sides so the butter doesn’t ooze out.

Start in a skillet on medium low (or on a grill over low heat) for the first 6 minutes. Finish off over medium high heat on a stove for 2 minutes a side, or until a meat thermometer reaches 160 degrees. Let rest 5 minutes to let the residual heat finish cooking the burger to 165. Top with cheese at the last minute to melt.

Notes

Serve with your favorite sides – on #teampurle that would be zero point corn and potatoes for me!

My whole dinner was 9 points, or 510 calories. #wortheveryone 😀

Since the burger is basically zero points (but has calories!) go ahead and splurge on a good bun.  This was a perfect summer dinner last week.  

So if you’ve been giving turkey burgers the stink eye for a while, give this one a try – pinky swear you’ll love it!

Until next time, be well!

 





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Diabetes & Sleep Apnea: Everything You Need to Know

By electricdiet / July 12, 2020


Sleep apnea is a far greater problem than it gets credit for — especially for people living with diabetes.

Difficult to diagnose without proper overnight testing, too many people struggling to breathe properly during their sleep may not have any idea they aren’t getting enough oxygen every night.

But sleep apnea can impact many aspects of your day-to-day life and overall health. And its link to diabetes is indisputable.

In this article, we’ll discuss what sleep apnea is, common causes and symptoms, its relationship with type 1 and type 2 diabetes, and today’s best treatment options.

Face of man sleeping with sleep apnea

What is sleep apnea?

Sleep apnea (also known as “obstructive sleep apnea” or “OSA”) is a condition defined by long pauses in breathing while you sleep. To qualify, the pause of breathing must be at least 10 seconds long, according to the National Sleep Foundation.

The pause in breathing is the result of muscles of the back of your throat closing partially or failing entirely to stay open for varying periods of time while you sleep.

The reason these pauses in breathing are worrisome and troublesome is that it can create a significant lack of oxygen in your blood which then leads to a variety of other problems.

Consequences of untreated sleep apnea

  • Daytime exhaustion and fogginess
  • High blood pressure
  • Cardiac arrhythmia
  • Congestive heart failure
  • Heart attack
  • Stroke
  • Depression and mood issues
  • Memory issues
  • Insulin resistance
  • Increased risk of type 2 diabetes
  • Drowsy driving

OSA can develop in children, too, although less common.

Causes of sleep apnea

While anyone could potentially develop sleep apnea, the following are characteristics or habits that increase your risk of developing the condition, according to Harvard:

  • Obesity: about 2/3s of people with OSA are overweight or obese
  • Family history of OSA or snoring
  • Abnormally smaller lower jaw or other abnormal facial characteristics
  • Recessed chin
  • Being male: far more people with OSA are male versus female
  • Smoking cigarettes
  • Large neck circumference
  • Large tonsils
  • Drinking alcohol before bedtime
  • Post-menopausal (for women)
  • Hypothyroidism (low levels of thyroid hormone)
  • Acromegaly (high levels of growth hormone)
  • Being over the age of 40 years old
  • Being African-American, Pacific-Islander, or Hispanic

While there is another type of sleep apnea that results from your brain failing to manage normal breathing, that type is rare.

The type most commonly experienced by the general population is obstructive sleep apnea and affects approximately 18 million people in the United States.

Symptoms

The signs and symptoms of sleep apnea are often easy to dismiss or easy to mistake as individual issues rather than many symptoms related to the same condition.

The National Sleep Foundation lists the following as common signs and symptoms of sleep apnea:

  • Chronic snoring
  • Constantly feeling sleep-deprived
  • Difficulty concentrating
  • Depression
  • Irritability
  • Sexual dysfunction
  • Learning and memory difficulties
  • Falling asleep during normal daytime activities
    Disturbed sleep

If you are a chronic snorer or suspect any of these symptoms may be regularly present in your life, talk to your primary care doctor about scheduling with a sleep specialist who can assess you for sleep apnea.

Sleep apnea and diabetes: how are they related?

Research has demonstrated over and over again that OSA and diabetes have an undeniable relationship, and are often found in the same patient.

Let’s take a look at some of the most significant research.

Sleep apnea increases blood sugar levels

OSA has been found to increase oxidative stress, inflammation, neuroendocrine dysregulation, and alter glucose homeostasis, according to this 2016 study from the American Diabetes Association.

The study’s finding urges healthcare professionals to assess every patient with type 2 diabetes for potential signs of sleep apnea, and vice versa — assessing patients with sleep apnea for high blood sugar levels.

“Early recognition and interventions for OSA can be expected to improve insulin sensitivity and control of hyperglycemia in many patients. Clinicians must remain vigilant for signs and symptoms of OSA and monitor compliance with CPAP along with weight management, diet control, and medication adherence in patients with type 2 diabetes.”

Sleep apnea linked to insulin resistance and type 2 diabetes

This 2018 report on OSA evaluated dozens of studies on the condition and its implications and connections with other conditions.

It was determined that patients with OSA had an increased risk of developing hypertension, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, dyslipidemia, and atherosclerosis.

Sleep apnea increases your risk of type 2 diabetes

This 2017 study from Taiwan determined that patients with OSA had a much higher likelihood of developing type 2 diabetes. In contrast, the study also determined that patients with type 2 diabetes did not necessarily have a higher likelihood of developing OSA.

This simply means that OSA seems to be a precursor for developing type 2 diabetes, but type 2 diabetes is not a precursor to developing OSA in patients who have not already developed this sleep condition.

A 2018 study from Japan echoed similar findings.

“OSA patients are more likely than non-OSA populations to develop type 2 diabetes, while more than half of type 2 diabetes patients suffer from OSA.”

Using a CPAP to treat OSA improves insulin resistance

A CPAP device — which stands for “continuous positive airway pressure” — is the primary method of treatment for OSA, and this 2018 study from Japan found that consistent use of a CPAP improves a patient’s levels of insulin resistance.

“CPAP improved glucose metabolism determined by the oral glucose tolerance test in OSA patients, and several studies have shown that CPAP improves insulin resistance, particularly in obese populations undergoing long-term CPAP.”

This is significant in terms of treating a patient with both OSA and type 2 diabetes. By treating the OSA, the patient may see modest to moderate improvements in their blood sugar levels and overall diabetes health, too.

Both type 2 diabetes and OSA increase risk of cardiovascular disease

“As both diabetes and OSA lead to cardiovascular disease, clinicians and healthcare professionals should be aware of the association between diabetes and OSA,” explains the same 2018 study from Japan.  

The study suggests that healthcare professionals should heavily consider treating patients with type 2 diabetes and/or OSA with a CPAP device to reduce the known stress both conditions have on a patient’s cardiovascular system.

OSA increases the risk of STDR (sight-threatening diabetic retinopathy)

This 2017 study from the United Kingdom found that patients with type 2 diabetes and existing diabetic retinopathy had a significantly increased risk of developing proliferative diabetic retinopathy, which is defined by the patient’s worsening vision.

Using a CPAP device to treat the OSA resulted in a reduction of the progression of the STDR in these patients, but it was determined that further studies are needed to focus more intensely on the benefits of treating OSA to inadvertently treat STDR.

Patients with type 1 diabetes have a higher risk of OSA

“The prevalence of asymptomatic OSA is high in a cohort of patients with type 1 diabetes,” determined a 2017 study from Denmark.

Other risk factors for the type 1 diabetes population included being older, overweight, and existing diagnosis of nephropathy (kidney disease).

“OSA was present in 32 percent of the patients with normal BMI, in 60 percent of overweight patients, and in 61% of obese patients,” explains the study.

Additionally, the study found that patients with type 1 diabetes and OSA showed very few symptoms, particularly very rarely reporting sleepiness compared to patients without OSA. This makes it harder to catch, diagnose, and treat.

Healthcare professionals treating patients with type 1 diabetes should keep in mind that this population should be potentially screened for OSA if they are also over the age of 40, overweight, and have nephropathy.

Treatment options

If you think you may have sleep apnea, the first place to start seeking help is through your primary care doctor.

Most likely, if you share your bed with a partner, it isn’t going to be news to you that you have a severely loud or disruptive snore. You might even want to try setting your phone up to record the sound of your own snore. This alone could reveal long gaps in breathing or very turbulent, inconsistent snore rhythms.

Your doctor will then recommend you partake in a sleep study which means you’ll stay overnight at a “sleep center” to have your breathing monitored for an entire night.

They will also monitor your eye movement, muscle activity, heart rate, respiratory effort, airflow, and the amount of oxygen in your blood.

This will give your healthcare team a clear understanding of whether or not you have sleep apnea, and how severe your sleep apnea may be based on just how little oxygen your body is getting while you sleep.

The number one treatment for sleep apnea, as mentioned earlier, is a CPAP device.

A CPAP looks more uncomfortable than it really is, which can deter patients from pursuing getting treated in the first place.

A CPAP is a mask that fits over your mouth and/or your nose, and it blows air into your airway to help keep it adequately open while you sleep.

Research has found that it is by far the most effective treatment for sleep apnea, but one tricky aspect of this method is getting patients to use it consistently.

The device itself also makes a light and soft noise when it’s turned on, which is similar to the sound of a noise machine. Ideally, the sound itself doesn’t interfere with your sleep and possibly improves your sleep by providing white noise.

What else can you do to treat sleep apnea? Let’s take a look at all of the options recommended by the National Sleep Foundation:

  • Continuous positive airway pressure (CPAP) device: A mask that covers your mouth and/or nose and delivers air to help keep your airway open while you sleep
  • Oral Pressure Therapy (OPT): Similar to a CPAP device but without the mask, this treatment is a mouthpiece that delivers air to help keep your throat properly open while you sleep.
  • Expiratory Positive Airway Pressure (EPAP): This device covers your nostrils with a disposable adhesive valve that opens and ensures your airway stays open.
  • Dental appliances to reposition jaw and tongue
  • Upper airway surgery to remove excess tissue: If you have an anatomical facial abnormality, it could be corrected with surgery and enable your jaw and throat to stay open properly during your sleep.
  • Lose weight: Weight-loss can have a significant impact on sleep apnea. If you’re reluctant to use a device, let sleep apnea be the motivation you need to lose weight.
  • Avoid, reduce, or limit alcohol intake
  • Quit smoking
  • Sleep on your side instead of on your back

While sleep apnea doesn’t sound terribly alarming at first, it can create a great deal of stress in the body and in your life if left untreated.

This easy-to-miss condition can put your longterm health in danger. Don’t hesitate to get tested if suspect you may be struggling with sleep apnea.



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Excitotoxicity and Overnutrition Additively Impair Metabolic Function and Identity of Pancreatic β-Cells

By electricdiet / July 10, 2020


Abstract

A sustained increase in intracellular Ca2+ concentration (referred to hereafter as excitotoxicity), brought on by chronic metabolic stress, may contribute to pancreatic β-cell failure. To determine the additive effects of excitotoxicity and overnutrition on β-cell function and gene expression, we analyzed the impact of a high-fat diet (HFD) on Abcc8 knockout mice. Excitotoxicity caused β-cells to be more susceptible to HFD-induced impairment of glucose homeostasis, and these effects were mitigated by verapamil, a Ca2+ channel blocker. Excitotoxicity, overnutrition, and the combination of both stresses caused similar but distinct alterations in the β-cell transcriptome, including additive increases in genes associated with mitochondrial energy metabolism, fatty acid β-oxidation, and mitochondrial biogenesis and their key regulator Ppargc1a. Overnutrition worsened excitotoxicity-induced mitochondrial dysfunction, increasing metabolic inflexibility and mitochondrial damage. In addition, excitotoxicity and overnutrition, individually and together, impaired both β-cell function and identity by reducing expression of genes important for insulin secretion, cell polarity, cell junction, cilia, cytoskeleton, vesicular trafficking, and regulation of β-cell epigenetic and transcriptional program. Sex had an impact on all β-cell responses, with male animals exhibiting greater metabolic stress-induced impairments than females. Together, these findings indicate that a sustained increase in intracellular Ca2+, by altering mitochondrial function and impairing β-cell identity, augments overnutrition-induced β-cell failure.

Introduction

The loss of β-mass and function in response to metabolic stress is a major determinant of type 2 diabetes (T2D) (1). While multiple mechanisms, including glucolipotoxicity, excitotoxicity, inflammation, endoplasmic reticulum (ER) stress, and oxidative stress (25), have been implicated in metabolic stress–induced β-cell failure, the molecular and cellular mechanisms that actually cause the loss of β-cell function and development of T2D are not understood.

Excitotoxicity refers to the pathological process in excitable cells in which overstimulation leads to a sustained increase in intracellular Ca2+ concentration ([Ca2+]i), resulting in disrupted homeostasis, loss of cell function, or cell death (6). In pancreatic β-cells, metabolic stress–induced increases in [Ca2+]i activate Ca2+/calmodulin-dependent kinases (CaMKs), calcineurin (a Ca2+-dependent phosphatase), and other Ca2+-dependent proteins, causing alterations in β-cell gene expression that negatively impact both β-cell mass and function (7). Increases in [Ca2+]i have been described in rat islets cultured in high glucose (8), in mouse islets from obese (db/db) mice (9), in islets of mice fed a high-fat diet (HFD) (10), and in β-cells that exhibit chronic membrane depolarization (11). Both the verapamil-induced blockage of Ca2+ influx (12) and genetic knockdown of Cavβ3, a Ca2+ channel subunit (13), by reducing [Ca2+]i, attenuate β-cell loss and diabetes in animal models, suggesting that an increase in [Ca2+]i is a fundamental determinant of stress-induced β-cell failure.

Overnutrition, by elevating circulating free fatty acids (FFAs), contributes to insulin resistance, increasing insulin biosynthesis and secretion (14). The prolonged exposure of β-cells to FFAs elicits multiple responses, including the activation of ER stress, oxidative stress, and inflammatory signaling pathways (15,16). Most notably, FFA-induced oxidative stress triggers the release of Ca2+ from ER stores, increasing [Ca2+]i, accentuating ER stress, and inducing apoptosis (17).

Sex also influences the response of β-cells to stress (18). Women are less likely than men to develop T2D and require a higher BMI to do so (19). Increased estrogen receptor signaling (20), sex-specific differences in islet DNA methylation status (21), and differences in the expression of islet-enriched transcription factors (TFs) and genes involved in cell cycle regulation (22) have all been suggested as causes for these differences.

