Hearts of Palm Tater Tots

By electricdiet / May 31, 2020


I am all about the tot life.  If it is on the menu, I will always get it.  If. I don’t see it on the menu, I ask anyway because you just never know!

Whenever there is a holiday I rarely do a meal plan that week because I know there will always be leftovers.  Jacob has been making his way through the leftover ribs, and I boiled enough boiled potatoes to feed a family of six.  But I love having leftover potatoes – they are already cooked and the possibilities are endless.

I found filet mignon in my deep freeze from my last Butcher Box – score!  I found one lone zucchini and was trying to decide on a side dish to go with the steak, and my mind went to steak and potatoes and then potatoes went to TOTS!

Shredding cooked potatoes is so cool.  The potato shreds go in the box grater and the skins stay on the outside.  I decided to get a lot more tots by adding hearts of palm pasta.  [See my disclaimer here]

Natural Heaven Hearts of Pasta is delicious, but let’s be clear, it doesn’t taste like pasta.  You can find it here on Amazon.  But I love hearts of palm so this is like the tofu of veggies – it really takes on the flavor of anything you put it with.

These literally took less than 10 minutes to mix and form and get in the freezer for 30 minutes before deep frying.  The potato has just enough moisture to hold their shape.  Freezing them for the 30 makes sure they keep their shape while frying.

Print

Hearts of Palm Tater Tots

Hearts of Palm pasta bulks up these tater tots – 24 tots are only 4 WW points on #teampurple!


Scale

Ingredients

8 ounces cooked potato, shredded
1 package @naturalheavenusa hearts of palm pasta, drained and chopped (I also pan fry mine in a hot skillet for 45 minutes to wick away any excess moisture)
2 tablespoons flour
1 teaspoon Italian seasoning
1/2 teaspoon pepper
3/4 teaspoon salt
1/2 teaspoon garlic powder
3/4 teaspoon cayenne (I like spicy!)
2 tablespoons panko bread crumbs

Instructions

Mix all the ingredients above.  Shape into tater tot shape.

After shaping, freeze for 30 minutes then deep fry for 5-6 minutes at 350. I served mine with steak and zucchini noodles, so my dinner came in at 9 points.

Notes

Next time I may leave out the panko bread crumbs to see if it makes a difference – then the only points would be the 2 points for the flour – nice!

I haven’t tried these in the air fryer, but I will test that out too.  To store for later, place leftover uncooked tots on a cookie sheet for an hour, then store in a ziplock bag – they won’t stick together that way.

So are you on #teamtots?  Let me know!  Until next time . . .





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Shredded Chicken Salad (Instant Pot)

By electricdiet / May 29, 2020


Tender juicy shredded chicken cooked in the Instant Pot makes a great addition to your salad for a quick and healthy lunch or dinner.

Plate of shredded chicken salad on a table with dressing on the side

The Instant Pot can be used to cook flavorful chicken very quickly. It gives the chicken breast a very tender texture without drying it out, which makes for great shredded chicken after just 15 minutes of cooking at high pressure.

The shredded chicken can then be used in several dishes for the next few days. One of my favorite uses for shredded chicken is in a healthy green salad.

Once you have everything ready and prepped for a salad, it is very easy to throw together, and very easy to stay on top of eating healthy.

How to make shredded chicken salad

Step 1: Season the chicken breast generously with salt, pepper, and garlic powder.

Seasoned chicken breast in the instant pot

Step 2: Turn the Instant Pot to sauté and add the vegetable oil. When the oil is hot, carefully place the chicken breast into the Instant Pot and allow to cook undisturbed for 5 minutes. This will create a nice brown crust. Use tongs to carefully flip the chicken breast, cook on the other side for 5 minutes. Press cancel to stop cooking.

Step 3: Add chicken stock or water to the Instant Pot. Cover with the lid and make sure the valve is in the “sealing” position. Set the Instant Pot to cook on manual setting at high pressure for 15 minutes. It will take about 5 minutes to come to pressure.

Close-up of instant pot settings

Step 4: Once it is done cooking, naturally release pressure for 5 minutes, and then turn the valve to “venting” to manually release pressure (make sure your fingers are not over the red button, so you don’t burn yourself with the steam!).

Step 5: Use two forks to shred the chicken. You can do this right in the Instant Pot to get all the delicious juices into the shredded chicken. Be careful not to burn your hands on the edges of the Instant Pot.

Shredded chicken in the instant pot

Step 6: While the chicken is cooking, prepare the salad dressing: In a small bowl, combine all the ingredients and gently stir. Allow the flavors to infuse while you prepare the salad and wait for the shredded chicken to cook.

Step 7: Prepare all the vegetables for the salad. Toss all the salad ingredients together in a large bowl, or divide between 2-4 plates. Top with warm shredded chicken. Pour the cilantro lime dressing over the salad right before serving.

Finished salad served on a white plate

Adjusting the carb count

Most of the carbohydrates in this recipe are from the chickpeas. I love to add the peas because of their great flavor and because they add some “crunch” to the salad, but you can cut them out if you prefer to keep your salad very low carb.

Why I use an Instant pot

The Instant Pot is one of my most used kitchen tools. I love that it can brown the chicken breast on the “saute” function and then cook it to tender perfection under high pressure all in one pot.

And I absolutely love using the saute function because the walls of the Instant Pot are so high that I don’t ever have to worry about splatter. Less splatter = less mess = less cleaning. Definitely a win in my book.

I shred the chicken right in the Instant Pot and it stays warm while I prep the salad.

Tip for making the perfect salad

I’ll share another tip for making great salad at home: use a salad spinner. I went so many years without a salad spinner because I just didn’t think it was a necessary kitchen tool. When I finally got one… GAME CHANGER.

Lettuce that is no longer dripping wet holds on to the dressing better. What this means for you is more flavor in every bite!

I wash and rinse the chopped lettuce, give it a quick spin, drain the extra water (it is amazing how much water comes off lettuce after spinning it), and then store the lettuce in the salad spinner until it’s ready to use.

Clean, dried lettuce also keeps in the fridge longer than lettuce brought home from the store, so that is another bonus. The clean, chopped lettuce is ready to use, which makes it easier to stick to healthy eating all through the week.

Give this Shredded Chicken Salad a try – it’s a classic salad that is so convenient to prepare with the help of the Instant Pot!

More healthy salad recipes

I am a huge fan of healthy and easy salads that taste great! Here are some of my favorite salads and dressing:

You can also check out my roundup of delicious Diabetic Salad Recipes for more inspiration.

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

Recipe Card

Shredded Chicken Salad (Instant Pot)

Tender juicy shredded chicken cooked in the Instant Pot makes a great addition to your salad for a quick and healthy lunch or dinner.

Prep Time:15 minutes

Cook Time:25 minutes

Total Time:40 minutes

Servings:4

Shredded Chicken Salad

Instructions

  • Season the chicken breast generously with salt, pepper, and garlic powder

  • Turn the Instant Pot to sauté and add the vegetable oil. When the oil is hot, carefully place the chicken breast into the Instant Pot and allow to cook undisturbed for 5 minutes. This will create a nice brown crust. Use tongs to carefully flip the chicken breast, cook on the other side for 5 minutes. Press cancel to stop cooking.
  • Add chicken stock or water to the Instant Pot. Cover with the lid and make sure the valve is in the “sealing” position. Set the Instant Pot to cook on manual setting at high pressure for 15 minutes. It will take about 5 minutes to come to pressure.

  • Once it is done cooking, naturally release pressure for 5 minutes, and then turn the valve to “venting” to manually release pressure (make sure your fingers are not over the red button, so you don’t burn yourself with the steam!).

  • Use two forks to shred the chicken. You can do this right in the Instant Pot to get all the delicious juices into the shredded chicken. Be careful not to burn your hands on the edges of the Instant Pot.

  • While the chicken is cooking, prepare the salad dressing: In a small bowl, combine all the ingredients and gently stir. Allow the flavors to infuse while you prepare the salad and wait for the shredded chicken to cook.

  • Prepare all the vegetables for the salad. Toss all the salad ingredients together in a large bowl, or divide between 2-4 plates. Top with warm shredded chicken. Pour the cilantro lime dressing over the salad right before serving.

Nutrition Info Per Serving

Nutrition Facts

Shredded Chicken Salad (Instant Pot)

Amount Per Serving

Calories 330 Calories from Fat 123

% Daily Value*

Fat 13.7g21%

Saturated Fat 1.5g8%

Trans Fat 0g

Polyunsaturated Fat 3.5g

Monounsaturated Fat 6.6g

Cholesterol 86.7mg29%

Sodium 192.9mg8%

Potassium 832.7mg24%

Carbohydrates 15.6g5%

Fiber 5.4g22%

Sugar 4.1g5%

Protein 37.4g75%

Net carbs 10.2g

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

Course: Salad

Cuisine: American

Keyword: instant pot salad, low-carb salad



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Fresh Corn Salad Recipe – Best Easy Summer Vegetable Recipes

By electricdiet / May 27, 2020


Corn on the Cob Makes BEST Summer Vegetable Recipes

Is there anything better than crisp summer vegetable recipes on a warm day? This fresh Corn Salad recipe from Guy’s Guide to Eating Well cookbook will be one of your favorite summer easy corn salad recipes. If you only eat corn on the cob and don’t use it in recipes, you are missing out!  Sweet summer fresh corn on the cob as an ingredient in easy summer salad recipes is amazing. Honestly, there isn’t much to it and it’s really is quick to do. Just get a sharp or serrated knife and scrape the corn off the cob lengthwise or check out the gadgets below that make it even easier.

Corn, Cucumber, and Tomato Salad
A bullet-proof, fresh and tasty recipe with crunchy cool cucumbers, ripe juicy tomatoes, creamy avocados and corn. An easy nutrition-loaded side dish to any meal.
Vegetarian, Diabetic, Gluten Free

    Servings5 2/3-cup servings
    Prep Time10 minutes

    Ingredients

    • 1cup


      corn

    • 1cup


      peeled chopped cucumber

    • 1cup


      chopped tomatoes

    • 1/4cup


      chopped red onion

    • 1/4cup


      chopped avocados

    • 2tablespoons


      lime juice

    • 1tablespoons


      nonfat plain Greek yogurt

    • 1/4cup


      chopped avocados

    • 2tablespoons


      lime juice

    • 1tablespoons


      nonfat plain Greek yogurt

    Instructions
    1. In large bowl, combine corn, cucumbers, tomatoes, onion, and avocado.

    2. In small bowl, combine lime juice and yogurt. Drizzle over salad. Refrigerate.

    Recipe Notes

    Calories 53, Calories from Fat 24%, Fat 2 g, Saturated Fat 0 g, Cholesterol 0 mg, Sodium 10 mg, Carbohydrates 10 g, Dietary Fiber 2 g, Total Sugars 4 g, Protein 2 g, Diabetic Exchanges: ½ starch, ½ fat

    Serving Suggestion: Pairs perfectly with grilled or baked fish on a warm summer day.