To obtain a systems-wide understanding of the effects of both excitotoxicity and overnutrition on β-cell function and gene expression, we used mice lacking Abcc8, a critical subunit of the ATP-dependent K+ channel (KATP). Previously, we have shown that β-cells from these mice exhibit chronic membrane depolarization and increases in [Ca2+]i that cause impairments in islet morphology, glucose tolerance, and β-cell identity (11,23). Because the loss of β-cell function in mice lacking Abcc8 develops slowly over several months (23), the individual and combined effects of excitotoxicity and overnutrition on β-cell function and gene expression were determined prior to the onset of hyperglycemia and glucotoxicity (11). Additionally, by using a recently described Ins2Apple allele, we avoided confounding effects of the MIP-GFP transgene (22).

Research Design and Methods

Mouse Lines and Husbandry

The Abcc8tm1.1Mgn (23) and Ins2Apple (22) alleles were bred into and maintained as C57BL/6J congenic lines (stock 000664; The Jackson Laboratory). At weaning (3–4 weeks of age), mice were fed either regular chow (RC) (4.5% fat content) (5L0D; PicoLab) or HFD (60% fat content) (D12492; Research Diets, Inc.) for 5 weeks. Verapamil (1 mg/mL) (V4629; Sigma-Aldrich) was administered through the drinking water during the period of HFD feeding. All animal experimentation was performed under the oversight of the Vanderbilt University Institutional Animal Care and Use Committee.

Glucose Homeostasis

Intraperitoneal glucose tolerance tests (GTTs) were performed following a 16-h overnight fast. Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 min after administering d-glucose (2 mg/g body mass). Insulin tolerance testing was performed following a 4-h morning fast by administering 0.1 units/mL insulin (in Dulbecco’s PBS) (Humulin R; Eli Lilly and Company) and measuring blood glucose concentrations at 0, 15, 30, 60, and 120 min.

Islet Isolation and Culture

Islets were isolated following injection of 0.6 mg/mL Collagenase P (Roche) into the pancreatic bile duct followed by Histopaque-1077 (Sigma-Aldrich) fractionation and handpicking. For FACS and RNA sequencing, islets from two to four mice were pooled for each sample. Islets were cultured in low-glucose DMEM (11966–025; Gibco) containing 1 g/L glucose and supplemented with 10% FBS and penicillin/streptomycin (100 mg/mL) (Gibco) at 37°C with 5% CO2 infusion and 95% humidity. Palmitic acid (PA) (P0500; Sigma-Aldrich) was diluted in 50% ethanol to 100 mmol/L and conjugated to an FA-free BSA (A6003; Sigma-Aldrich) to generate a 5 mmol/L PA/5% FA-free BSA stock solution. Experimental media concentrations of the compounds used were 100 μmol/L for tolbutamide (T0891; Sigma-Aldrich), 0.5 mmol/L for PA, and 50 μmol/L for verapamil (V4629; Sigma-Aldrich).

β-Cell Isolation, RNA Isolation, and Quantitative PCR

Purified β-cells were obtained as previously described (15) in which live cells expressing red fluorescence were sorted with a 100-μm nozzle using the FACSAria II instrument (BD Biosciences). Cells were collected in chilled Homogenization Solution from the Maxwell 16 LEV simplyRNA Tissue Kit (TM351; Promega), and RNA was isolated as directed. For quantitative PCR (qPCR), reverse transcription was done using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). A total of 2 ng cDNA was used in real-time qPCR with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) using a CFX96 Real-Time PCR system (Bio-Rad Laboratories). Primers are listed in Supplementary Table 1.

RNA Sequencing and Data Analysis

RNA samples were analyzed using an Agilent 2100 Bioanalyzer, and only those samples with an RNA integrity number of seven or above were used. cDNA synthesis and amplification were performed using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio, Inc.) using 10 cycles of PCR. cDNA libraries were constructed using the Low Input Library Prep Kit (Takara Bio, Inc.). An Illumina NovaSeq 6000 instrument was used to produce paired-end, 150-nucleotide reads for each RNA sample. Paired-end sequencing of 31 samples produced ∼1.55 billion raw sequencing reads. The Spliced Transcripts Alignment to a Reference (STAR) application (16) was used to perform sequence alignments to the mm10 (GRCm38) mouse genome reference and GENCODE comprehensive gene annotations (release M17). Overall, 80–88% of the raw sequencing reads were uniquely mapped to genomic sites, resulting in 1.3 billion usable reads. HTSeq was used for counting reads mapped to genomic features (17), and DESeq2 was used for differential gene expression analysis (18). Padj <0.05 cutoff was used to define differentially expressed genes. Gene ontology (GO) analysis of differentially expressed genes was performed using Metascape (19).

Mitochondrial Respirometry and mtDNA Copy Number

Oxygen consumption rates (OCRs) of isolated islets were determined using a Seahorse XF96 respirometer (Agilent Technologies), as described (24). A total of 10–20 islets/well were loaded onto a Cell-Tak (Corning) precoated XF96 spheroid plate (Agilent Technologies) and preincubated for 2 h at 37°C without CO2 in a Seahorse assay DMEM (Agilent Technologies) supplemented with 3 mmol/L glucose, 1 mmol/L pyruvate, and 2 mmol/L glutamine. After measuring basal OCR, glucose (20 mmol/L), oligomycin (5 mmol/L), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (1 mmol/L), or antimycin A/rotenone (2.5 mmol/L) was added at indicated time points to modulate mitochondrial OCR response. For mtDNA copy number measurements, islet DNA was isolated with a DNeasy kit (Qiagen) and analyzed by qPCR with primers for mtDNA-encoded gene mt-CO1 and nuclear gene Ndufv1. Relative copy number was calculated as 2^([Ct(mtDNA) * E] − [Ct(nuclear DNA) * E]), where E is efficiency of corresponding qPCR determined from standard curves and Ct is threshold cycle.

Results

Excitotoxicity and Overnutrition Additively Impair Glucose Tolerance

To compare the effects of excitotoxicity and overnutrition on pancreatic β-cells, we fed C57BL/6J (wild-type [WT]) and Abcc8 knockout (KO) mice either RC or HFD for 5 weeks. Both groups contained male and female animals (n = 7–8 of each sex), and all animals gained weight on HFD (Fig. 1A). Blood glucose measurements after 5 weeks showed that the KO animals had lower fasting blood glucose in comparison with WT mice (Fig. 1B), consistent with previous observations that Abcc8 KO mice have impaired glucagon secretion (25). In contrast, fed blood glucose levels were higher in the HFD-KO mice compared with the RC-KO and HFD-WT animals (Fig. 1C). Treatment with verapamil, a Ca2+ channel blocker, during HFD lowered the fed blood glucose concentration in both the HFD-KO and HFD-WT mice (Fig. 1C).

Figure 1
Figure 1

Abcc8 KO mice exhibit impaired β-cell function after 5 weeks on HFD that is improved with the addition of verapamil. A: Weight gain on HFD for 5 weeks was similar for both WT and Abcc8 KO animals. Fasting (B) and fed (C) blood glucose measurements for RC- and HFD-fed mice at 8–9 weeks. KO mice have lower fasting blood glucose than WT mice. Fed blood glucose is elevated in HFD-KO and is lowered with the addition of verapamil (+ver). D: Intraperitoneal GTT results comparing RC- and HFD-fed mice at 8–9 weeks. HFD-KO mice have impaired glucose tolerance in comparison with WT and RC-KO mice. *P ≤ 0.05, **P ≤ 0.01: HFD-WT vs. HFD-KO; #P ≤ 0.05, ##P ≤ 0.01: HFD-KO vs. RC-KO. E: GTT results comparing HFD- and HFD+ver–fed mice at 8–9 weeks. n = 14–16. *P ≤ 0.05: HFD-WT vs. HFD-WT+ver; #P ≤ 0.05: HFD-KO vs. HFD-KO+ver. F: GTT area under the curve (AUC) measurements. Addition of verapamil improves glucose tolerance of HFD-WT and HFD-KO mice. n = 14–16 (7–8 males and 7–8 females) for each condition. Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001 (determined by ANOVA).

After 5 weeks, HFD-KO mice exhibited greater glucose intolerance compared with the RC-KO, RC-WT, and HFD-WT mice (Fig. 1D and F). Verapamil treatment improved glucose tolerance in the HFD-WT mice, while in HFD-KO mice, the effect of the drug was apparent only at 120 min after glucose administration (Fig. 1E and F). Both the RC-KO and HFD-KO animals had increased insulin sensitivity compared with the RC-WT and HFD-WT mice, respectively (Supplementary Fig. 1A and C). However, while verapamil increased insulin sensitivity of HFD-WT compared with the RC-WT mice, it had no effect on the HFD-KO mice (Supplementary Fig. 1B and C). These findings indicate that Abcc8 KO mice are more insulin sensitive, have a higher fed blood glucose concentration, and are more intolerant of glucose on an HFD than are WT animals. Interestingly, coadministration of verapamil during HFD feeding normalized blood glucose concentration in the KO animals independent of improvements in insulin sensitivity. Together, these findings indicate that excitotoxicity increases the susceptibility of β-cells to the negative effects of overnutrition.

Effects of Excitotoxicity and Overnutrition on β-Cell Gene Expression

To determine how excitotoxicity, overnutrition, and both stresses affect gene expression, we performed RNA sequencing on FACS-purified β-cells. All mice were identical in strain (C57BL/6J) and age (8–9 weeks old), but differed by sex, the presence or absence of Abcc8, and diet (RC vs. HFD). Sample clustering by principal component analyses of data from 31 samples (Supplementary Table 2) showed clear separation of WT and KO samples and indicated that the effect of the Abcc8 KO was much greater than that of the HFD (Supplementary Fig. 2). After pooling the data from both sexes, we performed three differential expression (DE) analyses, as summarized in Supplementary Table 3.

Excitotoxicity

To determine effects of excitotoxicity, we first compared the RC-KO and RC-WT data sets. Similar to our previous report, which used an MIP-GFP transgene (11), we observed a profound alteration in the β-cell transcriptome with a total of 7,393 genes being affected (3,957 upregulated genes [URGs] and 3,436 downregulated genes [DRGs]) (Fig. 2A and B and Supplementary Table 4). The magnitude of gene dysregulation was similar to our prior study (R = 0.72) (Supplementary Fig. 3) with differences in dysregulated gene sets attributed to leaky growth hormone from the MIP-GFP transgene (22).

Figure 2
Figure 2

β-Cell transcriptome changes in response to excitotoxicity in Abcc8 KO mice. A: Volcano plot showing distribution of differentially expressed genes (Log2FC over P value) in the RCKO vs. RC-WT RNA-sequencing comparison. Top 10 differentially expressed genes are indicated by names, and total numbers of URGs and DRGs are shown (Padj < 0.05). B: Distribution of dysregulated genes by biotype. C: Functional enrichment analysis of URGs and DRGs. Select top enriched pathways are shown. D: DE levels of select top URGs (top) and DRGs (bottom), with colors indicating gene functional associations. ECM, extracellular matrix; FDR, false discovery rate; Log2FC, log2 fold change of gene expression values in normalized counts in RC-KO vs. RC-WT comparison; TCA, tricarboxylic acid.

GO term and pathway enrichment analysis of protein coding URGs and DRGs revealed that the URGs were enriched in mitochondrial genes involved in oxidative phosphorylation, mitochondrial organization, multiple metabolic pathways, and lysosomal genes (Fig. 2C and Supplementary Table 5). In contrast, DRGs were associated with microtubule cytoskeleton, insulin secretion, chromatin organization, transcription, FoxO and Mapk signaling, and cell junction organization. Several of the top URGs are critical for neural and β-cell development, including TFs (Ascl1, Fev, and Neurog3) and growth factors (Nog, Wif1, and Igf2) (Fig. 2D). Other top URGs include gastrin (Gast), a putative marker of dedifferentiating β-cells (26), the EF-hand domain Ca2+-binding protein S100a6, voltage-gated K+ channels (Kcnc2 and Kcns3), and Ca2+ channels (Slc24a33 and Cacng3), which are likely involved in compensatory regulation of ion flow in the absence of functional KATP channels (Fig. 2D). Top DRGs are involved in insulin secretion (Nnat and Ins1), cell junction formation (Cldn8 and Pcdh15), potassium ion transport (Trpm5 and Hcn1), response to vascular endothelial growth factor (Kdr and Flt1), and gene transcription (Npas4 and Egr4). These results indicate that β-cell excitotoxicity increases expression of many developmentally important TFs and genes required for mitochondrial energy production while also broadly downregulating genes involved in insulin secretion, chromatin maintenance, and cytoskeletal function.

Overnutrition

Next, we determined the effects of overnutrition on WT β-cells by comparing the HFD-WT and RC-WT data sets. This analysis revealed 2,372 affected genes (1,320 URGs and 1,052 DRGs) (Fig. 3A and B and Supplementary Table 4). Functional enrichment analysis indicated that URGs were involved in ER protein processing, ER stress, unfolded protein responses, glycan biosynthesis, and cell cycle regulation. In contrast, DRGs were involved in chromatin organization and response to hormone stimulus, as well as mammalian target of rapamycin signaling pathways (Fig. 3C and Supplementary Table 5). The top URGs included hormone receptors (Ptger3 and Oxtr), immune cell surface proteins (Cd74 and H2-Eb2), and cell cycle regulators (Cdc20 and Ccnb1) (Fig. 3D). Top DRGs included receptors (Erbb2 and Hspg2), extracellular matrix proteins (Olfm2 and Hspg2), secreted growth factors (Igfbp5 and Angptl7), and TFs (Trnp1 and Epas1). These results indicate that overnutrition causes increased expression of genes involved in ER protein processing and β-cell proliferation and downregulation of genes involved in mammalian target of rapamycin signaling and chromatin maintenance.

Figure 3
Figure 3

β-Cell transcriptome changes in response to overnutrition (HFD) in WT mice. A: Volcano plot showing distribution of differentially expressed genes (Log2FC over P value) in HFD-WT vs. RC-WT RNA-sequencing comparison. Top 10 differentially expressed genes are indicated by names, and total numbers of URGs and DRGs are provided (Padj < 0.05). B: Distribution of dysregulated genes by biotype. C: Functional enrichment analysis of URGs and DRGs. Select top enriched pathways are shown. D: DE levels of select top URGs (top) and DRGs (bottom), with colors indicating gene functional associations. ECM, extracellular matrix; ERAD, ER-associated protein degradation; FDR, false discovery rate; Log2FC, log2 fold change of normalized gene expression between HFD-WT and RC-WT samples; mTOR, mammalian target of rapamycin.

Excitotoxicity and Overnutrition

To determine the combined effects of excitotoxicity and overnutrition, we compared the HFD-KO and RC-WT data sets and identified 8,836 dysregulated genes (4,322 URGs and 4,514 DRGs) (Fig. 4A and B and Supplementary Table 4). URGs were involved in oxidative phosphorylation, the citric acid cycle, and nucleotide metabolism, whereas DRGs were involved in cytoskeleton, insulin secretion, cell projection and cell junction organization, and chromatin and transcriptional regulation (Fig. 4C and Supplementary Table 5). Many of the top URGs were also increased in the RC-KO (Ascl1 and Stc2) and HFD-WT (Cd74 and Cdc20) mice (Fig. 4D). Interestingly, genes involved in lipid uptake (Fabp3 and Apoe), stimulation of ketogenesis, and impairment of glycolysis (Hmgcs2 and Pdk4) were only upregulated in response to the combined stresses. The top DRGs included genes involved in cell adhesion (Gjd4 and Dlagap2), acid transporters (Slc28a2 and Slc7a11), and many genes that were also downregulated in either RC-KO (Npy and Nnat) or HFD-WT (Igfpb5 and Atf5) mice. These findings indicate that the combination of excitotoxicity and overnutrition causes a further increase in the expression of β-cell genes involved in energy metabolism and ATP production, as well as genes linked to a decrease in glucose and an increase in FA-derived ketone utilization as an energy source. At the same time, genes associated with cytoskeleton and cell junction organization are downregulated.

Figure 4
Figure 4

β-Cell transcriptome changes in response to excitotoxicity and overnutrition (HFD) in Abcc8 KO mice. A: Volcano plot showing distribution of differentially expressed genes (Log2FC over P value) in HFD-KO vs. RC-WT RNA-sequencing comparison. Top 10 differentially expressed genes are indicated by names, and total numbers of URGs and DRGs are provided (Padj < 0.05). B: Distribution of dysregulated genes by biotype. C: Functional enrichment analysis of URGs and DRGs. Select top enriched pathways are shown. D: DE of select top URGs (top) and DRGs (bottom). Colors indicate gene functional associations. FDR, false discovery rate; Log2FC, log2 fold change HFD-KO vs. RC-WT; TCA, tricarboxylic acid.

Excitotoxicity and Overnutrition Affects Many of the Same Genes and Pathways

To better categorize the many different transcriptional responses, we performed a meta-analysis of genes dysregulated in three comparisons (Fig. 5A). Major functional categories shared among URGs included oxidative phosphorylation, mitochondrial organization, metabolic pathways, and oxidative stress response, and shared downregulated pathways were chromatin organization, cytoskeleton and cell organization, and DNA damage response (Fig. 5B and C). The large overlap in genes dysregulated in response to both excitotoxicity and overnutrition (Fig. 6A) suggests that Ca2+-mediated nuclear responses are involved in many of the responses of β-cells to overnutrition. Of the 620 URGs that were similarly affected by excitotoxicity (RC-KO vs. RC-WT), overnutrition (HFD-WT vs. RC-WT), or excitotoxicity and overnutrition (HFD-KO vs. RC-WT), the most highly affected genes were Mc5r, a melanocortin receptor, Aldh1a3, an oxidoreductase for which expression correlates with β-cell failure (27), and Gabra4, a GABA receptor subunit that potentiates insulin secretion (28). In contrast, 522 DRGs were shared among all three comparisons with Trnp1, a regulator of cell cycle progression (29), tribbles pseudokinase 3 (Trib3), a multifunctional signaling protein involved in coordinating stress-adaptive metabolic responses (30), and glucagon receptor (Gcgr) being the most highly downregulated.

Figure 5
Figure 5

GO terms and pathways common for β-cell genes dysregulated in excitotoxicity, overnutrition, and the combination of both stresses. A: Cord diagrams show genes (purple curves) and GO terms/pathways (blue curves) shared among lists of URGs and DRGs from three comparisons. Excitotoxicity (blue, RC-KO vs. RC-WT comparison), overnutrition (green, HFD-WT vs. RC-WT comparison), and excitotoxicity and overnutrition (green, HFD-KO vs. RC-WT comparison). Enrichment network visualization of GO terms/pathways shared among URGs (B) and DRGs (C) from the three comparisons. Node size is proportional to the number of genes in GO category, with pie charts indicating a proportion of genes from each comparison. Intensity of a node border color indicates GO category enrichment P value (from 10−48 to 10−2). AA, amino acid; TCA, tricarboxylic acid.

Figure 6
Figure 6

Overlapping β-cell genes dysregulated in excitotoxicity, overnutrition, and the combination of both stresses. A: Venn diagrams indicating overlap between DRGs or DRGs (Padj < 0.05) identified from each pairwise comparison, with select top dysregulated genes indicated for each overlap. Excitotoxicity (blue, RC-KO vs. RC-WT comparison), overnutrition (light green, HFD-WT vs. RC-WT comparison), and excitotoxicity and overnutrition (pink, HFD-KO vs. RC-WT comparison). Genes in the circle that overlap among all three comparisons are overlapping stress URGs or DRGs. B: Functional enrichment analysis of URGs and DRGs. Select top enriched pathways are shown. ERAD, ER-associated protein degradation; TCA, tricarboxylic acid.

Functional gene enrichment analysis showed that overlapping stress URGs are involved in ER protein processing, glycan biosynthesis, metabolic pathways, oxidative phosphorylation, and lysosomes (Fig. 6B and Supplementary Table 5). Included were genes involved in carbohydrate metabolism (Me3 and Mdh1), amino acid metabolism (Gatm and Oat), FA β-oxidation (Acad11 and Acadvl), components of complexes I–V of mitochondrial electron transport chain (Uqcrfs1, Atp5d, Cox6b1, and Ndufc2), and mitochondrial rRNA proteins (Mrps12 andMrpl51) (Supplementary Fig. 4). Common URGs also include oxidoreductases (Ald1a3 and Aass), secreted proteins (Gc and Vgf), redox homeostasis maintenance genes (Gsto2 and Gpx3), lysosome (Ctsh and Dapl1), ER protein folding (Ppib and Selenos), and vesicle traffic (Rgs8) genes. Notably, genes involved in Ca2+ signaling (Camk1d and Mapkapk3) and TFs that are activated by Ca2+ signaling (Mef1c and Nfatc1) are among common URGs (Supplementary Fig. 4). Other upregulated TFs include Fev, Bach2, Etv1, Ppargc1a, and Bhlha15. Overlapping stress-induced URGs also contain genes involved in DNA damage cell-cycle checkpoint (Check1 and Ccng1), apoptosis regulation (Bcl2 and Endog), potassium ion transport (Kcnk13 and Slc12a2), receptors (Gabra4 and Gfra4), extracellular matrix (Col8a2 and P3h2), and immune response (H2-Eb1 and Tnfrsf11b).

DRGs common to all three comparisons are involved in transcription, chromatin modification, protein phosphorylation, as well as adherens junctions and FoxO1 signaling pathways (Fig. 6B and Supplementary Table 5). Downregulated TFs included known regulators of β-cell identity and function (Myt1, Thra, Myt1l, and Stat5a) and many for which the role in β-cells has not been studied (Mesp2, Phf21b, Otub2, and Chd7) (Supplementary Fig. 5). Over 40 proteins involved in epigenetic regulation were decreased, including chromatin-modifying enzymes (Kdm6b, Jmjd1c, and Kat2b), DNA methylation enzymes (Dnmt3a and Tet3), and miRNA-processing proteins (Ago1 and Tnrc6c). Common DRGs also included those involved in regulation of circadian rhythms (Prkab, Prkag2, and Nr1d1), the DNA damage response (Plk3, Taok1, and Primpol), Bmp and Wnt signaling (Acvr1c, Bmpr2, and Amer1/2), cell junction and polarity (Nectin1, Cldn4, Dlgap3, and Pard3), and cilia morphogenesis (Alms1, Cep162, Rfx3, and Ulk4) (Supplementary Fig. 5). In addition, common DRGs were for receptors (Ffar1 and Trpc1), kinases (Prkab2 and Jak), the phosphatidylinositol 3-kinase/AKT/FoxO1 signaling pathway (Akt3, Insr, and Foxo1), Ca2+ transport (Trpc1 and Grin2c), and proteins involved in amino acid transport (Slc7a11 and Slc36a1). A total of 114 long noncoding RNAs (lncRNAs) were also reduced in all three comparisons (Supplementary Fig. 6).

Analysis of genes that were dysregulated only in the presence of both stresses (1,098 URGs and 1,407 DRGs) (Fig. 6A) showed a further increase in oxidative phosphorylation, translation, and nucleotide metabolism genes and a decrease in microtubule-based processes, RNA transport, cilia, and nuclear pore organization genes (Supplementary Fig. 7 and Supplementary Table 5). Importantly, an increase in genes that impair glucose utilization for energy production (Hmgcs2 and Pdk4) was observed only when excitotoxicity and overnutrition were combined, suggesting that the two stresses additively cause metabolic inflexibility.

Sex Influences the Responses to Excitotoxicity and Overnutrition

To determine how sex affects the response of β-cells to excitotoxicity and overnutrition, we reanalyzed our glucose homeostasis measurements and transcriptome data to extract these differences. As shown in Supplementary Figs. 8 and 9, male WT and KO mice gained more weight and had higher fed glucose concentrations and greater glucose intolerance than females of the same genotypes after 5 weeks on HFD. Verapamil treatment during HFD improved glucose tolerance in both the WT and KO males, but not in females of the same genotypes (Supplementary Fig. 9). However, while verapamil improved insulin tolerance in both the WT males and females, it had no effect on the KO mice (Supplementary Fig. 10). These findings indicate that male mice are more susceptible to negative effects of increased [Ca2+]i than are female animals.

To determine how sex affects β-cell stress responses, we performed four different female-versus-male pairwise comparisons on the RNA-sequencing data sets (Supplementary Tables 3 and 4). Comparison of the RC-WT, RC-KO, and HFD-KO data sets in this manner yielded 140, 36, and 126 sex-specific genes, respectively (Padj < 0.05) (Supplementary Table 3). The lower number of sex-specific changes in the RC-KO and HFD-KO mice, compared with the RC-WT mice, suggests that the marked perturbation of the β-cell transcriptome that occurs in Abcc8 KO mice hinders the detection of sex-related differences. However, 2,618 were differentially expressed between female and male HFD-WT data sets. Overlaying all of the differentially expressed genes from the four pairwise comparisons revealed seven core sex-enriched genes, with Xist, Fmo1, and Kdm6a being female enriched and Kdm5d, Uty, Eif2s3y, and Ddx3y being male enriched (Supplementary Fig. 11A).

GO analysis of sex-enriched genes from HFD-WT comparison revealed that female-enriched pathways included oxidative phosphorylation, proteasome degradation, spliceosome, glutathione metabolism, and adrenergic signaling. Male-enriched pathways included ER to Golgi vesicle traffic, autophagy, and cell cycle (Supplementary Fig. 11B and Supplementary Table 5). Among the top genes expressed higher in females on HFD were neuropeptides (Npy and Pyy), hormones (Gcg and Sst), as well as developmental endocrine TFs (Neurog3, Mafb, Fev, Arx, and Hhex) (Supplementary Fig. 11C). In males, the more abundantly expressed genes included Mc5r and Aldh1a3, cell proliferation genes (Mki76 and Ccna), DNA damage-response genes (Pole and Fanca), and transcriptional regulators (Chd5, Bach2, and Txnip). Overall, transcriptional response of male β-cells to HFD indicates a greater increase in β-cell proliferation, secretory function, autophagy, and associated DNA repair and ER-associated protein degradation pathways. The transcriptional response of female β-cells to HFD shows an increase in mitochondrial function, glutathione antioxidant defense, adrenergic signaling, and changes in β-cell identity.

Dysregulated Transcription Factor Gene Expression Is Modulated by Increased [Ca2+]i and PA In Vitro

Because our in vivo analyses revealed the modulation of genes involved in mitochondrial energy production and the maintenance of β-cell identity, we further analyzed Ppargc1a, Bach2, Thra, and Myt1 in cultured islets by RT-PCR (Fig. 7A). Ppargc1a is a transcriptional coregulator that is central to activation of mitochondrial energy metabolism, FA β-oxidation, and mitochondrial biogenesis (31). Bach2 belongs to a family of TFs induced by oxidative stress that may play a role in immune-mediated β-cell apoptosis (32). Myt1 and Thra, a thyroid hormone nuclear receptor, both contribute to the function of mature β-cells (33,34). Cultured WT mouse islets were treated with tolbutamide, a KATP channel inhibitor, PA, or a combination of both agents alone and with verapamil. After 24 h, the expression of Ppargc1a and Bach2 was increased, and Thra and Myt1 decreased, in response to tolbutamide alone. These changes were greatly accentuated when tolbutamide and PA were combined (Fig. 7B) and largely negated with the addition of verapamil. While the effect of PA by itself was generally small, its combination with tolbutamide was strongly additive, particularly for Ppargc1a. These findings provide additional evidence for rapid changes in key β-cell TFs and FA signaling in response to a rise in [Ca2+]i.

Figure 7
Figure 7

Expression of overlapping stress-dysregulated TFs is modulated by increased [Ca2+]i and PA in vitro. A: Heat map of expression of TF genes Ppargc1a, Bach2, Thra, and Myt1 in β-cells from RC-WT, HFD-WT, RC-KO, and HFD-KO mice. RNA-sequencing data were normalized across all data sets, with color intensity indicating relative gene expression level within each row. B: RT-qPCR results using whole-islet RNA from WT islets treated for 24 h with 100 μmol/L tolbutamide (Tol), 0.5 mmol/L PA, a combination of both (Tol+PA), and with 50 μmol/L verapamil (Tol+PA+Ver). Expression of Ppargc1a and Bach2 is upregulated and expression of Thra and Myt1 is downregulated in response to drug treatment, with the most pronounced effect occurring in response to PA. The effects are negated when Ca2+ influx is blocked with verapamil. Data from five experiments containing both male and female samples were averaged for a total of 10 different islet samples. Error bars: ± SEM. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (determined by ANOVA). Cont, control; max, maximum; min, minimum.