    Harry Connick Jr LOVED this Fresh Corn Salad On The Harry Show

    When Holly was deciding what to prepare for her appearance on the Harry Connick show, she knew anyone can create this summer masterpiece by tossing the fresh produce into a bowl.  Holly put Harry Connick to work and made him whisk together the light dressing and he loved it. It really is simple to do and nothing beats fresh summer grilled corn in recipes.

    Guy’s Guide to Eating Well – Men’s Cookbook and So Much More!

    This refreshing Corn, Cucumber and Tomato Salad from Guy’s Guide to Eating Well cookbook is just one of the summer vegetable recipes Holly prepared on The Harry Connick Show cooking segment. You can make it year round, but fresh corn recipes are especially good in the summer.  This men’s health cookbook is full of realistic, approachable recipes any man (or woman!) can cook and will love to eat.

    Throughout the book, Holly provides delicious good-for-you recipes and it is divided into chapters that focus on common men’s health concerns, such as Heart Disease, Obesity-Diabetes,Weekend Warrior Arthritis, and Cancer with realistic simple preventive healthy recipes.

    Corn Gadgets To Make Fresh Corn Salad

     Chef’n Cob Corn Stripper, Yellow Kuhn Rikon Corn Zipper Kernel Kutter (Sweet Corn Cutter, Stripper) with Bit Corn Stripper, EverPlus Corn Cob Cutter Corn Peeler

    Left Over Ears of Corn Turn Into Easy Corn Salad Recipes

    You can add your favorite garden ingredients to Holly’s recipe!  There is only a little lime juice and olive oil to coat the salad. Season to taste and enjoy these wonderfully fresh summer ingredients that make a summer statement.

    You can make this fresh corn salad recipe easily with frozen corn but I promise this time of year treat yourself to fresh. It does make a difference. You’ll always cook extra for these easy corn recipes throughout the summer because it is so sweet this time of year!

    Get Holly’s Favorite Top 10 Summer Recipes Download with Shopping List!

    summer recipes

    For even more of the BEST Summer recipes you have to check out Holly’s Download only 99 cents! Simplify summer with Holly’s top healthy summer recipes with a shopping list plus tips and hints. It can be overwhelming what to cook so these quick summer recipes will guide you in the kitchen from appetizer to dessert.

    Download it now for only 99 cents and you will have all you need to refresh your healthy summer menu at your fingertips!

    Get All of Holly’s Healthy Easy Cookbooks

    The post Fresh Corn Salad Recipe – Best Easy Summer Vegetable Recipes appeared first on The Healthy Cooking Blog.



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    MicroRNA Networks in Pancreatic Islet Cells: Normal Function and Type 2 Diabetes

    By electricdiet / May 25, 2020


    Abstract

    Impaired insulin secretion from the pancreatic β-cells is central in the pathogenesis of type 2 diabetes (T2D), and microRNAs (miRNAs) are fundamental regulatory factors in this process. Differential expression of miRNAs contributes to β-cell adaptation to compensate for increased insulin resistance, but deregulation of miRNA expression can also directly cause β-cell impairment during the development of T2D. miRNAs are small noncoding RNAs that posttranscriptionally reduce gene expression through translational inhibition or mRNA destabilization. The nature of miRNA targeting implies the presence of complex and large miRNA–mRNA regulatory networks in every cell, including the insulin-secreting β-cell. Here we exemplify one such network using our own data on differential miRNA expression in the islets of T2D Goto-Kakizaki rat model. Several biological processes are influenced by multiple miRNAs in the β-cell, but so far most studies have focused on dissecting the mechanism of action of individual miRNAs. In this Perspective we present key islet miRNA families involved in T2D pathogenesis including miR-200, miR-7, miR-184, miR-212/miR-132, and miR-130a/b/miR-152. Finally, we highlight four challenges and opportunities within islet miRNA research, ending with a discussion on how miRNAs can be utilized as therapeutic targets contributing to personalized T2D treatment strategies.

    Introduction

    Currently, there are 2,654 mature microRNAs (miRNAs) in the human genome (mirbase.org, release 22.1) (1). Mature miRNAs are small (∼19–23 nucleotides) noncoding RNAs involved in posttranscriptional gene regulation, fundamentally required to specify cell identity and to adjust cell function. Therefore, perturbed miRNA expression is often associated with development of human diseases (2), including type 2 diabetes (T2D) and associated complications.

    Canonical miRNAs may be encoded as individual genes (monocistronic), as gene clusters (polycistronic), or within introns of a host gene and are transcribed by RNA polymerase II to form primary miRNAs (see top of Fig. 1). These are processed by the microprocessor complex containing Drosha and DGCR8 to cleave the primary miRNAs into hairpin-structured pre-miRNAs (3). Once exported into the cytosol by Exportin 5, the RNase III-type enzyme Dicer produces double-stranded miRNAs that are handed over to Argonaute (AGO), which selects one strand to become the mature miRNA. AGO together with the mature miRNA forms the RNA-induced silencing complex (RISC). Generally, nucleotides at positions 2–7 of the miRNAs known as the seed region pair with target mRNA, most commonly in the 3′ untranslated region (UTR). This causes posttranscriptional repression through mRNA cleavage, translational repression, and/or mRNA destabilization. Although the majority of the miRNA binding sites occur in the 3′ UTR, recent studies using CLIP-based techniques show miRNA target sites also present in protein-coding sequences (CDSs) (4), which opens new possibilities for miRNA regulation.

    Figure 1
    Figure 1

    Summary of future challenges and opportunities in islet miRNA research. The diagram illustrates miRNA biogenesis from gene to the mature form, the miRNA–mRNA interaction network, and the processes influenced by miRNAs in the β-cell (β-cell identity, β-cell proliferation/apoptosis, and β-cell physiology). For each field of these biological processes involving miRNAs, we have added the four areas of challenges and opportunities discussed in the text. pri-miRNA, primary miRNA.

    The pairing of miRNAs to their target mRNA is central to the function of miRNAs. Due to the short seed region, one miRNA can have hundreds of different targets. On the other hand, one target can be influenced by several miRNAs. Although miRNAs themselves are highly conserved among species, their targets can vary. Considering this, it is important to discuss the concepts of miRNA cluster and miRNA family. A miRNA cluster refers to a group of miRNAs transcribed from the same gene cluster, whereas miRNAs belonging to the same family share the same seed sequence and hence also share the same targets but do not necessarily need to be transcribed from the same genomic location (3). Examples of a miRNA family abundant in the pancreatic islet cells are the miR-200 family (miR-200a, miR-200b, and miR-429 on chromosome 1 and miR-200c and miR-141 on chromosome 12) (5). Several computational target prediction tools are available primarily based on seed sequence complementarity and RNA folding energy minimization (6). However, a remaining challenge with genome-wide miRNA target searches is to minimize false-positive predictions.

    The pancreas contains ∼1 million islets of Langerhans spread throughout the organ. Although the total islet mass only constitutes 1–2% of the whole pancreas, the islets are central for our survival, as they control blood glucose homeostasis. The main cell types within the pancreatic islets are the insulin-secreting β-cells and glucagon-secreting α-cells. A contributing factor in diabetes pathogenesis is when the balance between insulin secretion during hyperglycemia and glucagon secretion during hypoglycemia is disturbed. The role of the pancreatic β-cell in diabetes development has been questioned, but recent studies support the view that classical T2D is a combination of increased insulin resistance in target tissues and impaired β-cell compensation (7). This was reinforced by the new classification derived from analyses of a large Swedish cohort (n = 8,980; All New Diabetics In Scania [ANDIS] cohort) comprising newly diagnosed diabetes patients (8). A major finding is that the patients may be classified into five distinct subgroups, wherein among the nonautoimmune diabetes, 80% exhibit reduced insulin secretion capacity. Again, this supports the idea that impaired β-cell function not only contributes to the progression of the disease but is the primary driver of T2D.

    It has been 15 years since the work on the first islet-abundant miRNA, miR-375, was published (9). Today we know that miR-375 has multiple functions in the islet β-cell including roles in development, proliferation, and secretion (912). In addition, many other miRNAs have been demonstrated to have central roles in the pancreatic β-cells (13,14). Thus, it is not surprising that several miRNAs are differentially expressed in islets from human T2D donors (1517) and in diabetic animal models (18,19). Levels of miRNAs can be regulated by metabolic substrates that are increased in the blood prior to diabetes development, such as glucose and fatty acids (20). Moreover, miRNAs have the ability to act as rheostats, changing expression of the target genes to an optimal level (21). Hence, miRNAs are ideal to be part of β-cell adjustments to compensate for an increased demand to secrete more insulin. Indeed, many described differentially expressed islet miRNAs in diabetes have this role. Moreover, changes in the expression of other miRNAs before or during diabetes development are caused by genetic and/or regulatory defects of the miRNA expression leading to the inability of β-cells to secrete enough insulin.

    In this Perspective we will first focus on the current views on how miRNAs are involved in the regulation of insulin secretion and development of T2D. Then we will try to summarize future challenges and opportunities in islet miRNA research.

    Importance of miRNAs in the Pancreatic β-Cells

    The significance of miRNAs in the development and function of pancreatic β-cells has been shown by studying β-cell–specific Dicer1 knockout mice. Several different models of β-cell–specific Dicer1 knockout mice have been generated, and they all provide different clues how miRNAs collectively operate in the β-cell (Table 1). Knockout of Dicer1 during early development reduces β-cell mass (22), whereas knockout in adulthood results in functional defects (23,24). When Dicer1 was deleted under the Ins1 promotor, the mice developed full-blown diabetes (25). The contribution of miRNAs in specifying cellular identity is clearly exemplified in the β-cell where miRNAs can repress the expression of “disallowed” genes. This was demonstrated in a study using tamoxifen-induced deletion of Dicer1 under the Pdx1 promotor (24). Together, these studies show the importance of miRNAs in multiple processes from development, cell fate, proliferation, and maintenance to insulin production and secretion.

    Table 1

    Summary of different mouse models used to delete Dicer1 and their phenotypes

    Global miRNA ablation upon deletion or knockdown of Dicer1 in β-cells unequivocally demonstrates that multiple pathways in β-cell biology are influenced by miRNAs. However, to obtain detailed information on specific miRNAs and their function in β-cells, the focus has been on single miRNAs or miRNA families and a few of their targets. The most abundant miRNA in the islet, miR-375, was also the first miRNA detected in pancreatic islet (9). In this pioneering study, overexpression of miR-375 resulted in reduced exocytosis and thereby reduced insulin secretion. Later miR-375 deletion resulted in a phenotype with reduced β-cell mass (12), whereas a mouse model with a mild overexpression had no obvious phenotype (11). Modulation of miR-375 in cell lines and primary rodent cells have suggested roles in proliferation, insulin biosynthesis, ion channel activity, and exocytosis (10,26,27). Also, human islet cells losing their identity could be recovered to a β-cell phenotype by addition of miR-375 (28). As previously hypothesized with regard to the fine-tuning function of miRNAs (21), miR-375 is a typical miRNA that needs to be expressed at optimal levels in the cell, i.e., too high or too low expression would be detrimental for cellular functions.

    Other highly abundant miRNAs in islets are the members of the miR-29 family (miR-29a/b/c). These miRNAs are transcribed by two different miRNA clusters and contain a common seed sequence but can exhibit differential regulation and therefore do not always have the same function (29). The miR-29 family members have multiple functions in the β-cell, and downregulation increases expression of the disallowed monocarboxylate transporter gene Mct1 (30), increases exocytosis through targeting Onecut2 (31) and Stx1 (32), and increases apoptosis by binding to Mcl-1 mRNA (31).

    The miR-7 and the miR-200 family are other examples of islet abundant miRNAs. miR-7 is highly conserved among species and is derived from three different precursors. β-cell–specific overexpression of miR-7a in mice results in reduced insulin secretion through targeting genes involved in vesicle fusion and SNARE activity such as Snca, Cspa, and Cplx1 (11). The miR-200 family consists of five members, and β-cell–specific deletion in mice of either miR-200a/miR-200b/miR-429 or miR-200c/miR-141 shows that these miRNAs strongly regulate β-cell survival (33). Overexpression of miR-200 induces apoptosis and development of T2D in mice and vice versa: deletion of miR-200 protects the β-cells from apoptosis and ameliorates T2D.

    The stimulus-secretion coupling, which describes how glucose uptake results in increased insulin secretion through increased metabolism, electrical activity, intracellular Ca2+ concentration, and exocytosis, is central in β-cell function (34). Previous reviews from our group have highlighted the importance of miRNAs in the different subprocesses involved in insulin secretion (13), while another review emphasized miRNA involvement in β-cell survival and β-cell development (35). In summary, we have good knowledge today of how single miRNAs and specific miRNA families impact the expression of specific genes in the β-cell and how this influences insulin secretion. Next, we need to get a grip on the complexity in which miRNAs influence β-cell function, and for that we need to investigate how groups of miRNAs can affect single and/or multiple biological pathway(s).

    miRNAs in Islets of Healthy Subjects and Subjects With Diabetes

    Few studies have investigated global differential expression of miRNAs in human islets between donors without diabetes (ND) and donors with T2D (16,17). In the work by Kameswaran et al. (16), high-throughput sequencing of small RNAs was performed on islets from relatively few individuals (3 ND and 4 T2D), with a follow-up by quantitative PCR of hits in a larger cohort (islets from ∼15 donors in each group or sorted α- or β-cells from 3–4 donors). The expression of 16 miRNAs was identified to be downregulated in T2D islets (of these, 7 were transcribed from the DLK1-MEG3 locus at chromosome 14q32, a known region for genomic imprinting) and was shown to be regulated by epigenetic changes. The majority of these miRNAs had higher expression in β-cells compared with α-cells. Target validation using AGO2 immunoprecipitation identified targets within pathways important for cell survival (16). In another study using global Taqman miRNA arrays, Locke et al. (17) found miR-187 expression to be differentially upregulated in human T2D islets (11 T2D vs. 9 ND), followed by a validation cohort of 10 in each group using individual Taqman miRNA quantitative PCR assays. This miRNA was also observed to be upregulated in the work by Kameswaran et al. (16). Interestingly, miR-187 expression correlated negatively with insulin secretion in human islets, and overexpression of miR-187 in rat islets resulted in reduction of insulin secretion (17).

    Due to the scarcity of human islets for research, valuable information regarding differential expression of miRNAs in T2D islets was instead derived from rodent models. Zhao et al. (19) compared miRNA expression in islets from diabetes-resistant (B6) and diabetes-susceptible (BTBR) ob/ob mice and found enrichment not only of miR-375 but also of miR-127, miR-153, and the miR-200 family in islets compared with liver and adipose tissue. Interestingly, in human tissue, miR-375 and miR-127 were also shown to be more enriched in islets than in liver and muscle (15). Data from the B6 and BTBR mice also suggest that miR-184 expression in islets is suppressed by increased obesity, whereas miR-132/miR-212, miR-133a, miR-185, miR-152, miR-126-5p, and miR-34a/b increased in expression (19). Expression of miR-184 was later shown to be reduced in human islets from T2D donors, and miR-184 was suggested to be part of β-cell compensation during development of the disease (36).

    In rats, a global miRNA expression analyses in the pancreatic islets of the T2D model Goto-Kakizaki (GK) rat showed predominantly upregulation of miRNAs in the GK islets compared with Wistar controls (18). Among the top upregulated miRNAs validated were miR-132/212, miR-142-3p/5p, miR-130a, miR-124, miR-335, miR-376a, miR-433, and miR-409-3p. Collectively, although these miRNAs predominantly influence the expression of genes involved in transport and secretory processes, many potential target genes are also present in a wide array of biological networks (Fig. 2). Indeed, mechanistic studies on some of the upregulated GK miRNAs and their target genes revealed not only regulation of exocytotic genes, e.g., miR-335 targeting of Snap25, syntaxin-binding protein 1 (Stxbp1), and synaptotagmin 11 (Syt11) (37), but also regulation of genes involved in energy metabolism, e.g., regulation of pyruvate dehydrogenase E1 α (Pdha1) and glucokinase (Gck) by miR-130a/miR130b/miR-152 (38).

    Figure 2
    Figure 2

    The collective predicted mRNA targets of upregulated miRNAs in the GK islets were filtered to include only glucose-regulated genes and were subjected to GO enrichment analysis (18). The miRNA-mediated negative regulation of genes within each term is exemplified by the black “T” symbol. Among the genes within enriched GO terms, the majority belong to transport and secretory processes; the unique network (created in Cytoscape, version 3.7.2 [75]) in which the 10 miRNAs (yellow hubs) negatively regulate target genes (light blue nodes) is shown.

    Several studies have investigated differential expression in T2D for specific miRNAs (17,36,39,40). In some cases, it has been a follow-up from global profiling in islets from different diabetic animal models. miR-184 is one of the miRNAs downregulated in human T2D islets (36). A reduced expression of miR-184 in rodent models had impact on the expression of Ago2 and miR-375, which improved β-cell proliferation and increased insulin secretion. Thus, miR-184 is a typical example of a miRNA that participates in β-cell compensation during diabetes development. Of note, compensatory miRNAs seem to primarily impact β-cell proliferation and/or apoptosis (20). Expectedly, some of these compensatory miRNAs were first discovered in experiments investigating expansion of β-cell mass during pregnancy. One such example is miR-338. This miRNA was shown by Jacovetti et al. (41) to be downregulated during pregnancy. Moreover, inhibition of miR-338 increases proliferation and protects against β-cell apoptosis. Finally, the authors could show that this miRNA is reduced in high-fat diet–fed and ob/ob mice, clearly demonstrating a compensatory role in diabetes development. Interestingly, DLK1 is upregulated during pregnancy (42). The genetic region coding for DLK1 encodes many miRNAs, and the region is highly methylated in T2D islets. Also, as mentioned above, expression of miRNAs from this region is downregulated in T2D islets (16). Interestingly, experiments performed in mouse insulinoma βTC6 cells in which DNA methyltransferases in this region were activated resulted in increased methylation, reduced miRNA expression, and increased sensitivity to cytokine-induced β-cell death (43). Altogether, the DLK1-MEG3 region and the miRNAs involved have the capacity to promote β-cell expansion and protect against β-cell death prior to diabetes development; failure leads to full-blown diabetes. To this group of compensatory miRNAs also belong miR-375 (12) and miR-132/miR-212 (44). Meanwhile, other differentially expressed miRNAs in diabetes contribute to the β-cell dysfunction (20), including miR-130a/b and miR-152 (38). This is also true for miR-7 (39), miR-204 (45), and miR-200c (33).

    Recent focus in the field has been to identify islet cell type–specific or enriched miRNAs, e.g., with function in β-cells and subsequent validation in insulin-secreting cell lines or primary single β-cells. Only few studies have focused on α-cell–specific miRNAs. Two studies using sorted human α- and β-cells detected very few α-cell–specific miRNAs (16,46). In a more recent work, the α- and β-cell miRNA expression profiles between high-fat diet–fed obese hyperglycemic mice and low-fat diet–fed controls were compared (47), wherein miR-132 was identified to be highly differentially expressed in the α-cells between the two groups. Since glucagon secretion is also impaired in T2D and there is a potential for α-cell to β-cell transdifferentiation (48), understanding the role of miRNAs in α-cell biology is of great interest. One of the difficulties facing this area of research is the lack of a satisfactory α-cell line model.

    Future Challenges and Opportunities in miRNA Research

    Today we have good knowledge of a small group of miRNAs that show up in several studies to be either islet abundant (e.g., miR-375, miR-200 family, miR-127) or differentially expressed in islets in T2D (e.g., miR-7, miR-184, miR-187, miR-132/212, miR-130a/b, and miR-152). Still, current knowledge about islet miRNAs is limited, and there are enormous challenges and opportunities in miRNA research. For instance, one of the limitations of current findings is that most of the knowledge we have on miRNAs and their targets is from highly abundant pancreatic islet cell miRNAs (14). Meanwhile, even low/moderately expressed miRNAs are also perturbed in diabetes (19,38,44). However, investigating individual effects of such less expressed miRNAs is expected to generate marginal phenotypes with small effect size. To circumnavigate this, a combinatorial approach in manipulating the levels of multiple differentially expressed miRNAs could provide a key in truly understanding the impact of low/moderately expressed miRNAs in islet cell pathophysiology. Finally, there are great possibilities for clinical utilization of miRNAs in therapeutics and as biomarkers, but this also comes with a few challenges.

    Below we exemplify four areas of interest for future islet miRNA research (also summarized in Fig. 1): 1) transcriptional regulation of miRNA expression, 2) nonconservation of miRNA families and variable targeting, 3) the complexity of miRNA–mRNA interaction network, and 4) use of miRNAs as therapeutic tools in diabetes treatment.