Excitotoxicity and Overnutrition Additively Impair Mitochondrial Function

In β-cells, mitochondrial metabolism is essential for coupling glucose metabolism to insulin secretion. To determine whether the observed increases in genes involved in mitochondrial respiration, organization, and FA β‐oxidation reflect actual changes in mitochondrial function, we compared mitochondrial OCR and mitochondrial biogenesis of RC-WT, RC-KO, HFD-WT, and HFD-KO islets. OCR measurements (Fig. 8A) showed that basal respiration rate reflecting the cell baseline metabolic energy production was progressively increased in HFD-WT, RC-KO, and HFD-KO islets (Fig. 8B). Similarly, metabolically stressed islets also had increased spare respiratory capacity, indicating increased ability to respond to increased energy demand (Fig. 8C) and increased mtDNA copy number (Fig. 8F). These results are consistent with corresponding increases in both mitochondrial respiration and biogenesis gene expression. However, the change in OCR in response to glucose is significantly decreased in RC-KO islets and almost completely ablated in HFD-KO islets (Fig. 8D), indicating that excitotoxicity and overnutrition additively impair mitochondrial glucose metabolism, most likely due to an increased FA β-oxidation, a rise in metabolic inflexibility, and mitochondrial damage. Consistent with an additive increase in mitochondrial damage, mitochondrial coupling efficiency is decreased and proton leak is increased in HFD-KO islets (Fig. 8E and F). These findings directly indicate that excitotoxicity and overnutrition additively impair mitochondrial glucose metabolic coupling function.

Figure 8
Figure 8

Excitotoxicity and overnutrition affect islet mitochondrial function. A: OCR profiles measured by Agilent Seahorse mitochondrial stress assay. Islets from RC-WT, HFD-WT, RC-KO, and HFD-KO male mice at 8–9 weeks of age were consecutively treated with 20 mmol/L glucose (20G), 5 mmol/L oligomycin A (Oligo), 1 mmol/L FCCP, and 2.5 mmol/L antimycin A/rotenone (AA/Rot). n = 12 wells for each condition. *P ≤ 0.05, RC-WT vs. HFD-KO; #P ≤ 0.05, RC-KO vs. HFD-WT. B: Basal respiration was increased in HFD-WT, RC-KO, and HFD-KO islets. Basal respiration rate was calculated by subtraction of nonmitochondrial respiration from basal respiration rate. C: Spare respiratory capacity, or the ratio of basal respiration to maximal respiration after FCCP injection (×100), was increased in RC-KO islets. D: Glucose-stimulated OCR response was decreased in RC-KO and HFD-KO islets. Glucose response was calculated by subtracting basal respiration rate from the OCR after glucose injection. E: Coupling efficiency, or the ratio of basal respiration to ATP production rate (×100), was decreased in HFD-KO islets. The ATP production rate was calculated by subtracting the minimal rate after oligomycin injection from the basal respiration rate. F: Proton leak, or the minimal rate after oligomycin injection minus nonmitochondrial respiration, was increased in RC-KO and HFD-KO islets. G: Relative mtDNA copy number was increased in HFD-WT, RC-KO, and HFD-KO islets. mtDNA to nuclear DNA (nDNA) using real-time PCR. Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (determined by ANOVA).

Discussion

To gain a systems-wide understanding of pancreatic β-cell failure, we explored the additive effects of excitotoxicity and overnutrition on β-cell function and gene expression. Among the many changes that occur in β-cells in response to metabolic stress, we identified several critical alterations that may be tipping points for the development of T2D.

Excitotoxicity and HFD Additively Impair β-Cell Function

It has long been known that C57BL/6J (WT) mice develop insulin resistance and impaired β-cell function on an HFD (35). Our studies strongly suggest that an increase in [Ca2+]i, as occurs in the Abcc8 KO β-cells, increases the propensity of β-cell to fail in response to HFD and, conversely, verapamil, which blocks calcium entry and protects against the loss of β-cell function. RC-KO mice, which are euglycemic at 8–9 weeks of age (11,23), exhibit greater insulin sensitivity than RC-WT mice, as has been reported for mice lacking Kcnj11, another essential KATP channel component (36). While HFD predictably caused insulin resistance in WT mice, Abcc8 KO mice, like Kcnj11 KO animals (37), remained insulin sensitive on HFD, indicating that observed impairments in glucose homeostasis are mostly due to loss of β-cell function. Furthermore, while verapamil had no effect on insulin sensitivity in the KO mice, it increased the insulin sensitivity of WT mice on an HFD. Thus, our results not only confirm that Ca2+ channel blockers attenuate the development of obesity-induced insulin resistance (38), but they also indicate that the protective effects of these agents extend to β-cells. Moreover, they also indicate that an increase in [Ca2+]i, besides contributing to obesity-induced insulin resistance (39), has a negative effect on β-cell function. Indeed, given the pleiotropic metabolic effects of dysregulated Ca2+ homeostasis, it is noteworthy that the effects of Ca2+ channel blockers in both type 1 diabetes and T2D are now being investigated (40,41).

β-Cell Transcriptome Changes in Response to Excitotoxicity, HFD, and a Combination of Both Stresses

Excitotoxicity and overnutrition each have a major impact on β-cell gene expression, and together, the two stresses affect the expression of 11,952 unique genes, or nearly three-quarters (72%) of all genes expressed in β-cells. We also observed an overlap between genes and pathways that were dysregulated in response to both stresses, suggesting that increased [Ca2+]i may be involved in mediating the nuclear responses of other metabolic stresses.

Energy Metabolism and Mitochondrial Function

The largest category of genes upregulated by excitotoxicity and/or overnutrition are those involved in mitochondrial function, metabolism, and oxidative phosphorylation. Mitochondrial metabolism is a major determinant of insulin secretion from pancreatic β-cells, and mitochondrial dysfunction plays a key role in development of T2D (42). Increased [Ca2+]i, in both normal and pathological states, invariably leads to increased mitochondrial Ca2+ uptake that stimulates mitochondrial Ca2+-sensitive metabolic enzymes and oxidative phosphorylation in the electron transport chain (43). However, a persistent increase in mitochondrial [Ca2+] and respiration may lead to an increase in reactive oxygen species production, collapse of the mitochondrial membrane potential, and mitochondrial dysfunction (6). Consistently, we observed that even though mitochondrial biogenesis and basal respiration are increased in KO islets, they also exhibit decreased coupling efficiency and increased proton leak upon addition of HFD, indicating mounting mitochondrial damage. Normally, damaged mitochondria are replaced by the combination of mitophagy, a lysosomal-based degradation process, and the production of new mitochondria that occurs through mitochondrial biogenesis. A balance between these processes is essential for normal β-cell function (44). Excitotoxicity increases the expression of multiple mitochondrial and lysosomal genes, as well as master regulators of both mitochondrial (Ppargc1a) and lysosomal (Tfeb) biogeneses, potentially maintaining biogenesis/mitophagy balance. However, the combination of both excitotoxicity and overnutrition shifts this balance, as there is a further increase in mitochondrial energy metabolism, oxidative stress, and DNA damage-response genes, indicating an increase in reactive oxygen species and mitochondrial damage, while expression of lysosomal genes is not further changed, and several mitophagy-associated genes, such as Clec16a and Prkn (45), become downregulated. These results suggest that the combination of excitotoxicity and overnutrition overwhelms the ability of β-cells to replace metabolically damaged mitochondria.

Ppargc1a, a central transcriptional regulator of mitochondrial biogenesis, energy metabolism, and FA β-oxidation, is also increased in β-cells in response to both excitotoxicity and overnutrition. PPARGC1A interacts with multiple TFs and chromatin modifiers to regulate metabolic reprogramming in response to diet and oxidative stress and is implicated in pathogenesis of T2D (46). Ppargc1a is necessary for normal β-cell function (47), and its overexpression causes β-cell dysfunction (48). In this study, we show that expression of Ppargc1a is induced in cultured WT islets in response to increases in [Ca2+]i and is further increased with the addition of PA. This finding further implicates [Ca2+ ]i as an important regulator of mitochondrial metabolism in β-cells and suggests that an increase in [Ca2+]i causes the rapid upregulation of Ppargc1a, most likely through the activation of Ca2+-dependent CaN/CaMK/MAPK/AMPK signaling pathways. Similar signaling pathways are activated in skeletal muscle cells in response to exercise (49). In support of this hypothesis, excitotoxicity and overnutrition both upregulate Mef2c, a known target of Ca2+ signaling and a known activator of Paprgc1a in muscle, several CaMK and MAPKs, and multiple Ppargc1a target genes (Supplementary Fig. 12A). Similar to the activation of Ppargc1a, we also confirm an additive effect of increased [Ca2+]i and PA on the upregulation of Bach2, an oxidative stress–responsive TF, and the downregulation of both Myt1 and Thra, two other TFs important for the function of mature β-cells. However, less is known about how these other important genes are regulated and how they may contribute to normal β-cell function and the maintenance of β-cell identity.

Coupling of glucose metabolism to insulin secretion is an important aspect of mitochondrial function in β-cells. We find that the combination of excitotoxicity and overnutrition increases expression of genes that contribute to metabolic inflexibility or the decreased ability to use glucose as an energy source, another key feature of β-cell failure (50). Similar to overnutrition alone, excitotoxicity alone causes an increase in genes involved in FA β-oxidation, indicating that β-cells partially switch to utilization of fat as a fuel in response to an increased [Ca2+]i, a process known as glucose sparing (Supplementary Fig. 12B). This switch is reflected in reduced ability of KO islets to increase mitochondrial respiration in response to glucose. However, in addition to FA β-oxidation, combination of excitotoxicity and overnutrition causes increases in Pdk4, a kinase that inhibits pyruvate flux into the tricarboxylic acid cycle, promoting FA and ketone body utilization (51), and Hmgcs2, a rate-limiting enzyme in ketone body production that is activated by FAs (52). Because these changes would be expected to impair glycolytic flux and cause metabolic inflexibility (Supplementary Fig. 12C), they may explain the observed collapse of mitochondrial glucose response in HFD-KO islets. Therefore, our data suggest a tipping point at which the combination of an increase in [Ca2+]i and an elevation in FAs causes the β-cell to cease relying on glycolysis and to switch to FAs and ketones as their fuel source, impairing their ability to sense and respond to changes in the blood glucose concentration.

ER Protein Folding and Protein Glycosylation

Glycosylation is a process during which glycans (mono- or oligosaccharides) are attached to proteins in the ER and Golgi that serve as a quality control signal in ER protein folding (53). In β-cells, increases in protein glycosylation cause ER stress, eventually leading to apoptosis (54). We found that overnutrition in particular increased expression of genes associated with ER protein folding and N– and O-linked protein glycosylation, suggesting that the stability, localization, trafficking, and function of many receptors, ion channels, nutrient transporters, and TFs are also adversely affected, likely contributing to development of T2D (55).

β-Cell Structure: Cytoskeleton, Cell Polarity, and Cell Adhesion

Excitotoxicity, overnutrition, and the combination of both stresses downregulate genes important for cell organization and secretory function of β-cells, including cell adhesion, cell junctions, cilia, cytoskeleton, and vesicular trafficking genes. We have previously identified impairments in islet architecture of Abcc8 KO mice (11), suggesting that critical cell-to-cell contacts, which are necessary for insulin secretion, may become impaired (56). The downregulation of synaptic vesicle-targeting proteins, GTPases, and cytoskeletal proteins has been shown to affect insulin exocytosis (57). Similarly, alterations in β-cell polarity may occur as genes associated with the apical domain (Pard3) and associated primary cilia (Alms1 and Cep162) and lateral domain (Dlgap3 and Nectin1) are downregulated, and genes associated with the vasculature-facing basal domain (Col8a2, Ntn4, and Ppfia3) are increased (58). These changes indicate that Ca2+ signaling in β-cells is crucial for maintaining cell polarity and cytoskeleton dynamics and that a chronic increase in [Ca2+]i impairs the ability of these cells to secrete insulin.

β-Cell Identity

Another important category of genes downregulated in metabolically stressed β-cells is those involved in transcriptional control. Besides decreases in many TFs that are necessary for function of mature β-cells (Myt1, Thra, Pbx1, Stat5a, and Nrf1), we also identified several other stress-inhibited TFs (Mesp2, Klf7, Nfia, Ikzf3, and Chd7) that may be critical for maintaining β-cell identity and function. Similarly, many chromatin modifiers, including histone methyltransferases and acetyltransferases, were downregulated in response to both stresses. Among these is Dnmt3a, a DNA methyltransferase important for silencing of developmental or “disallowed” metabolic genes in mature β-cells (59). Consistent with this, several disallowed genes (60) (Slc16a, Oat, and Aldob) are upregulated in response to excitotoxicity and/or overnutrition. Finally, maintenance of the epigenetic and transcriptional landscape of β-cells is also regulated by lncRNAs (61), and we identified multiple lncRNAs that are downregulated in response to these two metabolic stresses.

Effects of Sex on β-Cell Stress Responses

Male rodents have a greater propensity for β-cell failure than do females (18). In this study, we analyzed the effects of excitotoxicity and/or HFD on β-cell function and gene expression in both sexes. We found that female animals (both WT and Abcc8 KOs) withstand overnutrition better than males. These results are consistent with previous data on HFD-WT mice (62) and may reflect the protective influence of female sex hormones on β-cell function and metabolism (63). Interestingly, verapamil improved insulin sensitivity and glucose clearance in both the HFD-WT and HFD-KO males, but had little effect in females, suggesting that males are more negatively affected by a stress-induced increase in [Ca2+]i than females. Testosterone is known to increase [Ca2+]i in multiple tissues (64) and may also predispose β-cells for dysfunction associated with further [Ca2+]i increase due to increased metabolic load.