    Transcriptional Regulation of miRNAs

    Several islet studies have shown different environmental stimuli that can regulate miRNA expression such as glucose (18,45,49), fatty acids (44), and cytokines (5052) via islet-specific transcription factors. Many intronic miRNAs are coregulated with their host coding genes. However, most miRNAs are under independent transcriptional control of specific transcription factors. Currently, a few detailed analyses of signaling pathways involved in miRNA transcriptional regulation in islets have been done. For miR-132/miR-212 cluster, we and others demonstrated that its transcriptional regulation is dependent on the presence of cAMP and is a PKA-dependent process involving the transcriptional coregulator CRTC1 together with CREB (53,54), as well as the novel transcription factor CAMTA1 (55). For other miRNAs, it has, e.g., been shown that miR-204 expression is increased through glucose-mediated regulation of thioredoxin-interacting protein (TXNIP) (45), while miR-184 expression is reduced through glucose-regulated changes in AMPK activity (49). Moreover, the transcription factor NeuroD1 cooperates with Pdx1 to regulate the transcription of miR-375 (56). Understanding detailed transcriptional regulation mechanisms of miRNAs is central for deeper understanding of their roles in diabetes development.

    Another aspect is how miRNA expression can be regulated by changes in the epigenome and expression of other noncoding RNAs. The finding that several miRNA genes present in the DLK1-MEG3 cluster are highly methylated in T2D (16) suggests epigenetic regulation of miRNA expression. Moreover, differential DNA methylation between sexes is associated with differential expression of miR-660 and miR-532 (57). Other types of noncoding RNAs have also been suggested to regulate miRNA expression. There is initial evidence that long noncoding RNA H19 might repress let-7 (58) and that the circular RNAs ciRS7 and circHIPK3 can sequester miR-7 (59,60). The mathematical integration of posttranscriptional networks (containing gene expression data, expression of noncoding RNAs including miRNAs and epigenetic data) will be one of the future challenges, but also an opportunity for a better understanding of the complex regulation of the pancreatic islet cells. However, a comprehensive and accurate understanding of biological processes may only be attained by careful validation using traditional molecular biology tools.

    Nonconservation of miRNA Families and Variable Targeting

    The miRNA families, just like any genetic element, arose from evolutionary processes over long timescales. Remarkably, while strong purifying selection associated with their regulatory roles resulted in gain of new functional miRNAs, their loss in different genomes has also been shown to be frequent (61). This heterogeneity in rates of miRNA evolution across phyla resulted in species specificity of many miRNA families. Indeed, comparing the well-characterized genomes of humans and mice, there are more than 600 mature miRNAs that can only be found in humans (1). An interesting example of a human-specific miRNA is miR-941, which was shown to be highly expressed in pluripotent stem cells and is involved in brain development and function (62). Characterization of regulatory functions of the nonconserved human-specific miRNAs in mouse and rat diabetes models is a challenge, similar to hurdles posed by primate-specific long noncoding RNAs in islet research (63). Another challenge comes from the nature of miRNA–target interactions in which variable targeting can arise from differences in 3′ UTR sequences of the same gene target in different organisms (64). Although most mammalian mRNAs are conserved miRNA targets (65), it was estimated that ∼50% of the predicted miRNA target sites in humans are not conserved in other organisms (66). In one large-scale study, many experimentally determined noncanonical and nonconserved sites were identified for a number of miRNAs including let-7c, miR-103, miR-106b, miR-141, miR-15a, miR-16, miR-17-5p, miR-192, miR-20, miR-200a, and miR-215 (67). In human and chicken primary chondrocytes, a significant number of nonconserved targets of miR-140 were identified using mRNA expression profiles after manipulation of miR-140 levels (68). Even in closely related species such as rats and mice, the highly enriched β-cell miR-375 modulates voltage-gated sodium channels differently resulting in variable shifts in steady‐state inactivation properties of the channel (27).

    Complexity of miRNA–mRNA Interaction Network

    The development of mathematics moving into life science comes with many possibilities to fully dissect complex cellular networks. miRNAs have been suggested to act as rheostats in biological systems (21), making them ideal entities in computational modeling for better understanding of their roles. The fact that a single miRNA may have many different targets and that a single mRNA may be targeted by multiple miRNAs means a complex regulatory network of miRNA–mRNA interactions. Remarkably, miRNAs are known to target multiple genes belonging to the same biological pathway (69). In our own pathway analysis of collective targets of the 10 upregulated miRNAs in the islets of GK rats (18), we found that Gene Ontology (GO) terms clustered as “transport and secretory related genes” were enriched (Fig. 2). However, we also found other enriched biological processes, each of which has its own unique set of miRNA–mRNA interaction networks. Consequently, the complexity of miRNA-mediated regulation of insulin secretion starts to emerge.

    In the future we need more of these networks in which we can also integrate genetic and epigenetic factors. This is a mathematical challenge encompassing a huge number of coregulatory processes, but with novel bioinformatics approaches there are great possibilities to solve this problem. However, one must always consider the quality of input data required in such endeavors, lest one only succumbs to the most common pitfall of “big” data analytics: “garbage in, garbage out.” For instance, the miRNA seed sequence is very short, giving rise to a high number of predicted false-positive targets, emphasizing why biological validation of targets is necessary for accurate network models.

    miRNAs as Therapeutic Tools in Diabetes Treatment

    Due to the fact that miRNAs can impact insulin secretion through multiple cellular pathways, they also hold promise to become excellent therapeutic targets. Currently several candidates are in phase 1 and phase 2 clinical trials, e.g., a locked nucleic acid (LNA)-based drug to inhibit miR-92 has potential in wound healing (70). In attempts to treat T2D, studies in animal models have used strategies that could be potentially translated to humans (7173). Different approaches to silence the miRNAs using chemically modified antagomirs have been employed in these studies. LNA molecules with modified backbones are more stable in blood (74), which might be the most favorable for future studies. However, like any drug-development strategies, a huge challenge is tissue-specific delivery of these RNA-based therapeutics. In our work, systemic injection of antagomir-132 resulted in its delivery in the pancreatic islets resulting in reduced miR-132 levels and subsequent glycemic improvement in the mice (72). Although a promising “proof-of-concept” work in the field, specific delivery into the relevant cells/tissue of RNA-based drugs is highly desired. Another great opportunity utilizing miRNAs as drug targets is the simple base-pairing mechanism of miRNA-mediated regulation, which allows for designing sequences tailored for specific gene variants. With that said, there are still many challenges with RNA-based inhibitors of miRNAs. Current complications with specificity and off-target effects, sensitivity of inhibitors, innate immune responses, and the exact tissue/cell delivery require further development of antagomirs before use. However, the concept of using miRNA inhibition offers a novel approach in personalized diagnostic and treatment strategies in diabetes and associated complications.

    Concluding Remarks

    We hope this Perspective will help put renewed focus on miRNAs in islet research and the possibility of using miRNA antagomirs in treatment of T2D. We wanted to highlight some of the challenges and opportunities that miRNA research in islets, and in general, will be facing in the coming years. During the 15 years since the first discovery of an islet-enriched miRNA, an immense amount of data and knowledge have been gathered. Let us hope for a new prosperous miRNA period, keeping in mind that we are also moving into the era of personalized medicine.

    Article Information

    Acknowledgments. The authors thank colleagues and friends for fruitful discussions in this area.

    Funding. The authors are supported by grants from the Swedish Foundation for Strategic Research (IRC-LUDC), the Swedish Research Council (project grant to L.E., SFO-EXODIAB), Region Skåne-ALF, the Swedish Diabetes Foundation, the Diabetes Wellness Network Sweden, Albert Påhlsson Foundation, Crafoord Foundation, Byggmästare Olle Engqvist Foundation, Syskonen Svenssons Fond, and the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 667191 (T2DSystems).

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

    • Received October 15, 2019.
    • Accepted December 20, 2019.



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    Spicy Beef Lettuce Cups with Peanut Noodle Salad

    By electricdiet / May 23, 2020


    My friend Bobby from FlavCity has SO many recipes.  I got his keto cookbook last year and I made a few dishes right off the bat.

    But like most things in life, it got put to the side and I recently rediscovered it.

    I made his Spicy Beef Lettuce Cups – I followed his recipe exactly for the beef filling (sans onions!) and made a different noodle salad on top using hearts of palm pasta.  You can find his recipe here.

    I had forgotten that he wrote this inscription in my copy 😀

    Here is the lineup for my “pasta” salad:

    If you are a condiment whore like me, you probably already have most of the ingredients.  Although I was out of rice vinegar and stocked up on that – I puffy heart that vinegar.  Also I used PB Fit in this recipe because that’s what I had on hand.

    Print

    Spicy Beef Lettuce Cups with Peanut Noodle Salad

    Spicy Beef Lettuce Cups with Peanut Salad – this recipe screams with flavor – I love the combo of the hot beef mixed with the cold noodle salad.

    The recipe for the beef can be found on Bobby’s blog – link above – this is the recipe for the noodle salad.

    • Author: Biz
    • Prep Time: 10
    • Total Time: 10
    • Yield: 4 servings 1x

    Scale

    Ingredients

    2 tablespoons powdered peanut butter mixed with 2 tablespoons water
    1 tablespoon soy sauce
    1 teaspoon sambal oelek (thai chili sauce)
    1 teaspoon minced garlic
    juice of 1 lime
    zest of 1 lime
    1 teaspoon sesame oil
    1 teaspoon rice wine vinegar
    1/2 cup diced red bell pepper
    1/2 cup chopped sugar snap peas
    1 package hearts of palm pasta

    Instructions

    First drain the pasta for 10 minutes in a strainer. Next, heat a skillet with nothing in it over medium low heat and cook for 6 minutes – this gets all the excess moisture out of the pasta. Add to a bowl.

    In the same pan, add the sesame oil and cook the peppers and peas for a few minutes. Toss in with the pasta. In a small bowl, whisk the peanut butter through rice wine vinegar and toss to coat. This is a cold salad. I made it a day ahead and it was way more flavorful the next day. I think I may use the filling to make wontons for dinner tonight.

    Notes

    Each wrap on all @ww plans is 3 points – I added 1/3 cup cooked rice to my wraps 🤩 Also, the whole noodle salad is only 2 points for the whole recipe – I barely used 1/4 of it in the wraps above, so am not counting points for that.

    This was hearty, healthy, bright and delicious! Give hearts of palm a try, you might be surprised you’ll like it!

    I’ve learned some things about this hearts of palm pasta since I’ve been using it.  It comes in a plastic bag that is pantry shelf stable, and I find that if I drain it for a bit, then pan “fry” it over low heat without anything in the pan, it evaporates all the moisture from the hearts of palm.  I like that texture much better.

    That being said, this is not going to taste like bucatini pasta from an Italian restaurant.  Its hearts of palm in the shape of pasta, so it tastes like hearts of palm, which is like the tofu of vegetables – not much flavor until you mix something with them.  Here is how it looks after its dried out.

    I made the noodle salad the day before I put this dinner together and it had WAY more flavor marinating overnight.  The veggies were all still crunchy which was a nice texture balance with the beef.

    Klassy as ever!  Ha!

    Here is what I thought of my dinner:

    Guess what?!  I am doing a live with Mariano’s tomorrow night!  The event is free, you just need to register to get the zoom link – I hope you’ll join me!