Analysis of sex differences on a transcriptome level in WT mice confirmed our previous findings that female β-cells express higher amounts of several TFs important for β-cell function including Mlxipl, Nkx2-2, and Hnf1b (22). Interestingly, we were only able to detect a few sex-related changes in β-cells from both RC- and HFD-Abcc8 KO mice, suggesting that the massive gene expression changes that occur in the Abcc8 KO mice may mask the detection of sex-related differences. However, overlaying the differentially expressed genes from all four female to male transcriptome comparisons allowed us to identify three core female-enriched β-cell genes (Xist, Fmo1, and Kdm6a) and four male-enriched genes (Kdm5d, Uty, Eif2s3y, and Ddx3y). All of the male-enriched genes are located on the Y chromosome, whereas only two of the female-enriched genes (Xist and Kdm6a) are located on the X chromosome. Kdm6a, Uty, and Kdm5d all code for histone demethylases that may be involved in sex-specific epigenetic regulation of gene expression (65,66). Our discovery that Fmo1, an autosome-located gene, is enriched in female β-cells is interesting, as this gene encodes a flavin-containing monooyxgenase 1, a drug-metabolizing enzyme that regulates energy balance (67). Another member of the same family, Fmo4, was also upregulated in females on HFD. Overall, the most profound sex differences revealed by our analysis were in the β-cell transcriptome on HFD (2,618 genes). Female β-cells express higher levels of genes involved in oxidative phosphorylation, suggesting they may have higher energy metabolism than do male β-cells. Genes involved in prevention of oxidative damage, such as glutathione peroxidases, are also higher in female cells, suggesting a mechanism enabling female β-cells to tolerate overnutrition better than males. Female β-cells also express more of neuropeptides Npy and Pyy, both of which could be protective against β-cell damage (68,69), and the adrenergic receptor Adrb2, signaling through which can increase insulin secretion. Consistent with this, it was recently reported that pancreas-specific loss of Adrb2 causes glucose intolerance and impaired glucose-stimulated insulin secretion only in female mice (70). Intriguingly, genes associated with endocrine progenitor (Neurog3 and Mafb) and α-cells (Gcg and Arx) and δ-cells (Sst and Hhex) are upregulated in HFD-fed female β-cells, suggesting changes in cell identity. Male mice fed HFD exhibit higher expression of β-cell genes involved in protein secretion and cell proliferation, consistent with the well-established fact that male β-cells exhibit greater proliferation when fed HFD (71).

Concluding Remarks

We have identified many different genes and pathways in β-cells that are affected by metabolic stress. The broad-based nature of the changes we observe suggests that β-cell failure in T2D is not due to the failure of a single cellular process, but instead is a complex, multifaceted, and additive process in which Ca2+ signaling plays a crucial role.

Article Information

Acknowledgments. The authors thank the Vanderbilt Technologies for Advanced Genomics (VANTAGE) Core and the Vanderbilt University Medical Center Flow Cytometry Shared Resource for assistance in performing cell sorting and RNA sequencing and the Vanderbilt Islet Procurement and Analysis Core for help with isolating islets.

Funding. This research was supported by institutional and philanthropic funds provided by Vanderbilt University. The Vanderbilt Islet Procurement and Analysis Core is supported by DK-020593. VANTAGE is supported by grants P30-CA-68485, P30-EY-08126, and G20-RR-030956. Vanderbilt University Medical Center Flow Cytometry Shared Resource is supported by grants P30-CA-68485 and DK-058404.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. A.B.O. and M.A.M. designed the study. A.B.O., J.S.S., and K.D.D. performed experiments. A.B.O. analyzed the data. J.-P.C. performed RNA-sequencing data processing, alignment, and DE analyses. A.B.O. and M.A.M. wrote and edited the manuscript. J.S.S. edited the manuscript. M.A.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.



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Butternut Squash Posole (Vegan, Gluten-free)

By electricdiet / July 8, 2020


This butternut squash posole combines sweet, spicy, and earthy flavors for a delicious vegan soup that will warm you right up!

Butternut Squash Posole in a bowl topped with chopped avocado and cilantro

This vegan butternut squash posole is a rich, hearty soup that’s easy to make and packed with amazing flavor!

It’s a little bit sweet, a little bit spicy, a little bit earthy, and perfect to warm you up on a chilly day.

Traditional posole (also spelled “pozole”) is a Mexican dish that features hominy, pork or chicken, chile peppers, and seasonings. This vegan recipe uses hearty vegetables and spices to get all that delicious flavor without the meat!

If you’re looking for a rich soup packed with tasty veggies to fill you up, definitely give this easy recipe a try.

How to make butternut squash posole

This posole comes together in one pot on the stove with just a few simple steps!

You can see how in this video and follow the step-by-step instructions below.

Step 1: In a large Dutch oven or heavy-bottom pot, heat the oil over medium-high heat.

Step 2: Stir in the chili powder, then immediately add the squash, chile peppers, oregano, cumin, garlic, and salt.

Step 3: Cook, stirring frequently, until the peppers soften and the spices are evenly distributed over the squash, about 5 minutes.

Step 4: Add water (or stock) and tomatoes, then cover and bring to a slow boil.

Step 5: Uncover, reduce the heat to low, and simmer until the squash is tender, about 20 minutes.

Step 6: Add the hominy and cook until warmed through, about 3 minutes.

Step 7: Top with avocado before serving.

Your delicious vegan posole is ready to enjoy! I like to garnish mine with cilantro for even more wonderful flavor.

To reduce the sodium in this recipe, you can use no-salt-added canned tomatoes or skip the additional Kosher salt.

What to serve with posole

What goes well with a dish that features dried corn kernels as a main ingredient?

More corn, of course!

I love to serve vegan posole with a few tortilla chips. They provide an excellent crunch to contrast the soft vegetables.

Or, if your carb count will allow it, a small piece of cornbread is fantastic for soaking up the soup’s flavor!

If you’re looking for something a bit lighter, a green salad with orange vinaigrette would also be nice.

Butternut Squash Posole in a bowl topped with chopped avocado and cilantro

Is posole a soup or a stew?

The main difference between a soup and a stew is how the dish is prepared. Specifically, a stew is made by stewing the ingredients — in other words, submerging the ingredients and simmering them in a covered pot until they are cooked through.

Usually, a stew uses just enough liquid to cover the other ingredients. As it cooks, the liquid reduces, so a stew becomes very thick.

This means that our vegan posole is probably more of a soup, which uses a good amount of liquid and simmers the ingredients to extract their flavor.

Mexican-inspired posole may seem like a stew because of all the hearty ingredients. If you want, you can always add more water or broth to create a thinner soup.

Storage

Any leftover posole can be stored covered in the refrigerator. For maximum freshness, you should eat the rest of your soup within 4-5 days.

You may notice that the flavors become even deeper and richer after sitting in the fridge overnight. That’s why I always try to make enough to have leftovers!

Other healthy vegan recipes

Vegan recipes can be so warm and comforting! If you’re looking for a few more hearty recipes that are packed with flavor and totally meat-free, here are some of my favorites that I know you’ll enjoy:

When you’ve tried this posole, please don’t forget to let me know how you liked it and rate the recipe in the comments below!

Recipe Card

Butternut Squash Posole (Vegan, Gluten-free)

Butternut Squash Posole

This butternut squash posole combines sweet, spicy, and earthy flavors for a delicious vegan soup that will warm you right up!

Prep Time:5 minutes

Cook Time:25 minutes

Total Time:30 minutes

Author:Shelby Kinnaird

Servings:4

Instructions

  • In a large Dutch oven or heavy-bottom pot, heat the oil over medium-high heat.

  • Stir in the chili powder, then immediately add the squash, chile peppers, oregano, cumin, garlic, and salt.

  • Cook, stirring frequently, until the peppers soften and the spices are evenly distributed over the squash, about 5 minutes.

  • Add water (or stock) and tomatoes, then cover and bring to a slow boil.

  • Uncover, reduce the heat to low, and simmer until the squash is tender, about 20 minutes.

  • Add the hominy and cook until warmed through, about 3 minutes.

  • Top with avocado before serving.

Recipe Notes

This recipe is for 4 servings of posole. To reduce the sodium, use no-salt-added canned tomatoes or skip the additional Kosher salt. Leftovers can be stored covered in the refrigerator for 4-5 days.

Nutrition Info Per Serving

Nutrition Facts

Butternut Squash Posole

Amount Per Serving (0 g)

Calories 270 Calories from Fat 131

% Daily Value*

Fat 14.5g22%

Saturated Fat 2.7g17%

Trans Fat 0g

Polyunsaturated Fat 1.8g

Monounsaturated Fat 9g

Cholesterol 0mg0%

Sodium 720.2mg31%

Potassium 769.9mg22%

Carbohydrates 31.2g10%

Fiber 9.3g39%

Sugar 7.6g8%

Protein 4.7g9%

Net carbs 21.9g

* Percent Daily Values are based on a 2000 calorie diet.

Course: Soups and Stews

Cuisine: Mexican

Diet: Diabetic, Gluten Free, Vegan

Keyword: butternut squash posole, vegan posole, vegan recipes



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Mock Mojito with Sugar Free Simple Syrup

By electricdiet / July 6, 2020


On July 1 I declared that July was going to be #dryjuly and give up alcohol for the month.  It corresponds with my DietBet that starts today (not too late to join! click here to get the details!)  and in order for me to lose 4%. of my body weight in four weeks (just over 7 pounds) I needed to ditch my beloved wine. 

So Friday night I went to my friend Jacky’s house and had a BBQ with her husband and their friend.  It wouldn’t be a BBQ without some cheese and snacks, right? Jacky made jerk shrimp and melon nachos and I made this mini cheese board from cheese from Mariano’s, only my favorite grocery store in the world. 😀

I knew I wanted to bring a mock tail and earlier in the day decided to see if I could make a sugar free simple syrup.  Only one tiny problem, is that sweeteners like Truvia dissolve, but they don’t really melt like sugar if you know what I mean.  So I decided to add corn starch to thicken it. 

Once I let the mixture cool, it looked . . .really gross.  The corn starch kind of separated on the top, but I did get a syrup!  So I thought, why not strain it?  Which worked perfectly!  Once cooled I added the lime juice and zest.  This will keep in the fridge up to a week.

Print

Mock Mojito with Sugar Free Simple Syrup

A great way to have a summer drink without alcohol.  My sugar free simple syrup was the perfect addition to lime zeltzer and fresh mint.


Scale

Ingredients

1/2 cup water
1/2 cup @truvia
1 tablespoon cornstarch
juice of 1 lime
zest on one lime

Instructions

In a pan over medium heat. Mix the cornstarch with the water. Add that to the Truvia and bring to a low boil, stirring constantly. It should thicken up in about 5 minutes. Let cool. It’s going to look gross, so strain it into a mason jar. Once completely cool, stir in the juice and lime zest.

Per drink: 2 teaspoons lime simple syrup to 1 can of lime seltzer. Add lime and fresh mint. Enjoy!

Notes

This drink is zero points on all WW plans.

I bought not one but TWO tomahawks steaks to cook up this evening.  I brought one to Jacky’s house.   I get a bit nervous cooking on other people’s grills because I don’t know the hot spots, etc.  But it turned out perfect if I do say so myself!

And not that I wasn’t happy to see Jacky, the best part was that she gave me her old bike!  She got a new one last year, and all I needed to do was to add air to the tires.  I rode for 30 minutes last night and it was awesome.  Thank you Jacky!

On Saturday my Mom, Hannah and Jacob came over for another BBQ and another steak.  This time I was able to cook in indirect heat.  I cooked the steak until 115 degrees, then reverse seared it over the hot coals until it reached 120 degrees.

My Mom slept over and we watched Hamilton on the big t.v.  – so good!  We slept in a bit and then Hannah and Jacob came over to mow my lawn and they brought the dogs over to visit.   I treated my Mom to a belated Mother’s Day gift pedicure and wow, did we need that!

My feet have never felt better!

After my Mom left, I took a 45 minute nap.  Um, someone (me) decided it was a good idea to drink coffee at 10:30 at night on Saturday while watching Hamilton (it would usually be wine!) so I had a hard time falling asleep.  

But, I got up, did laundry, cleaned the kitchen and get ready for the work week.  Last week I drove to Chicago for the day to go to the office.  Um, I think I like my 10 second commute better though.

Happy Monday friends.  I hope you had an amazing long weekend, and it’s never too late to make better choices.  I am proud that I have tracked everything, have been moving my body more.  It’s consistency not perfection that will get you to your goal – don’t forget that!

Until next time….hugs!





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10 Healthy Chia Seed Pudding Recipes

By electricdiet / July 4, 2020


Looking for a nutritious breakfast or snack to make ahead? Try one of these healthy chia seed pudding recipes! They’re easy to whip up and come in so many delicious flavors.

Collage of chia seed pudding images

Is chia seed pudding good for you?

Puddings made with chia seeds are becoming more and more popular, which we love to see!

After all, chia seeds are known for giving you a boost of antioxidants and nutrients. Plus, they’re very low in both calories and net carbs while delivering a healthy serving of fiber and plant-based protein.

In fact, almost all of the carbs in chia seeds come from fiber, which your body digests differently than sugar and starches. This means that chia seeds will provide a steady supply of energy without blood sugar spikes.

However, it’s important to pay attention to the other ingredients in your pudding. While chia seeds themselves are quite healthy, you can easily undo those benefits by loading up your pudding with other sugars and carbs!

The key is to find puddings made with healthy low-carb ingredients.

Delicious pudding with chia seeds

If you’ve been looking for new ways to use chia seeds in tasty pudding recipes, you’re in luck! There are so many delicious options that deliver all the benefits of chia seeds without spiking your blood sugar.

From light and fresh puddings made with fruit to rich and creamy chocolate puddings that feel like you’re eating dessert, there’s a recipe for everyone.

Whip up any of these tasty recipes and enjoy a wonderful nutrient-packed pudding for a satisfying breakfast or perfect snack on the go!

With so many different flavors to try, you may never run out of delicious ways to enjoy chia seeds!

More healthy recipe roundups

It’s always fun diving into a collection of healthy recipes. If you’re looking for more inspiration, here are a few collections I think you’ll enjoy:

If you try any of these recipes, don’t forget to leave a comment below and let me know how you liked them!