    Wine Wednesday! Creating the perfect ceviche with Belinda Chang & Biz Velatini of @MyBizzyKitchen

    About this Event

    Keep calm, stay home, drink wine with friends, and learn how to whip up the perfect ceviche! There is no corkage fee at this virtual gathering! Join Mariano’s Tastemaker and sommelier Belinda Chang and Biz Velatini of @MyBizzyKitchen as they share the perfect ceviche recipe and what wines to sip while making it!

    It’s at 6:30 p.m. tomorrow night – hope to see you there!





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    Keto Tomato Sauce Recipe (Sugar-Free)

    By electricdiet / May 21, 2020


    This recipe for sugar-free keto tomato sauce is full of flavor and can be used in so many recipes. All while only using 5 ingredients!

    keto tomato sauce in glass jar

    While tomatoes are keto-friendly, store-bought versions of tomato sauce can be full of hidden sugars, carbs and who knows what else that isn’t good for you.

    This is a simple keto tomato sauce recipe that you can use in any way you like. Use this sauce on a cauliflower pizza crust, low-carb eggplant parmesan, or a keto pizza casserole.

    The sauce can easily be prepared ahead, saving you time in the kitchen.

    How to make keto tomato sauce

    Step 1: In a small saucepan over medium-high heat, saute the garlic in the olive oil. Saute for a minute until the garlic is fragrant. Make sure the garlic doesn’t brown and become burnt.

    garlic being fried in olive oil

    Step 2: Turn the heat down and add the tomatoes, oregano, vinegar, and salt to the saucepan. Bring to a simmer and allow to cook for 30 – 40 minutes, allowing the sauce to reduce and thicken.

    sauce being cooked until thick with wooden spoon

    Step 3: Use the sauce immediately if desired or store for later use.

    Make a batch and store it!

    You can use this sugar-free tomato sauce immediately if desired or it can be stored for later use. To store the sauce, allow it to cool to room temperature first. Once cooled, transfer to an airtight container and refrigerate for up to 5 days. 

    You can also freeze the sauce after it has cooled to room temperature. Freeze in bags or a container for up to 6 weeks. Defrost before use. 

    tomato sauce in pot with spoon

    Spicing it up!

    Because this keto marinara sauce only uses 5 simple ingredients, it’s a great base that you can add other flavors to.

    It tastes fantastic as it is, of course, but you can change the flavor profile by adding additional spices such as basil or parsley. You can also adjust the heat by adding red pepper flakes or even chopped chili peppers.

    A sweet sugar-free tomato sauce?

    The fact that this sauce is sugar-free doesn’t mean that it can’t be a little sweet! There are many no-carb sweeteners that you can eat on a keto diet.

    I have made this sauce with a little bit of Stevia sweetener and it turned out great. Monkfruit sweetener is also a great option. Just be careful and add a little at a time as both sweeteners are quite strong.

    Other healthy low-carb recipes to try

    If you liked this recipe, here are some other low-carb recipes you might enjoy: 

    You can also check out the roundup I created of Healthy Dinner Recipes for Diabetics for even more great recipe ideas.

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

    Recipe Card

    Keto Tomato Sauce

    This is the best recipe for a keto tomato sauce that is full of flavor and can be used in so many recipes. All while only using 5 ingredients!

    Prep Time:5 minutes

    Cook Time:30 minutes

    Total Time:35 minutes

    Servings:10 servings

    featured image for tomato sauce recipe

    Ingredients

    • 1/4 cup extra virgin olive oil
    • 3 cloves garlic finely diced
    • 1 28 ounce can diced tomatoes
    • 1/2 tsp. dried oregano
    • 1 tsp. white vinegar
    • 1/2 tsp. salt

    Instructions

    • In a small saucepan over medium-high heat, saute the garlic in the olive oil. Saute for a minute until the garlic is fragrant. Make sure the garlic doesn’t brown and become burnt.

    • Turn the heat down and add the tomatoes, oregano, vinegar, and salt to the saucepan. Bring to a simmer and allow to cook for 30 – 40 minutes, allowing the sauce to reduce and thicken.

    Recipe Notes

    You can use this tomato sauce immediately if desired or it can be stored for later use. To store the sauce, allow it to cool to room temperature first. Once cooled, transfer to an airtight container and refrigerate for up to 5 days.  You can also freeze the sauce after it has cooled to room temperature. Freeze in bags or a container for up to 6 weeks. Defrost before use. 

    Nutrition Info Per Serving

    Nutrition Facts

    Keto Tomato Sauce

    Amount Per Serving

    Calories 58 Calories from Fat 52

    % Daily Value*

    Fat 5.8g9%

    Saturated Fat 0.9g5%

    Trans Fat 0g

    Polyunsaturated Fat 0.6g

    Monounsaturated Fat 4g

    Cholesterol 0mg0%

    Sodium 6mg0%

    Potassium 128.2mg4%

    Carbohydrates 1.5g1%

    Fiber 0.5g2%

    Sugar 1g1%

    Protein 0.7g1%

    Vitamin A 0IU0%

    Net carbs 1g

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

    Course: Side Dish

    Cuisine: American

    Keyword: keto marinara sauce, Keto tomato sauce



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    Hacienda Chicken – No More Boring Chicken Dinners!

    By electricdiet / May 19, 2020


    No Boring Chicken Recipes Here

    Why get bored of chicken when you can take it to new heights with Holly’s Hacienda Chicken recipe from Guy’s Guide to Eating Well cookbook ? Found in the Heart Disease chapter of Holly’s latest cookbook, this recipe is the epitome of casual cooking! Sprinkle chicken with a smoky cumin seasoning in a simple salsa sauce and have yourself an effortless yet flavorful, protein-packed dinner in under a hour. Good news, it is also, is a freezer-friendly and diabetic recipe.

    Hacienda Chicken
    Easy and effortless, chicken with a smoky cumin seasoning in a simple salsa sauce makes a quick, flavorful nightly dinner.
    Freezer friendly, Diabetic

      Servings4 servings
      Prep Time5 minutes
      Cook Time30 minutes

      Ingredients

      • 1 1/2pounds


        bonelessskinless chicken breasts

      • 1tablespoon


        ground cumin

      • 1


        onionthinly sliced

      • 1cup


        picante sauce or salsa

      • 1/2cup


        plain fat-free Greek yogurt

      • 1tablespoons


        all-purpose flour

      • 1/2cup


        chopped green onion

      Instructions
      1. Sprinkle chicken with cumin. In large nonstick skillet coated with nonstick cooking spray, brown chicken on both sides. Top with sliced onions and picante sauce.

      2. Cook, covered, over medium-low heat 25-30 minutes or until chicken is tender.

      3. In small bowl mix yogurt and flour. Lower heat and gradually add yogurt to pan, heating but do not boil. Sprinkle with green onions.

      Recipe Notes

      Calories 260kcal, Calories from Fat 18%, Fat 5g, Saturated Fat 1g, Cholesterol 109mg, Sodium 460mg, Carbohydrates 13g, Dietary Fiber 1g, Total Sugars 7g, Protein 39g, Dietary Exchanges: 2 vegetable, 5 lean meat

      Serving Suggestion: Serve with yellow rice.

      Terrific Tip: Pick different flavored salsas to switch up the flavor.

      Hacienda Chicken from Heart Disease Chapter In Men’s Cookbook

      When Holly was writing her easy men’s cookbook, she knew she wanted good-for-you foods in creative ways. Throughout the book, Holly provides delicious good-for-you recipes like low saturated fat skinless chicken breast to lower heart disease risk. lean meats and Holly included men’s favorite recipes but made them healthier.  This book is a great resource of information as this chapter gives you the foods to fight inflammation. Plus, this cookbook entices men in the kitchen.

      Get All of Holly’s Healthy Easy Cookbooks

      The post Hacienda Chicken – No More Boring Chicken Dinners! appeared first on The Healthy Cooking Blog.



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      A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of β-Cell Glucolipotoxicity

      By electricdiet / May 17, 2020


      Abstract

      Type 2 diabetes (T2D) is caused by loss of pancreatic β-cell mass and failure of the remaining β-cells to deliver sufficient insulin to meet demand. β-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on β-cell function and survival, contributes to T2D-associated β-cell failure. Drugs and mechanisms that protect β-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected β-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured β-cell–protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve β-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

      Introduction

      Diabetes is caused by inability of the pancreas to meet metabolic demand for insulin due to a shortage of functional insulin-secreting β-cells (1). In type 2 diabetes (T2D), elevated levels of circulating glucose and fatty acids contribute to insulin resistance in peripheral tissues, which leads to an increased demand for insulin as well as direct effects on the β-cell, such as stress and apoptosis. This concept of toxic energy excess, termed glucolipotoxicity (GLT), has been extensively studied in the last two decades (2,3).

      GLT conditions are generally associated with increased oxidative and endoplasmic reticulum (ER) stress, calcium dysregulation, and inflammasome activation (413). In the pancreas, GLT conditions result in increased β-cell death, low insulin content, and reduced glucose-stimulated insulin secretion (GSIS) (1417). GLT conditions have been shown to lead to reduced insulin gene expression via dysregulation of its upstream transcription factor PDX-1 (3). Decreased PDX-1 mRNA levels and nuclear exclusion of PDX-1 were observed in β-cells under GLT conditions and in islets from humans and rodents with diabetes (1821). ER stress and ER calcium dysregulation have also been implicated in GLT-induced β-cell dysfunction (22). Free fatty acids can induce calcium influx from the ER in β-cells via activation of Gq-coupled receptors such as G-protein–coupled receptor (GPR) 120, GPR40, and inositol 1,4,5-trisphosphate receptor (IP3R). High glucose stimulation of β-cells results in membrane depolarization, leading to calcium influx through the voltage-gated L-type calcium channels on the plasma membrane and, as a result, calcium-induced calcium release from the ER via the ryanodine receptor and IP3R. The influx of calcium into the cytosol is necessary for the glucose-stimulated insulin release, but excess stimulation with high levels of glucose and fatty acids depletes ER calcium stores, which can also contribute to the reduced insulin secretion under GLT (23). Moreover, several recent reports support a role for cytosolic calcium overload in pathogenesis of diabetes. In mice expressing a mutant form of Abcc8, a key component of the β-cell KATP channel showing constant depolarization similar to that observed with nutrient overload, intracellular calcium is constantly elevated, leading to diabetes (24). Furthermore, mouse islet cells exposed to diabetic serum show hyperactivation of L-type calcium channels (25).

      To date, there are no efficient therapies protecting patients with diabetes from β-cell death and loss of β-cell mass. Glucagon-like peptide 1 (GLP-1) signaling has been reported to exert direct β-cell–protective effects (26,27). However, despite many years of research, whether the beneficial effects of GLP-1–based therapy on β-cell mass observed in animal studies are applicable to humans is still unclear (28).