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Central α-Klotho Suppresses NPY/AgRP Neuron Activity and Regulates Metabolism in Mice

By electricdiet / July 2, 2020


Introduction

α-Klotho, a well-documented antiaging protein primarily produced in the kidney and choroid plexus (1,2), has recently been observed to have therapeutic potential in rodent models of metabolic disease (36). Studies show α-klotho promotes lipid oxidation, protects pancreatic β-cells from oxidative damage, increases energy expenditure, and facilitates insulin release (37). Furthermore, circulating α-klotho concentrations are decreased in patients with obesity and diabetes (8), suggesting a possible direct role in the pathophysiology of metabolic disorders. Notably, studies have primarily investigated peripheral α-klotho, which neglects the central function of α-klotho due to its impermeability to the blood-brain barrier (9). The few studies investigating centrally circulating α-klotho demonstrate that α-klotho has antioxidative and anti-inflammatory properties (10), is involved in myelination (11), and can be therapeutic in models of hypertension (12). However, the role of central α-klotho in the regulation of metabolism remains unexplored.

Neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing neurons are located within the arcuate nucleus (ARC) of the hypothalamus and are critical to homeostatic regulation of metabolism. NPY/AgRP neurons sense nutritional changes in the cerebrospinal fluid (CSF) to regulate feeding behavior (13), energy expenditure (14), and glucose metabolism (1517). However, disordered overactivity of these neurons results in phenotypes resembling diabetes and obesity (13,15). Some circulating factors, such as leptin and insulin, also modulate NPY/AgRP neurons (18,19), but in metabolic disease states, signaling of these hormones is disrupted. Therefore, identification of novel regulators of this neuron population could facilitate the development of therapeutic tools for the prevention and treatment of metabolic disease.

Recent studies have identified several fibroblast growth factor (FGF) hormones that activate FGF receptor (FGFR)–phosphatidylinositol 3 kinase (PI3K) signaling to elicit antidiabetic effects and regulate NPY/AgRP neurons (2025). Interestingly, α-klotho serves as a critical scaffolding protein to the FGF23-FGFR complex to promote FGFR activity (26,27). The current study investigates the novel role of central α-klotho in the regulation of NPY/AgRP neurons and whole-body metabolism via an FGFR/PI3K mechanism.

Research Design and Methods

Cell Culture

Cell culture experiments were performed on immortal hypothalamic GT1-7 cells cultured in high-glucose (4.5 mg/dL) DMEM, 10% FBS, and 1% penicillin-streptomycin. Cells were treated with 3.65 mmol/L α-klotho (R&D Systems) (10,11,28), treated with 100 ng/mL FGF23 (R&D Systems) (28), and/or pretreated with 10 nmol/L FGFR1 antagonist PD173074 (Fisher Scientific) (29) or 50 nmol/L PI3K inhibitor wortmannin (Fisher Scientific) (25). All experiments used vehicle-treated cells as controls.

Experimental Animals

C57BL/6 and B6.Tg(NPY-hrGFP)1Lowl/J (NPY-GFP reporter) mice were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and experimental protocols were approved by Institutional Animal Care and Use Committees of East Carolina University. Mice were housed at 20–22°C with a 12-h light-dark cycle.

High-Fat Diet–Induced Obesity

The 6-week-old male C57BL/6 mice were given ad libitum access to a high-fat diet with a kilocalorie composition of 58%, 25%, and 17% of fat, carbohydrate, and protein, respectively, for 10 weeks (D12331; Research Diets, New Brunswick, NJ) before undergoing intracerebroventricular (ICV) cannulation.

ICV Cannulation

Prior to the procedure, mice were given oral analgesic meloxicam and anesthetized with intraperitoneal (i.p.) injection of ketamine and xylazine. Mice were placed on a stereotaxic device, and a midline incision was made on the head. A hole was drilled (1.0 mm lateral, −0.5 mm posterior, 2.5 mm deep to the bregma), and a cannula was placed into the lateral ventricle (Supplementary Fig. 1). Another hole was drilled, and a screw was placed approximately at the ipsilateral lambdoid structure to aid in supporting the cannula in the skull with 3M carboxylate dental cement. Mice recovered for 14 days before immunohistochemical experiments and 7 days before all other experiments. All ICV treatments were administered via Hamilton syringe as 2.0 μL between 6:30 and 7:30 p.m.

The 12-Day ICV Injection Timeline

In high-fat diet–induced obesity (DIO) mice, central administration of either 2.0 μg recombinant α-klotho (R&D Systems) alone, 25.0 μg PD173074 alone, 25.0 μg PD173074 10 min before 2.0 μg α-klotho (20), or vehicle was performed daily. On day 7, glucose tolerance tests (GTTs) or insulin tolerance tests (ITTs) were performed, and on day 12, a body composition analysis was performed using EchoMRI (Echo Medical Systems, Houston, TX). Mice were then euthanized, and tissues were collected. Food intake and body weight data were analyzed from the first 7 days to prevent confounding effects from additional assays.

Single ICV Injection Timeline

In DIO mice, ICV administration of either 2.0 μg recombinant α-klotho (R&D Systems), 10 ng wortmannin, 10 ng wortmannin 1 h before 2.0 μg α-klotho (30), or vehicle was performed the night before a GTT or ITT. Food intake was measured for 4 days after the injection.

Streptozotocin-Induced Diabetes

The 8- to 9-week-old chow-fed male mice underwent ICV cannulation before receiving 3 days i.p. injection of 100 mg/kg streptozotocin (STZ). The dose was determined by pilot studies yielding consistently elevated fed glucose levels between 250 and 600 mg/dL (Supplementary Fig. 2A). At 7 days after STZ injection, mice received 7 days ICV treatment with either 2.0 μg recombinant α-klotho (n = 10) or vehicle (n = 9). Food intake, body weight, and fed glucose levels were monitored daily. To identify the effects of α-klotho treatment on glucose levels independent from food intake, a pair-fed experiment was performed using the same protocols, and on day 7, fasting glucose levels were measured (n = 8–9/group).

Central α-Klotho Inhibition via ICV Anti–α-Klotho Antibody

The 9-week-old male, chow-fed mice were ICV treated with 1.0 μg anti–α-klotho (ab-α-klotho) (R&D Systems) or vehicle (n = 8/group). Treatments were performed on the evening of day 1 and morning of day 2, while subsequent treatments were performed between 6:30 and 7:30 p.m. for 7 days. GTTs were performed on day 3, and on day 7, mice were euthanized, followed by tissue collection for further assays.

Food Intake Measurements

Food intake was measured daily by weighing food (8–9 g) and subtracting from the total food. Bedding was inspected thoroughly for residual bits of food, which were included in measurements. On day 4 in one cohort of DIO mice, food was removed from cages during the light phase and replenished at the beginning of the dark phase (7:30 p.m.). Food intake was measured at 0.5, 1, 1.5, 2, 3, 4, 8, 14, and 24 h after food reintroduction.

GTTs and ITTs

For GTTs, 20% glucose solution (1.0 g/kg body weight) was i.p. injected after an overnight fast, and for ITTs, 0.6 units/kg insulin were i.p. injected after a 4-h fast. Tail blood samples were collected 15, 30, 60, 90, and 120 min after injections for analysis using a glucose meter (ReliOn Prime Blood Glucose Monitoring System; ARKRAY Inc., Kyoto, Japan). Serum was isolated from clotted blood spun at 4°C and 2,000g for 30 min. Insulin levels were quantified using an insulin ELISA kit (Crystal Chem).

Insulin-Stimulated Signaling

On day 12 of ICV treatment, a weight-matched cohort of DIO mice was i.p. injected with 10 units/kg insulin or saline. At 7 min after the injection, hypothalamus, epididymal adipose tissue (eWAT), liver, and hindlimb skeletal muscle were flash frozen for future Western blot analysis.

Immunohistochemistry

For immunofluorescent analysis, mice were intracardially perfused with PBS followed by 10% formalin before immunohistochemistry was performed as described previously (31). Briefly, brains were sliced into 20-μm coronal sections using a freezing microtome (VT1000 S; Leica, Wetzlar, Germany) and incubated overnight in antibody to phosphorylated ERK (1:500; Cell Signaling Technology, Danvers, MA) or cFOS (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with Alexa-fluorophore secondary antibody for 2 h. Stains were photographed using an optical microscope (DM6000; Leica), followed by blind analysis using ImageJ. At least three anatomically matched images per mouse were quantified.

Western Blot

Western blot was performed as described previously (31). Briefly, equal protein samples were loaded into a 4–20% HCL gel, transferred to a nitrocellulose membrane, and incubated overnight in 1:500–1:1,000 antibody dilutions in 5% milk with Tris-buffered saline with Tween for phosphorylated (p)ATser473, total AKT, pFOXO1ser256, total FOXO1, pERKthr202/thr204 (Cell Signaling Technology), pIRtyr972 (Invitrogen), and total ERK (Santa Cruz). Image J software was used to quantify mean intensity of equal-area sections representing each sample.

Quantitative PCR

Cell and tissue RNA was extracted by Trizol (Thermo Fisher Scientific, Waltham, MA). The expressions of specific mRNA were analyzed using quantitative real-time PCR (RT-qPCR) (Power SYBR Green PCR Master Mix; Applied Biosystems, Foster City, CA). Reactions were performed in triplicate for each sample, while GAPDH was used as a reference gene for normalization.

Patch Clamp Electrophysiology

We conducted cell-attached voltage clamp recordings of NPY/AgRP neurons as described previously (32,33). Briefly, mice were deeply anesthetized by isoflurane followed by intracardial perfusion with chilled N-methyl-d-glucamine solution and sliced into 200- to 300-μm sections. Slices recovered for an hour in HEPES recovery solution, and recordings were conducted in a normal artificial cerebrospinal fluid (aCSF) bath. For whole cell recordings, gigaohm seals were obtained, and the cells were broken into using negative pressure. Data were sampled at 10 kHz. Current clamp recordings were stabilized for repeated firing under the baseline condition. Equivalent length periods (0.5–5 min) were set within each recording during perfusion of aCSF or α-klotho (3.65 mmol/L). Firing rate (Hz) was calculated by dividing the number of events by the number of seconds. Bath application of terodotoxin (TTX) (1.0 μmol/L) prior to α-klotho treatment was used to determine action potential–independent effects on membrane potential. Voltage clamp whole cell recordings were conducted at a −70 mV holding potential and a high KCl intracellular solution (130 mmol/L KCl, 5 mmol/L CaCl2, 10 mmol/L EGTA, 10 mmol/L HEPES, 2 mmol/L magnesium adenosine triphosphate, 0.5 mmol/L sodium guanosine triphosphate, and 5 mmol/L phosphocreatine) was used. For voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs), glutamatergic blockade was induced using NMDA receptor blocker AP5 (50 μmol/L) and AMPA receptor blocker cyanquixaline (10 nmol/L), followed by α-klotho administration, and then currents were abolished with picrotoxin (100 μmol/L) (34).

Statistical Analysis

Unpaired t tests were used for in vivo mouse experiments to compare differences between groups in food intake, body weight, and body composition. To compare pre-post within-group changes over the course of the experiment, paired t tests were performed. To compare differences at GTT or ITT time points, two-way ANOVA with repeated measures for time and Bonferroni corrections for multiple comparisons were used. Unpaired t tests or one-way ANOVA with Tukey correction for multiple comparisons were used in cell culture experiments when appropriate. Paired t tests or repeated-measures ANOVA with Tukey correction for multiple comparisons was used in patch clamp electrophysiology experiments when appropriate. All analyses were performed using GraphPad Prism statistics software, and a P value <0.05 was considered statistically significant.

Data and Availability Statement

The data sets generated during the current study are available from the corresponding author on reasonable request.

Results

Seven Days of Central Administration of α-Klotho Results in Weight Loss, Suppressed Food Intake, and Improved Glucose Regulation in DIO Mice

A 7-day ICV α-klotho treatment in DIO mice significantly reduced body weight (4.9%) compared with vehicle-treated controls (Fig. 1A and B). These changes were, at least in part, due to decreased food intake both daily (14.8%) and after a daytime food restriction (11.4%) (Fig. 1EH). ICV α-klotho treatment also improved glucose clearance and insulin release during a GTT, as well as insulin sensitivity during an ITT (Fig. 1IN).

Figure 1
Figure 1

Seven days of central administration of α-klotho results in weight loss, suppressed food intake, and improved glucose regulation in DIO mice. A: Body weight. B: Changes in body weight. C: Fat mass. D: Lean mass. E: Average daily food intake. F: Timeline of daily food intake. G: Cumulative food intake after a daytime food restriction. H: Timeline of food intake after a daytime food restriction. I: Blood glucose levels during a GTT. J: Area under the curve (AUC) of the GTT. K: Serum insulin levels 30 min into the GTT. L: Blood glucose during an ITT. M: AUC of the ITT. N: Fasting serum insulin. AN: In 17- to 18-week-old male DIO mice after 7 days ICV α-klotho or vehicle injections (n = 8–13/group). Data represented as mean ± SEM. *P < 0.05 vs. ICV control.

A Single ICV α-Klotho Injection Improves Glucose Clearance and Suppresses Food Intake in DIO Mice

To determine if the effects of central α-klotho on glucose metabolism were independent from changes in body weight, a single ICV α-klotho injection was performed in DIO mice the night before a GTT or ITT. Acute ICV α-klotho treatment improved glucose clearance during a GTT (Fig. 2AC) and even decreased food intake the following day (Fig. 2D). Interestingly, acute ICV α-klotho treatment had no effects on insulin sensitivity (Fig. 2EH). These data suggest central α-klotho regulates glucose metabolism independent from changes in body weight and insulin sensitivity. Supporting this hypothesis, 12 days of ICV α-klotho had no effects on insulin-stimulated signaling in hypothalamus, skeletal muscle, eWAT, or liver in weight-matched DIO mice (Supplementary Fig. 3).

Figure 2
Figure 2

Acute central administration of α-klotho improves glucose clearance and suppresses food intake independent of body weight in DIO mice. A: Blood glucose during a GTT. B: Area under the curve (AUC) of the GTT. C: Body weight before the GTT. D: Daily food intake (including overnight fast before GTT on day 1). E: Blood glucose during an ITT. F: AUC of the ITT. G: Food intake the night before the ITT. H: Body weight before the ITT. AH: In 17- to 18-week-old male DIO mice after a single ICV α-klotho or vehicle injection (n = 7–10/group). Data represented as mean ± SEM. *P < 0.05 vs. ICV control.