      Here we report discovery of a chemical series that protects β-cells and islets from GLT-induced apoptosis and dysfunction. Mechanism of action (MOA) studies revealed that this series protects β-cells by inhibiting GLT-induced calcium influx. Our work suggests a central role for cytosolic calcium overload in β-cell GLT.

      Research Design and Methods

      Cellular Apoptosis and Viability Assays

      For more details including cell culture, materials and screening, please see Supplementary Methods. The GLT apoptosis assay was performed in INS1E cells as follows. After overnight incubation in low-serum culture media (RPMI plus 0.5% BSA), cells were preincubated with compounds for 1 h and then challenged with BSA-conjugated palmitate (800 μmol/L palmitate; palmitic acid [PA]:BSA ratio 6:1) (#P9767; Sigma-Aldrich). Apoptosis was measured by caspase 3/7 Glo (CaspGlo, #G8091; Promega) 24 h after the palmitate challenge. Cell viability was measured by Cell Titer Glo (#G7570; Promega) 48 h after the palmitate challenge, according to the manufacturer’s instructions, and luminescence was detected by EnVision Plate Reader (Perkin Elmer). For both CaspGlo and Cell Titer Glo assays, the percentage reduction and percentage protection, respectively, were calculated using relative luciferase units (RLU) as [(RLU of DMSO control + PA − RLU of compound + PA)/RLU of DMSO + PA]∗100%.

      For TNF-related apoptosis-inducing ligand (TRAIL) apoptosis assays, Jurkat cells (#TIB-152; ATCC) were treated with compounds for 1 h, followed by treatment with human recombinant TRAIL protein (100 μmol/L final concentration) (#375-TL-010; R&D Systems), and apoptosis was measured as described above using CaspGlo kit 24 h after the TRAIL challenge.

      For high-throughput screening, the final BSA-palmitate concentration was 1 mmol/L (PA:BSA ratio was maintained at 6:1).

      Islet Apoptosis and GSIS Assays

      Rat pancreatic islets were isolated from Sprague Dawley rats after perfusion with Liberase TL Research Grade (#5401020001; Sigma-Aldrich) as previously described (29). For apoptosis evaluation, islets were dispersed to single cells using a papain dissociation kit (#LK003150; Worthington) and plated in laminin-5–coated plates. After 48 h recovery, islet cells were cotreated with GLT media (0.5% fatty acid-free BSA, 500 μmol/L palmitate in RPMI, PA:BSA ratio 6:1, 16 mmol/L glucose) and compounds of interest for 72 h. For determination of cell death, islet cells were stained with 1:1,000 Hoechst, 1:1,000 propidium iodide (PI), and 1:20 annexin V Alexa Fluor 488. The staining was analyzed using the Cellomics ArrayScan (Cellomics, Pittsburgh, PA). See the Supplementary Data for additional details and calculations of apoptosis protection.

      Human islets were obtained from Prodo Laboratories (Aliso Viejo, CA), which provided donors’ demographic and clinical data, HbA1c level, and islet isolation parameters. Human islets were cultured for at least 24 h in PIM(S) human islet-specific medium (Prodo Laboratories), containing 5% human AB serum and 5.8 mmol/L glucose before assay.

      For GLT-GSIS assays, rat or human islets were incubated in high-glucose, palmitate-containing media (300 μmol/L PA:BSA, 27 mmol/L glucose) overnight at 37°C in 5% CO2. Islets were washed with Krebs-Ringer bicarbonate buffer (KRBB) containing 2.8 mmol/L glucose, and three size-matched islets per well were picked into a 96-well plate by stereomicroscope observation. For each experiment, four to eight wells (three islets each) per condition were assayed. After 2 h of incubation in KRBB at low glucose (2.8 mmol/L) condition, islets were stimulated with high glucose (16.7 mmol/L) for 1 h. The culture media were filtered to remove cell debris and stored at −20°C for analysis. Intracellular insulin was extracted overnight in lysis buffer (Cyquant kit, #C7026; Invitrogen) at −20°C. Insulin concentration in culture media and islet lysates was determined using the Cisbio insulin ultrasensitive assay kit (62IN2PEG; Cisbio) and/or the Mercodia insulin ELISA kits (rat: Mercodia #10-1,124-10; human: Mercodia #10-1,113-01) with appropriate sample dilutions. All GSIS experiments were performed three times or more. For rat islets, pancreata from eight rats were pooled and combined for each islet isolation. For human islets, islets from two donors without diabetes were used individually. For human islet assays, secreted insulin and insulin content were normalized by DNA content in human islets lysates (Cyquant DNA proliferation kit, #C7026; Invitrogen). We used GraphPad Prism 8.1.2 software for data analyses and ordinary one-way ANOVA for statistical significance assessment.

      Sample Preparation for Quantitative PCR and RNA Sequencing

      Isolated islets from eight rats were divided into seven groups (∼1,000 islet equivalents/sample) as follows: two control groups cultured in regular media (RPMI with 11 mmol/L glucose) with DMSO for 3 and 24 h, and two treated with GLT media (27 mmol/L glucose and 300 μmol/L PA:BSA in RPMI) and DMSO, or GLT media with 300 nmol/L compound D or compound E for 3 and/or 24 h. This experiment was performed three times with different islet preparations to obtain three independent biological repeats. Total RNA was prepared using the RNeasy kit (Qiagen). RNA quantity and quality were assessed using an Agilent 2100 Bioanalyzer and 1 μg total RNA was sent to the Beijing Genome Institute for reverse transcription, library preparation, and sequence analysis (Hi-Seq2000; Illumina). RNA sequencing (RNAseq) reads (100 base pairs) from paired-ends were mapped to the rat genome (rn5) with TopHat2 (version 2.0.3) and Bowtie2 (version 2.0.0) (30,31). Gene expression values were summarized as raw counts by running high-throughput sequencing (HTSeq) software (32). The variance-stabilizing transformation from DESEq software (33) was applied to the raw sequencing count data before statistical analyses. See Supplementary Data for the statistical models to identify significant gene expression changes. For PDX-1 gene expression analyses by quantitative PCR we used the TaqMan probe set Rn01423448_m1 from Thermo Fisher.

      Calcium Flux Assay in Islet Cells

      For calcium flux assay, we used a Fluorescent Imaging Plate Reader (FLIPR) Tetra High-Throughput Cellular Screening System. Papain-dissociated islets were plated on laminin-coated 384-well plates (354663; BD). After 48 h of recovery, islets were pretreated with compounds in GLT media (16 mmol/L glucose, 500 μmol/L PA:BSA in RPMI) for 1 h at 37°C in 5% CO2. FLIPR calcium dye 6 was then resuspended in GLT media and added with appropriate compound dilutions for 2 h at 37°C in 5% CO2. Calcium influx was measured for the next 10 min at 1-s intervals. For chronic calcium load measurements, readings were taken every minute over 10 consecutive hours after GLT treatment, and the cumulative calcium signal was calculated based on the sum of individual measurements per assay condition. Analyses were done using Molecular Devices software, with the amplitude of the response (Fig. 6A, ΔF/F maximum-minimum fluorescent signal) as the main reported readout.

      Measurement of Recombinant L-Type Calcium Channel Activity

      Measurements of compound activities on L-type calcium channel current was performed on the IonWorks Barracuda system (Molecular Devices, LLC, San Jose, CA) by ChanTest Corporation (Cleveland, OH). Cav1.2 channel (cloned Cav1.2/β2/α2δ1) was expressed in CHO cells (ATCC), and stable cell lines were generated. For Cav1.2 measurements, CHO cells stably expressing Cav1.2 were treated with compounds diluted in a calcium-free HEPES-buffered physiological saline (composition in mmol/L): NaCl, 137; KCl, 4.0; MgCl2, 2.8; HEPES, 10; glucose, 10; and BaCl, 5.0 (pH 7.4). The current recordings were performed on IonWorks Barracuda at ambient temperature, before compound application to the cells (baseline) and 5 min after the application. Before digitization, the current records were low-pass filtered at one-fifth of the sampling frequency (10 kHz). Cav1.2 current was measured using a stimulus voltage pattern consisting of a depolarizing test pulse to 10 mV for 300 ms from a −90 mV holding potential. Peak current was measured during the step to 0 mV. Data acquisition and analyses were performed using the IonWorks Barracuda system operation software (version 2.0.2). The decrease in current amplitude after test article application was used to calculate the percentage block relative to control. Results for each compound concentration (n ≥ 2) were averaged, and mean and SD values were calculated and used to generate dose-response curves.

      The blocking effect was calculated as: %Block = (1 − Icpd/IControl) × 100%, where IControl and Icpd are the currents measured before addition of compound and in the presence of a compound, respectively. Concentration-response data for inhibitors were fit to an equation of the following form: %Block = %VC + {(%PC − % VC)/[1 + (cpd/IC50)N]}, where cpd indicates compound concentration, N is the Hill coefficient, %VC is the percentage of the current run-down, and %Block is the mean value of the percentage of ion channel current inhibited at each concentration of compound. Nonlinear least squares fits were solved with the XLfit add-in for Excel 2003 software (Microsoft, Redmond, WA).

      Data and Resource Availability

      The RNAseq data reported in this study are available in the Gene Expression Omnibus repository (GSE#134272). No applicable resources were generated or analyzed during the current study.

      Results

      Identification of a Molecule That Selectively Protects β-Cells From GLT-Induced Apoptosis

      To identify compounds that protect β-cells from GLT-induced apoptosis, we designed a low-molecular-weight compound phenotypic screen (Fig. 1A). Caspase 3/7 activation was measured in INS1E cells cultured in the presence of glucose and palmitate (GLT conditions) to identify compounds that reduced GLT-induced apoptosis. A total of 312,000 compounds were screened in 1,536-well plate format. The average Z′ for the primary high-throughput screen was 0.59, and the average RZ′ was 0.72. To verify that reduced caspase activation was not a result of cell death, compounds from the primary screen were tested in a general cell viability assay that measured ATP levels in GLT-treated INS1E cells. Compounds that reduced caspase activation but did not cause general cytotoxicity were further profiled in an apoptosis assay in Jurkat cells to eliminate general apoptosis inhibitors. Six scaffolds (chemical classes) that selectively protected β-cells from GLT-induced apoptosis remained and were further profiled in a primary rat islet cell apoptosis assay to select compounds that protect primary islet cells. Multiple readouts were used to measure apoptosis and general cell death in islets (annexin 5 and PI staining, respectively), to ensure specificity of the observed effects. Finally, compounds were tested for prevention of GLT-induced reduction of GSIS.

      Figure 1
      Figure 1

      An unbiased chemical screen for β-cell protection from GLT identifies compound (Cpd) A. A: Screen flowchart. For screen design and experimental flow, see the first section of results, Identification of a Molecule That Selectively Protects β-Cells From GLT-Induced Apoptosis. B: Structure of the screening hit compound A. *Rac, chiral center on the racemic allene compound.

      Compound A (Fig. 1B), a racemic mixture, inhibited GLT-induced caspase activation in INS1E cells but did not inhibit Jurkat cell apoptosis (Fig. 2A) and was selected for further characterization. The racemic compound A was separated into two enantiomers, more active B and less active C (Supplementary Fig. 1), and enantiomer B was further optimized. As a result of medicinal chemistry efforts, compound D was developed; it protected pancreatic islets from GLT-induced apoptosis at nmol/L concentrations (Fig. 2B and C). Further, a structurally related inactive tool, compound E (Fig. 2B), was used as a negative control in follow-up MOA experiments.

      Figure 2
      Figure 2

      Compound (Cpd) A and its derivatives protect INS1E cells and primary islet cells from GLT-induced apoptosis. A: INS1E cells or Jurkat cells were preincubated with compound A for 1 h, followed by apoptosis stimulus. INS1E cells were treated with GLT-conditioned media (800 μmol/L palmitate [PA:BSA, 6:1], 11 mmol/L glucose in RPMI). Jurkat cell apoptosis was induced with 100 μmol/L TRAIL. Apoptosis was measured in both cell lines after 24 h (n = 4–8 wells/condition). B: Structure of compounds B, D, and the inactive analog E. C: Dissociated rat islet cells were cotreated with compound D and GLT media (RPMI supplemented with 15 mmol/L glucose and 500 μmol/L PA:BSA). Apoptosis was detected after 72 h using annexin-V Alexa Fluor 488 staining, and the percentage of apoptosis protection was calculated as described in research design and methods.

      To determine whether the islet cells that were protected from apoptosis maintained their function, we tested whether compounds B and D could promote insulin production and secretion in response to glucose in rat and human islets compromised by GLT. As previously reported (18), a significant reduction of insulin content and insulin secretion after GLT treatment was observed in rat islets. Treatment with compounds B or D rescued both reduction in insulin content and GSIS in GLT-stressed rat islets (Fig. 3A and B). Exendin-4, a clinically used GLP-1 receptor agonist and a known stimulator of insulin secretion, served as the positive control in these experiments and increased GSIS under GLT conditions (Fig. 3B). Interestingly, unlike compounds B and D, exendin-4 did not alter islet insulin content (Fig. 3A).

      Figure 3
      Figure 3

      Compound (Cpd) B and compound D increased insulin content and GSIS in rat and human islets under GLT conditions. A and B: Rat islets (three islets/well, six to eight wells/condition) were incubated in GLT media (RPMI with 300 μmol/L PA and 27 mmol/L glucose) with indicated compounds for 24 h, and then GSIS assay was performed in the presence of the indicated compounds. After 2 h of preincubation in KRBB buffer with 2.8 mmol/L glucose, islets were stimulated with 16.7 mmol/L glucose in KRBB. Secreted insulin in media was measured after 1 h of stimulation (B), and the islets were then lysed to measure total insulin content (A). Islets from eight rats were pooled for each experiment. C and D: Human islet insulin secretion and content reduced by GLT are restored by compounds B and D. Human islets were incubated with GLT media and the GLP-1R agonist exendin-4 (Ex-4; 100 nmol/L), compound B, or compound D (300 nmol/L) for 24 h, followed by GSIS and insulin content measurement as for rat islets above. Compounds B and D preserved GSIS and insulin content in GLT rat islets, whereas exendin-4 increased GSIS without affecting insulin content. Experiments were repeated with islets from two donors without diabetes. E: Compound D did not increase GSIS in nonstressed rat islets. Rat islets were preincubated with compound D in normal media (RPMI media containing 11 mmol/L glucose and no PA) for 24 h and then stimulated with glucose for GSIS assay, followed by GSIS as above. *P < 0.05 vs. GLT. #P < 0.05 vs. control (CON), by one-way ANOVA.

      Compound D also protected human islets from GLT-induced dysfunction (i.e., increased insulin content and secretion under GLT) (Fig. 3C and D). Notably, compound D did not increase insulin secretion under normal culture conditions, suggesting the effects on islet function were specific to the GLT condition (Fig. 3E). Inhibition of insulin secretion occurred with compound D treatment at μmol/L concentrations and was initially attributed to scaffold toxicity (Fig. 3E). We then initiated studies to gain more insights into the bell-shaped effects of compound D on GSIS and to identify its MOA.

      Compound D Targets a Central Regulator of Islet GLT

      To determine which GLT-regulated genes might be affected by compound D, we performed an RNAseq analysis of rat islets treated ex vivo, with or without GLT and compound D, for 3 h and 24 h, respectively. The expected suppression of β-cell function/specification induction genes, such as PDX-1 and INS1, and induction of stress-response genes was observed after 24-h GLT treatment (Supplementary Tables 1 and 2). Treatment with compound D prevented close to 80% of the gene expression changes caused by a 24-h GLT treatment (Fig. 4 and Supplementary Table 3), suggesting that compound D protects islets by regulating a central node of GLT stress response. A closely related inactive compound from the same scaffold, compound E (Fig. 2B), did not reverse gene expression changes caused by GLT (Fig. 4; see Supplementary Table 4 for all gene expression changes and statistical models used in this study).

      Figure 4
      Figure 4

      RNAseq analyses of GLT-stressed rat islets reveal that most of the GLT-induced gene expression changes are prevented by treatment with compound (cpd) D. Total RNA was prepared from rat islets (1,000 islet equivalents/sample) treated with control or GLT-containing media (27 mmol/L glucose, 300 μmol/L PA:BSA) for 3 or 24 h, with DMSO, cpd D, or cpd E, an inactive compound with similar chemical structure, at 300 nmol/L. For control (Ctrl) groups, islets were collected after incubation in normal media (no PA, 11 mmol/L glucose) and DMSO. The effects of cpd D and cpd E are shown on the top 190 GLT-induced gene expression changes. Each column represents an independent treatment/repeat. Red, increased expression; green, decreased expression.

      Compound D Decreases Cytosolic Calcium Overload Induced by GLT

      To explore the MOA of compound D, we initially tested whether compound D may target the GLP-1 receptor, which has been reported to protect β-cells and improve their function; however, compound D failed to activate GLP-1 receptor signaling (data not shown). Moreover, compound D protected islets from GLT-induced loss of insulin content, whereas exendin-4 did not (Fig. 3A), suggesting distinct mechanisms.

      GLT depletes ER calcium stores and causes ER stress (13). We hypothesized that restoration of calcium homeostasis might be the potential MOA of compound D; therefore, we tested whether calcium flux modulators could phenocopy the effects of compound D in islets. We found that L-type calcium channel blockers nifedipine and verapamil reduced GLT-triggered islet apoptosis similar to compound D, whereas the calcium channel opener FPL64176 had the opposite effect, further boosting apoptosis under GLT conditions (Fig. 5A). We also tested whether modulators of ER calcium release and reuptake can mimic the effects of compound D on apoptosis. We tested the inhibitors of ryanodine receptor-mediated calcium release, ryanodine, dantrolene, and xestospongin, which inhibit IP3R-mediated calcium release. None of these three inhibitors had any effect on apoptosis under our assay conditions (Fig. 5A). Thapsigargin, which inhibits sarco-ER calcium ATPase and calcium reuptake from the cytosol into the ER, greatly increased GLT-induced apoptosis, consistent with the published role of ER stress in GLT (Fig. 5A). The calcium channel blocker nifedipine also prevented GLT-induced reduction in insulin content, and this effect was similar to and nonadditive with that of compound D. Furthermore, the L-type calcium channel opener FPL64176 blocked the protective effects of compound D, suggesting that compound D protection requires functional L-type calcium channels (Fig. 5B). Reduction in islet insulin content under GLT stress can be partly due to the suppressed expression of PDX-1 that we and others have observed under GLT. Nifedipine treatment partially prevented the reduction in PDX-1 expression, again mimicking the compound D effect (Fig. 5C).

      Figure 5
      Figure 5

      L-type calcium channel blockers mimic many of the effects of compound (Cpd) D in rat islets. A: Dissociated rat islet cells were cotreated with modulators of Ca2+ channels and GLT media (RPMI supplemented with 15 mmol/L glucose and 500 μmol/L PA:BSA). Apoptosis was detected after 72 h using annexin-V Alexa Fluor 488 staining, and the percentage of apoptosis protection was calculated as described in research design and methods. L-type calcium channel blockers but not ER calcium release inhibitors reduced GLT-induced apoptosis in primary rat islets. B: Nifedipine (Nif) increased insulin content reduced by GLT, similar and nonadditive to compound D, whereas the L-type Ca2+ channel opener FPL1642 (Fpl) reversed compound D effects on islet insulin content. Rat islets (three islets/well, six to eight wells/condition) were incubated in GLT-containing media (RPMI with 300 μmol/L PA:BSA and 27 mmol/L glucose) with compounds as indicated for 24 h. Total insulin content was measured as described in Fig. 3B. C: Rat islets were cotreated with GLT media (RPMI with 300 μmol/L PA:BSA and 27 mmol/L glucose) and nifedipine or compound D at 0.01, 0.1, and 1 μmol/L for 24 h. Islet mRNA was then isolated, and Pdx-1 expression measured by quantitative PCR. Similar to compound D, nifedipine dose-dependently restored expression of Pdx-1 reduced by 24-h GLT treatment. CON, control; Dnt, dantrolene (10 μmol/L); Ry, ryanodine (10 μmol/L); Tg, thapsigargin; Ver, verapamil; Xs, xestospongin (10 μmol/L); -, DMSO. Unless stated otherwise, compounds were tested at 1 μmol/L. *P < 0.05 vs. GLT by one-way ANOVA.

      Compounds from this scaffold were then tested for ability to directly regulate calcium flux in islet cells. Using a fluorescent cell-permeable calcium dye, we monitored cytosolic calcium in islet cells under GLT conditions (Fig. 6A). Interestingly, the GLT-induced increase in calcium influx into the cytosol was biphasic, with a sharp initial peak within seconds of GLT media addition, followed by a second sustained plateau. To explore the nature of the two calcium peaks, we pretreated islet cells with calcium modulators with known MOA. L-type calcium channel modulators and compound D dose-dependently reduced the second calcium peak (Fig. 6B and Supplementary Fig. 2), while depletion of sarco-ER calcium with thapsigargin eliminated the first calcium peak (Supplementary Fig. 2). Although the above experiments measure acute response to GLT, extended treatment of islets with GLT media led to sustained accumulation of cytosolic calcium over the course of 10 h, and compound D prevented this accumulation, reducing calcium to baseline levels (Fig. 6C). These results suggest that acute inhibition of GLT-induced calcium influx by compound D translates into chronic suppression of GLT-induced calcium overload, and this effect may be responsible for the protective effects of the scaffold. Indeed, using diverse molecules generated as part of the medicinal chemistry activities around this scaffold, we observed a tight linear association of calcium flux inhibition with apoptosis protection within the series (Fig. 6D), which suggests molecules from this series protect β-cells from GLT by modulation of calcium influx.