To begin to investigate alternative peripheral mechanisms through which central α-klotho improves glucose regulation, basal hepatic gene expression was analyzed. Hepatic PEPCK mRNA was significantly reduced (0.75-fold reduction) in DIO mice treated with α-klotho for 12 days, suggesting attenuated hepatic gluconeogenesis, despite no changes in pyruvate kinase, glucose-6-phsophatase, or glucokinase. α-Klotho–treated mice also had reduced hepatic lipid accumulation and upregulated ACC1 and ACC2 mRNA (Supplementary Fig. 4).

Seven Days of Central α-Klotho Administration Attenuates the Progression of Diabetes in STZ-Treated Mice

The therapeutic potential of α-klotho was also investigated in a model of type 1 diabetes induced by STZ treatment. Similar to DIO mice, ICV α-klotho decreased body weight (5.3%), suppressed food intake (27.8%), and attenuated hyperglycemia (20.2% reduction in fed glucose levels) in STZ-treated mice compared with vehicle-treated controls (Fig. 3AG). Even in pair-fed STZ-treated mice, ICV α-klotho attenuated hyperglycemia and trended to improve fasting glucose levels (Fig. 3KM). These data further demonstrate glucoregulatory and anorexic action of central α-klotho in both type 1 and type 2 diabetes models.

Figure 3
Figure 3

Seven days of central α-klotho administration attenuates the progression of diabetes in STZ-treated mice. A: Body weight. B: Changes in body weight. C: Average daily food intake. D: Timeline of food intake. E: Fed blood glucose levels. F: Timeline of fed blood glucose. G: Change in fed blood glucose levels. AG: In 9- to 10-week-old, STZ-treated, ad libitum fed mice after 7 days ICV α-klotho or vehicle injections (n = 9–10/group). H: Body weight. I: Changes in body weight. J: Average daily food intake. K: Fed blood glucose levels. L: Change in fed blood glucose levels. M: Fasting blood glucose levels. HM: In pair-fed, STZ-treated mice (n = 8–9/group). Data represented as mean ± SEM. *P < 0.05 vs. ICV control.

Central α-Klotho Inhibition Impairs Glucose Tolerance

To determine the effects of central α-klotho inhibition on energy and glucose homeostasis, we performed central administration of ab-α-klotho antibody. A 2-day ab-α-klotho treatment significantly impaired glucose tolerance compared with vehicle-treated controls despite similar body weights (Fig. 4AC). There were no differences in liver PEPCK or glucose-6-phsophatase. However, gene expression of glucokinase and pyruvate kinase trended to be lower in ab-α-klotho mice (P = 0.14 and 0.15, respectively) (Supplementary Fig. 5D). Surprisingly, 7 days of ab-α-klotho significantly decreased body weight with no changes in food intake (Fig. 4DG). Taken together with ICV α-klotho experiments, these data suggest a distinct glucoregulatory role of central α-klotho independent of body weight and food intake.

Figure 4
Figure 4

Central α-klotho inhibition impairs glucose tolerance. A: Blood glucose levels during a GTT. B: Area under the curve (AUC) of the GTT. C: Fasting glucose levels. D: Body weight. E: Changes in body weight. F: Daily food intake. G: Timeline of food intake. AG: In 9-week-old chow-fed male mice treated with ab-α-klotho antibody compared with vehicle-treated controls (n = 8/group). Data represented as mean ± SEM. *P < 0.05 vs. ICV control.

α-Klotho Suppresses NPY/AgRP Neuron Activity, at Least in Part, by Enhancing mIPSCs

We next aimed to investigate the effects of α-klotho on NPY/AgRP neurons considering their critical role in energy homeostasis. A single ICV α-klotho injection in NPY-GFP reporter mice before an overnight fast significantly reduced cFOS colocalization with NPY/AgRP neurons by 49.0% (Fig. 5AC). Furthermore, electrophysiological recordings revealed α-klotho treatment decreases NPY/AgRP neuron firing rate and membrane potential (0.79 vs. 0.22 Hz and −52.7 vs. −57.8 mV, respectively) (Fig. 5DF).

Figure 5
Figure 5

α-Klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing mIPSCs. A: Representative image of cFOS (red) colocalization with NPY/AgRP (green). B: Number of NPY neurons. C: Number of NPY neurons with cFOS colocalization in the ARC. AC: Mice ICV treated with 2.0 μL α-klotho or vehicle before an overnight fast (n = 4 mice/group). DF: Representative cell-attached recording of an NPY/AgRP neuron (D), calculated firing rate (Hz) (E), and membrane potential (mV) (F) during α-klotho administration (n = 8 neurons from four male mice). G and H: Representative current clamp trace of an NPY/AgRP neuron (G) and mean membrane potential (H) induced by TTX or TTX and α-klotho. I: Representative whole cell recording tracers with α-klotho, glutamatergic blockade, and GABA-A receptor antagonist picrotoxin. JM: Mean amplitude (J and K), differences in cumulative probability of mIPSC amplitude (L), and mean frequency (M) of IPSCs under glutamatergic blockade with and without α-klotho treatment (n = 5 neurons from three male mice). Data represented as mean ± SEM. *P < 0.05 vs. aCSF.

To determine if α-klotho’s suppressive effects on NPY/AgRP neurons are due to pre- or postsynaptic events, brain slices were pretreated with TTX (1.0 μmol/L) to block action potentials. In the presence of TTX, α-klotho still decreased membrane potential (−53.4 vs. −58.4 mV), suggesting postsynaptic action of α-klotho on NPY/AgRP neurons (Fig. 5G and H). We also observed α-klotho to increase the magnitude, but not the frequency, of mIPSCs in NPY/AgRP neurons under glutamatergic blockade (25.9 vs. 34.4 pA) (Fig. 5IM), indicating α-klotho is directly antagonizing NPY/AgRP neurons by modulating receptor availability or intracellular signals (35). Overall, these experiments illustrate that α-klotho directly suppresses NPY/AgRP neuron activity by, at least in part, increasing receptor-mediated inhibitory signals.

α-Klotho Induces Cell Signaling, Alters Gene Expression, and Decreases NPY/AgRP Neuron Activity via FGFRs

To investigate the potential of α-klotho to alter cell signaling and gene expression in the hypothalamus, we first used the GT1-7 immortal hypothalamic cell line (36). A 30-min α-klotho treatment increased phosphorylation of ERKthr202/tyr204, AKTser473, and FOXO1ser256 (Supplementary Fig. 6A). Additionally, α-klotho treatment during both overnight and 2 h of serum starvation significantly reduced AgRP mRNA (by 28.5% and 30.3%, respectively), suggesting hormonal action of α-klotho in GT1-7 cells (Supplementary Fig. 6C). To investigate if α-klotho has hormonal action in the hypothalamus in vivo, we performed acute ICV α-klotho administration in healthy, fed mice and observed elevated phosphorylated ERK after 90 min in the ARC compared with vehicle-treated controls (Supplementary Fig. 6B).

Previous studies demonstrate the importance of α-klotho as a scaffolding protein increasing the affinity of FGF23 to FGFR1 (26,27). In hypothalamic GT1-7 cells, 30-min FGF23 (100 ng/mL) treatment had no effects on phosphorylated ERK or AKT (Fig. 6AC), while cotreatment with FGF23 and α-klotho had no synergistic effect compared with α-klotho alone. This suggests, at least in hypothalamic GT1-7 cells, α-klotho is independent of exogenous FGF23-mediated signaling. When cells were pretreated with FGFR1 inhibitor PD173074 (10 nmol/L), α-klotho–mediated cell signaling and suppression of AgRP mRNA were abolished (Fig. 6AD), indicating hypothalamic α-klotho action is dependent on FGFR1 activity. Moreover, immunofluorescent staining of cFOS revealed that ICV pretreatment with PD173074 also inhibited the ability of α-klotho to decrease NPY/AgRP neuron activity in vivo (Fig. 6EG).

Figure 6
Figure 6

α-Klotho–mediated cell signaling and regulation of NPY/AgRP neurons in the hypothalamus is dependent on FGFRs. A: Representative Western blot image. B: Phosphorylation of ERK. C: Phosphorylation of AKT. D: AgRP mRNA expression. AC: In GT1-7 cells treated with α-klotho, FGF23, PD173074, and/or wortmannin (n = 5–10/group). E: Representative image of cFOS (red) colocalization with NPY/AgRP neurons (green). F: Number of NPY neurons. G: Number of NPY neurons colocalized with cFOS. DG: In the ARC of the hypothalamus of mice ICV treated with vehicle, FGFR inhibitor with vehicle, or FGFR inhibitor with α-klotho before an overnight fast (n = 3 mice/group). Data represented as mean ± SEM. *P < 0.05 vs. controls. tAkt, total Akt; tERK, total ERK.

PI3K is a downstream mediator of FGFR1 and is also an important negative regulator of NPY/AgRP neurons (25,37). PI3K inhibition using wortmannin (50 nmol/L) also eliminated α-klotho’s ability to suppress AgRP gene expression (Fig. 6D). Taken together, these data demonstrate the importance of FGFR1/PI3K signaling in hypothalamic α-klotho function.

Seven Days of Central α-Klotho Treatment Suppresses Food Intake and Reduces Body Weight via FGFR and PI3K Signaling in DIO Mice

To determine if the therapeutic effects of α-klotho in DIO mice were dependent on FGFRs, we centrally injected PD173074 to inhibit endogenous FGFR function. Mice receiving α-klotho treatment alone experienced significantly decreased food intake and body weight compared with all groups (Fig. 7AD), while PD173074 treatment alone induced weight gain (Fig. 7A and B). PD173074 treatment blunted α-klotho–mediated reductions in food intake and body weight, suggesting the effects of central α-klotho on energy balance are mediated by FGFR signaling. Surprisingly, both α-klotho with an FGFR inhibitor and α-klotho alone groups experienced improved glucose clearance compared with vehicle-treated controls (Fig. 7E and F), suggesting FGFRs may not be involved in central α-klotho–mediated glucose regulation.

Figure 7
Figure 7

Central inhibition of FGFR1 blunts the therapeutic effects of 7 days α-klotho in DIO mice. A: Body weight. B: Changes in body weight. C: Average daily food intake. D: Timeline of food intake. E: Blood glucose levels during GTT. F: Area under the curve (AUC). AF: In DIO mice receiving 7 days ICV injection with vehicle, α-klotho, FGFR inhibitor, or inhibitor with α-klotho (n = 7–11/group). Data represented as mean ± SEM. *P < 0.05 vs. ICV vehicle.

Similar to FGFR antagonism, central inhibition of PI3K abolished α-klotho’s ability to suppress food intake and improve glucose clearance (Fig. 8). These data indicate that PI3K is critical to α-klotho–mediated regulation of food intake and glucose metabolism. Overall, coupled with in vitro cell signaling experiments, these data demonstrate a novel α-klotho/FGFR/PI3K mechanism in the central regulation of metabolism.

Figure 8
Figure 8

Central inhibition of PI3K negates the therapeutic effects of a single α-klotho injection in DIO mice. A: Timeline of food intake (including overnight fast before GTT on day 1). B: Average 48-h food intake. C: Blood glucose during a GTT. D: Area under the curve (AUC) of the GTT. AD: In DIO mice receiving a single injection with vehicle, α-klotho, wortmannin, or wortmannin with α-klotho (n = 9–10/group). Data represented as mean ± SEM. *P < 0.05.

Discussion

To our knowledge, this is the first study to provide evidence that α-klotho functions as a hypothalamic hormonal agent. ICV α-klotho administration improved glucose regulation, suppressed food intake, and reduced body weight in mouse models of type 1 and 2 diabetes, illustrating the therapeutic potential of central α-klotho in metabolic disease states. Although deeper investigation is required to identify the direct connection between central α-klotho activity and peripheral glucose metabolism, the current study determined that the glucose-lowering effects of α-klotho are independent from insulin sensitivity but, rather, are mediated by augmented insulin secretion during a glucose challenge. However, STZ experiments revealed that some of the glucoregulatory actions of α-klotho are independent of insulin altogether. Basal hepatic PEPCK mRNA was decreased in α-klotho-treated DIO mice, suggesting decreased hepatic glucose output may be an alternative mechanism.

The central and peripheral pools of α-klotho have distinct, independent functions due to α-klotho’s inability to cross the blood-brain barrier (9). While recent publications have demonstrated metabolic roles of α-klotho in the blood, including long-term α-klotho injection improving adiposity in DIO mice (6) and ameliorating diabetic cardiomyopathies in STZ-treated mice (38), the current study identifies distinct differences between central and peripheral α-klotho–mediated metabolic regulation. For example, α-klotho’s effects on food intake and glucose metabolism seem to be mainly via central mechanisms, while peripherally circulating α-klotho regulates gene expression to promote lipid oxidation and energy expenditure (6,38). Notably, whole-body α-klotho knockout and knockdown models have been previously utilized to investigate α-klotho’s functions (1,7), but these approaches do not distinguish between peripheral and central α-klotho function.

ICV ab-α-klotho was used in this study as a novel approach specifically impairing central α-klotho signaling, and as expected, ab-α-klotho treatment impaired glucose clearance. Although central α-klotho concentrations have yet to be quantified in patients with diabetes, past studies show blood α-klotho concentrations to be decreased in some populations with diabetes (8,39). Thus, our data connecting central α-klotho impairment and disordered glucose regulation may provide new insight into the pathophysiology of metabolic disorders.

Contrary to our hypotheses, central α-klotho inhibition resulted in decreased body weight with no differences in food intake. α-Klotho knockout mice also experience weight loss, primarily due to atrophy of metabolically active organs, resulting in premature death (1,7). These findings highlight the complicated and diverse metabolic functions of α-klotho. For example, while evidence from the current study and past literature describes α-klotho as an antidiabetic agent (36,38), overexpression of α-klotho has been shown to elicit insulin resistance (1). Notably, α-klotho–overexpressing mice do not experience hyperglycemia, adiposity, or hyperphagia associated with clinical insulin resistance (1). Moreover, α-klotho is an important negative modulator of insulin and IGF-I signaling to regulate apoptosis and ROS buffering (10,11). The many complex physiological roles of α-klotho may explain the unexpected results in response to central α-klotho inhibition.

The current study identified central α-klotho as a novel antagonist of NPY/AgRP neurons. Considering NPY/AgRP neuron overactivity is associated with disordered feeding, body weight, and glucose regulation (13,40), our data provides encouraging evidence of α-klotho as a potential therapeutic target in metabolic disease prevention. At the present, it is unclear if NPY/AgRP neurons are the primary mediators of central α-klotho’s regulation of metabolism, underscoring the importance of further investigation into the specific neuronal effectors and cell signaling involved. However, the observed ICV α-klotho phenotype has many similarities to the previously described effects of NPY/AgRP neuron inhibition, including suppressed food intake, reduced body weight, improved glucose clearance and insulin release, and decreased hepatic gluconeogenic gene expression (13,1517,41,42).

Similar to findings in studies using hippocampal and oligodendrocyte progenitor cells (10,11,28), α-klotho induced phosphorylation of ERKthr202/tyr204, AKTser473, and FOXO1ser256 in hypothalamic GT1-7 cells—all of which are established signaling molecules involved in downregulating NPY/AgRP gene transcription and activity (18,43). Furthermore, the observed ICV α-klotho phenotype resembles FGFR activation, which also results in suppressed food intake, improved glucose regulation, attenuated NPY/AgRP neuron activity, and decreased liver gluconeogenic gene expression (2024). α-Klotho serves as a nonenzymatic scaffold to increase FGF23 affinity to FGFR1 (27). Thus, we investigated the potential importance of a hypothalamic α-klotho–FGFR1 signaling mechanism. Similar to previous studies in hippocampal cells, our results show that hypothalamic α-klotho–mediated signaling and AgRP mRNA regulation in GT1-7 cells were abolished with pretreatment with FGFR1 antagonist PD173074 (28). Additional experiments determined that PI3K signaling, a downstream mediator of FGFR1 (37) and potent regulator of NPY/AgRP neurons (25), was also required for α-klotho–mediated AgRP mRNA suppression. Future studies should further investigate the possible involvement of a novel α-klotho–FGFR1–PI3K axis in the homeostatic modulation of NPY/AgRP neurons.

We further investigated the involvement of FGFR/PI3K signaling to central α-klotho–mediated regulation of metabolism. Central FGFR or PI3K inhibition blunted ICV α-klotho’s effects on food intake and body weight, while only PI3K inhibition affected α-klotho–mediated glucose regulation. Overall, these data support the hypothesis that central FGFR-PI3K signaling is critical to α-klotho–mediated regulation of metabolism. However, studies investigating the function of central FGFRs in metabolism yield mixed results depending on animal model and experimental approach. ICV PD173074 (FGFR inhibitor) impairs glucose clearance in healthy rats, but it is described as stress related (23,44). ICV PD173074 in DIO mice elicits no phenotype (21,24). Furthermore, antibody-mediated inhibition of FGFR1 in rodents and monkeys increases energy expenditure, decreases food intake, and reduces body weight, while genetic deletion of FGFR1 in NPY/AgRP neurons also results in no metabolic phenotype (4547). Additionally, the specificity for PD173074 in vivo is unclear; thus, it likely has nonspecific antagonism of other FGFRs. Future studies should investigate the specific roles of FGFRs, their isoforms, and their neuronal effectors in central regulation of metabolism by performing selective deletion of FGFRs in specific neurons of mature mice using the inducible Cre-LoxP system.

In addition to FGFR-PI3K signaling, there are likely unknown concurrent mechanisms underlying central α-klotho–mediated metabolic regulation. Other neuron populations, such as proopiomelanocortin (POMC) neurons, which are closely associated with NPY/AgRP neurons, may be involved. Our cell culture and immunohistochemistry data also may suggest ERK as an additional cell signaling mechanism of hypothalamic α-klotho action. ERK signaling is downstream of α-klotho, negatively regulates NPY/AgRP neurons, possibly via Kruppel-like factor 4, and is involved in hypothalamic FGF1- and FGF19-mediated glucose lowering (21,43,48).

To summarize, this study identifies α-klotho as a novel antagonist of NPY/AgRP neurons and demonstrates α-klotho’s importance to central regulation of metabolism via an α-klotho–FGFR1–PI3K signaling axis. Our data revealed central administration of α-klotho to yield various therapeutic effects in models of type 1 and 2 diabetes, including improved glucose regulation, suppressed food intake, and reduced body weight. To our knowledge, this study provides the first evidence of α-klotho as a novel hypothalamic regulator of energy balance and glucose metabolism, thus providing new insight into the pathophysiology of metabolic disease.



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Roasted Zucchini Fries with Parmesan

By electricdiet / June 30, 2020


These roasted zucchini fries with Parmesan are oven-baked to crispy perfection. And you only need a few ingredients to make this delicious summer side dish!

Garlic zucchini fries on an oven pan

Looking for something crispy and easy to serve with your meal?

These roasted zucchini fries with Parmesan are one of my favorite side dishes, especially for the summer! They’re the perfect way to sneak some healthy veggies onto the menu for your next cook out.

And they’re so easy to make. Just slice the zucchini, coat the sticks in Parmesan and garlic pepper, drizzle with olive oil, and throw them in the oven until they’re ready!

I’ve seen a lot of zucchini fry recipes made with bread crumbs to give them crunch. Unfortunately, this adds a lot of carbs and calories.

These zucchini fries, on the other hand, are made with Parmesan to give them that satisfying crispy coating without all the carbs. That means you can enjoy these healthy fries without the guilt!

So whether you have summer zucchini you need to use or just want a delicious and healthy side dish, definitely give this recipe a try!

How to make roasted zucchini fries with Parmesan

These crispy sticks are easy to prep and only take about 20 minutes to bake in the oven.

Step 1: Preheat oven to 425°F. Line a baking sheet with parchment paper.

Step 2: Cut the two zucchini into 16 sticks. To do this, start by trimming the ends of both zucchini. Next, quarter both zucchini lengthwise so you end up with 8 long pieces. Finally, cut each piece in half crosswise to get 16 shorter triangular-shaped pieces.

Step 3: Beat the egg in a shallow bowl. You want to use a bowl that will be large enough to dip the zucchini pieces.

Step 4: Combine the Parmesan and garlic pepper in a second shallow bowl, again ensuring that it’s large enough to dip the zucchini pieces.

Step 5: Take one zucchini strip and dip the cut sides into the egg. Once the cut sides are coated, dip the zucchini into the Parmesan mixture until well-coated.

Step 6: Place the zucchini strip on the baking sheet with the skin side down.

Step 7: Repeat with the remaining zucchini.

Step 8: Once all the zucchini has been coated and placed on the baking sheet, drizzle with olive oil.

Step 9: Place the baking sheet in the oven and roast until the zucchini sticks are golden, about 15 to 20 minutes.

That’s it! Once the exterior is nice and crispy, your zucchini fries are ready to serve.

Cutting zucchini into sticks

Zucchini has recently become popular as a healthy alternative to high-carb and starchy foods like french fries. The trick is to make sure the zucchini turns out crispy and not soggy.

I’ve seen a few recipes for zucchini cooked similar to this one, but it was sliced into rounds instead of sticks.

When I tried that method, the rounds turned out a bit too soggy for my taste.

This triangular shape seemed to get much more crispy. That’s why I prefer to quarter the zucchini and then cut each long piece in half. I also like that they look a bit like house-cut fries this way!

Storing zucchini fries

I recommend serving this dish immediately. The fries will be best when they are hot out of the oven.

If you have leftovers, you can store them covered in the refrigerator for 3-4 days. However, the moisture from the zucchini will make the exterior less crispy than when they’re fresh.

To reheat, I recommend either pan-roasting or sticking them back in the oven. This will help the exterior get crispy again.

Zucchini fries on a wooden plate

Other healthy zucchini recipes

Zucchini is such a versatile vegetable. It’s amazing how many different kinds of recipes you can make with it! Plus, it’s so easy to find in the summertime.

If you’re looking for a few more fun recipes to use up your zucchini, here are some of my favorites that I think you’ll enjoy:

When you’ve tried these zucchini fries, please don’t forget to let me know how you liked it and rate the recipe in the comments below!

Recipe Card

Roasted Zucchini Fries with Parmesan

Roasted Zucchini Fries with Parmesan

These roasted zucchini fries with parmesan are oven-baked to crispy perfection. And you only need a few ingredients to make this delicious summer side dish!

Prep Time:10 minutes

Cook Time:20 minutes

Total Time:30 minutes

Author:Shelby Kinnaird

Servings:4

Instructions

  • Preheat oven to 425°F. Line a baking sheet with parchment paper.

  • Cut the two zucchini into 16 sticks. To do this, start by trimming the ends of both zucchini. Next, quarter both zucchini lengthwise so you end up with 8 long pieces. Finally, cut each piece in half crosswise to get 16 shorter triangular-shaped pieces.

  • Beat the egg in a shallow bowl. You want to use a bowl that will be large enough to dip the zucchini pieces.

  • Combine the Parmesan and garlic pepper in a second shallow bowl, again ensuring that it’s large enough to dip the zucchini pieces.

  • Take one zucchini strip and dip the cut sides into the egg. Once the cut sides are coated, dip the zucchini into the Parmesan mixture until well-coated.

  • Place the zucchini strip on the baking sheet with the skin side down.

  • Repeat with the remaining zucchini.

  • Once all the zucchini has been coated and placed on the baking sheet, drizzle with olive oil.

  • Place the baking sheet in the oven and roast until the zucchini sticks are golden, about 15 to 20 minutes.

Recipe Notes

This recipe is for 4 servings of zucchini fries. If you cut both zucchini into 8 pieces, then one serving will be 4 zucchini fries. This dish is best served immediately. If you have leftovers, they can be stored covered in the refrigerator for 3-4 days. The exterior will lose some of its crispiness, but you can pan roast or bake the fries to reheat and try to re-crisp the outsides.

Nutrition Info Per Serving

Nutrition Facts

Roasted Zucchini Fries with Parmesan

Amount Per Serving (4 pieces)

Calories 127 Calories from Fat 81

% Daily Value*

Fat 9g14%

Saturated Fat 3.5g22%

Trans Fat 0g

Polyunsaturated Fat 2.2g

Monounsaturated Fat 3g

Cholesterol 57.3mg19%

Sodium 297.5mg13%

Potassium 287.6mg8%

Carbohydrates 3.6g1%

Fiber 1g4%

Sugar 2.5g3%

Protein 8.4g17%

Vitamin A 400IU8%

Vitamin C 38mg46%

Calcium 150mg15%

Iron 0.7mg4%

Net carbs 2.6g

* Percent Daily Values are based on a 2000 calorie diet.

Course: Side Dishes

Cuisine: American

Diet: Diabetic, Low Fat

Keyword: low-carb, roasted zucchini fries, zucchini, zucchini fries



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Back on Track – My Bizzy Kitchen

By electricdiet / June 28, 2020


If I were to search my blog and see how many times I’ve said “hit the reset button!” or “the switch is now on!” I would probably have a million dollars and could have retired by now.  But, such is the case.  Since working from home and so close to my kitchen, I’ve been making all the things.  The only problem with that is that I normally only had time on a weeknight pre-COVID to make dinner, do a couple things around the house, watch a 30 minute t.v. show and go to bed.

Now I clock out at 5 and I can easily recipe develop two dishes, taste test and THEN eat dinner.

A week ago Saturday I got on the scale and was like “um, excuse me?”  Last week I walked more, drank water rather than wine, but didn’t go crazy with trying to be perfect.  I was mindful.  And I lost 1.4 pounds.  And it wasn’t that hard.  And I know this.  Yet somehow I still get back on that hamster wheel thinking that the way I used to do things (that got me to 190 pounds) will somehow work in my favor.  But I know that doesn’t work.

I really don’t know what all those numbers mean, but what I realized during the week I first stepped on it, and 7 days later – is that because numbers fluctuate so much, I only stepped on the scale a week later.  My weigh in is normally on Saturday’s and there have been some weeks where I step on the scale on Thursday and see I am down 2.0 pounds and think “well, I guess I can have an extra glass of wine and some pizza!”

So I am being mindful.  Nothing more and nothing less.

+++++++++++

I had a great weekend.  It was really the first full weekend by myself in the house without Hannah, Jacob or my Mom over.  It was . . . quiet.  And weird.  And quiet.  But guess what?  I could listen to music all day long without air pods – that was wonderful!  I also got up early on Saturday morning and made a pot of coffee, did my WW zoom in the kitchen and didn’t have to worry about being too noisy to wake anyone up.

After my WW meeting I hit up the local farmers market for first time this summer.  Farm fresh eggs will forever be worth the $5 a dozen.  Side note:  save these eggs for scrambled eggs or omelets – don’t waste these in a recipe because you won’t tell.

I spoke to a lovely young man at this farmstand – Waypoint Farm.  Each bag of little gem romaine was only $2!  I quickly grabbed two.  He was so passionate about his produce – #love.   I hope to spend more time talking with him next week – it was about to storm as I grabbed my two bags of romaine.

I made another shitty video on my YouTube channel!  This time I showed how to make my KFC chicken nuggets.  So so good.

My new column for The Daily Herald will be posted the week of 4th of July, but my article shows how you can host a BBQ for 8 people on a budget – I made chicken legs, corn salad, grilled potato fries and a strawberry tomato salsa – all for $23.11.  Here is a sneak peak for that article.

It’s funny but since Sunday was just a regular day for me – no father’s day celebrations, I made this dinner for 8 thinking I would just bring it over to my neighbors house.  How rude would that have been to barge in on their father’s day!  So I’ll be having leftovers this week.  Not that I am complaining because this was delicious.

All in all it was a great weekend.  Getting used to this new normal of living by myself.  The longest I’ve lived alone is three weeks before Hannah and Jacob moved in after my husband died in December 2014.  In another week I’ll break that record – ha!

I’ve yet to walk around my house naked, but I have walked from my bedroom to the bathroom in just my underwear and bra, so that’s a start!

Come back tomorrow for “What I Eat in A Day” on #teampurple – it was a delicious day!  Until then, be well.

 



Sell Unused Diabetic Strips Today!

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