      Figure 6
      Figure 6

      Compound D and its analogs protect islets by reducing calcium influx. A: Calcium influx was measured in dispersed islet cells incubated with cell-permeable fluorescent calcium dye 6 after treatment with GLT media (27 mmol/L glucose, 500 μmol/L PA:BSA). Readings were taken every 2 s for 10 min after GLT addition. Maximum amplitude of the second peak (ΔF/F max-min) is indicated by the arrow. B: Calcium influx (ΔF/F max-min of the second peak) was measured in dispersed rat islet cells treated with compound (Cpd) D or nifedipine (Nif) at increasing concentrations (1 nmol/L–10 μmol/L) for 10 min after glucose and lipid addition. C: Calcium influx was measured as above but with readings taken every minute for 10 h after glucose and lipid addition. Cumulative calcium in islets under normal or GLT conditions, with or without compound D at 300 nmol/L, was calculated by summing all readings over the course of 10 h. D: Potency of calcium flux inhibition was tightly associated with GLT-apoptosis protection potency (R2 = 0.87) by molecules from this chemical series (each point represents one compound).

      To determine whether compound D is a bona fide L-type calcium channel blocker, its effect on recombinantly expressed L-type calcium channel Cav1.2 was examined in CHO cells. Nifedipine robustly blocked the Cav1.2- induced response, whereas compound D had no effect (Fig. 7A). In addition, unlike compound D, which consistently increased insulin secretion under GLT conditions, enhanced GSIS under GLT conditions was not observed with nifedipine, which resulted in further worsening of GSIS (Fig. 7B). These data imply that compound D regulates calcium influx by a mechanism that is distinct from that of nifedipine.

      Figure 7
      Figure 7

      Compound D does not block the recombinant L-type Ca2+ channel. A: Inhibition of calcium current was measured on the IonWorks Barracuda system in CHO cells expressing recombinant Cav1.2 after treatment with nifedipine (Nif) and compound (Cpd) D. The current recordings were performed at ambient temperature, before compound application to the cells (baseline) and 5 min after the application. Cav1.2 current was measured using a stimulus voltage pattern consisting of a depolarizing test pulse to 10 mV for 300 ms from a −90 mV holding potential. Peak current was measured during the step to 0 mV. Data acquisition and analyses were performed using the IonWorks Barracuda system operation software. The decrease in current amplitude after test article application was used to calculate the percentage block relative to control. B: Unlike scaffold compounds, nifedipine did not restore GSIS in GLT-stressed rat islets. GLT-GSIS was performed as in Fig. 3. CON, control; Ex-4, exendin-4 GLP-1 receptor agonist; -, DMSO. *P < 0.05 vs. GLT.

      Discussion

      Here we report the design, execution, and analysis of a phenotypic screen aimed to identify molecules that protect pancreatic β-cells from nutrient overload-associated GLT. We used an in vitro INS1E GLT assay to screen a library of compounds for reduction of GLT-induced apoptosis. Our goal was to identify compounds that specifically protect from GLT-induced toxicity in β-cells. We used several counterscreens to eliminate general apoptosis inhibitors and toxic molecules and identified a few selective β-cell–protective hits.

      GLT is associated with reduced insulin gene expression, reduced islet insulin content, and reduced insulin secretion (2). In rats, glucose and intralipid infusion decreases insulin biosynthesis, GSIS, and expression of β-cell genes despite increased β-cell mass, suggesting that β-cell mass per se cannot always predict the functional outcome (18). We therefore assessed the effects of our hits on primary islet function under GLT stress. Several observations indicated that the islet system recapitulates β-cell stress observed in human diabetes. First, we observed a modest increase in cell death, in line with an increased apoptotic index reported for islets from humans with diabetes (34). Second, we observed the expected reduction of INS and PDX-1 gene expression and induction of ER stress genes (Fig. 4 and Supplementary Table 4). We also observed reduction of islet insulin content (Fig. 3), which is a feature of T2D (21,34,35). Finally, we observed a GLT-induced reduction in insulin secretion that, as expected, could be reversed by a GLP-1 receptor agonist (Fig. 3), an approved diabetes drug (27).

      We identified a chemical scaffold that prevented apoptosis and improved insulin secretion in rodent and human primary islets under GLT stress. MOA studies pointed to the suppression of cytosolic calcium influx as a primary mechanism responsible for islet protection from GLT-induced apoptosis. We generated a series of compounds structurally related to our initial hit and observed a tight association of calcium flux modulation with apoptosis protection within the series. Unfortunately, the undesired pharmacokinetic properties prevented us from comprehensive testing of these molecules in vivo in a T2D animal model.

      The discovery that our compounds inhibit calcium flux was unexpected. Although calcium dysregulation by excess fatty acids is well documented (13) and apoptosis protection has been described for calcium channel blockers (36), the increased insulin secretion we observed was contrary to the well-established inhibition of insulin secretion with calcium channel blockers. Indeed, we have not observed inhibition of recombinant L-type channel with compound D or improvement of insulin secretion with the canonical calcium antagonist nifedipine. We hypothesize that compound D regulates calcium influx by a mechanism that is distinct from that of nifedipine and is a state-dependent L-type calcium channel antagonist or represents a novel class of calcium antagonists.

      The calcium antagonist described here could largely prevent gene expression changes induced by GLT, suggesting that intracellular calcium overload is the central mechanism underlying GLT-induced apoptosis and dysfunction. Several recent reports showed a link between calcium dysregulation and diabetes (37). Interestingly, we did not observe apoptosis protection for ER calcium release modulators but did observe protection with diverse L-type calcium channel blockers. The antidiabetic activity of verapamil was recently confirmed in human studies, where patients on verapamil showed decreased incidence of newly diagnosed T2D (38) and improvement in diabetes in a small clinical study (39). These human data support a role for calcium overload in diabetes pathology.

      Our results are consistent with a model (Fig. 8) where GLT conditions cause an increase in cytosolic calcium influx, which can induce apoptosis as well as reduction in gene expression of critical β-cell function genes, such as PDX-1 and insulin, followed by reduction in islet insulin content and GSIS. Molecules identified in the β-cell protection screen described here can reduce the excessive calcium influx by restoring nearly optimal calcium levels and protect islets from all of the above detriments. Unlike the canonical L-type calcium channel blocker nifedipine, these molecules allow insulin secretion under conditions of calcium channel overstimulation. Calcium antagonists with such properties may prove to be a novel class of diabetes treatment and prevention drugs.

      Figure 8
      Figure 8

      A model for protective MOA of compound D. Ins, insulin. See discussion for details.

      Article Information

      Acknowledgments. The authors thank Faye Zhao for uploading RNAseq data into Gene Expression Omnibus, Yunshan Peng, David Parker, Katsumasa Nakajima, Timothy Rasmusson, Toshio Kawanami, T.R. Vedananda, and Yongjin Gong for synthesizing analogs of compound D, Jeffrey A. Brown for insulin measurements, and Martin G. Waters and Jovita Marcinkeviciene (all from Novartis Institutes for BioMedical Research at the time of this work) for discussions and critical reading of the manuscript.

      Duality of Interest. All authors were employees of the Novartis Institutes for BioMedical Research at the time of the reported studies. No other potential conflicts of interest relevant to this article were reported.

      Author Contributions. J.V., J.Y., L.S., S.X.W., R.Z., S.F., C.-H.C., A.C.W., and B.D. performed experiments and analyzed data, A.C., F.Z., G.T., T.M.S, B.D., D.M.R., X.C., and A.B. contributed to discussions and data analyses. H.X. analyzed RNAseq data. A.B. wrote the manuscript. All authors reviewed and edited the manuscript. A.B. 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.

      • Received August 15, 2019.
      • Accepted February 7, 2020.



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      Asian Meatballs – My Bizzy Kitchen

      By electricdiet / May 15, 2020


      With everything that is going on in the world, I did a poll on my Instagram about what types of recipes you want to see, and the surprising response was making take-out dishes at home.

      I loved the idea of using the new calorie-free @Truvia Sweet Complete™ to make a sweet and savory dish, so I made Asian sweet and spicy meatballs with white rice and sugar snap peas.

      It is quick, delicious, and the meatballs can be made ahead of time as well as the rice, so the actual cooking time would be less than 15 minutes from beginning to end with a bit of planning. And, because Sweet Complete™ bakes and browns in recipes and sweetens and measures like sugar, I didn’t need to think twice about changing up the recipe.  Makes 4 servings. 

      Print

      Asian Meatballs

      These sweet and spicy Asian meatballs will be a hit with your whole family!

      • Author: Biz
      • Prep Time: 5
      • Cook Time: 10
      • Total Time: 15
      • Cuisine: Asian

      Scale

      Ingredients

      For the meatballs:

      • 1 pound ground sirloin
      • 1/2 cup panko bread crumbs
      • 1 egg
      • 1 teaspoon garlic powder
      • 1 teaspoon fresh ginger
      • 1/2 teaspoon salt
      • 1/2 teaspoon Truvia® Sweet Complete™
      • 1/2 teaspoon pepper

      Shape the meatballs in as big a shape as you like – I prefer small meatballs, so mine were about the size of a marble.  Heat skillet over medium heat, and cook the meatballs until they are about 80% cooked – about 4-5 minutes.

      For the glaze:

      • 1 teaspoon sesame oil
      • 2 tablespoons minced garlic
      • 1 teaspoon fresh ginger, minced
      • 1/4 cup soy sauce
      • 2 teaspoons cornstarch
      • 1/2 cup water
      • 2 tablespoons Truvia® Sweet Complete™
      • 1 tablespoon hoisin sauce
      • 1 tablespoon sugar free orange marmalade
      • 1 tablespoon orange zest
      • 1 teaspoon chili garlic sauce

       

      Instructions

      While the meatballs cook, place the glaze ingredients in a sauce pan and cook over medium heat, stirring constantly for 4 minutes or until thickened.  Toss in the meatballs and cook for 1 more minute.  Serve over rice garnish with what you have on hand – I used sliced sugar snap peas. 

      This is an easy recipe – pinky swear! This recipe is also done before you would even have the chance to call in a take-out order.  

      Notes

      This recipe is 8 WW points, no matter which plan you are on – that is just for the beef.  Completely worth it!!

      Keywords: Asian Meatballs

      While this post is sponsored by @Truvia Sweet Complete™, all opinions are my own.  You can read my full disclosure policy here.

       



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      Diabetes and Itching – Causes and Treatment Options

      By electricdiet / May 13, 2020


      The way your blood sugars affect your skin is so slow and so gradual, it can be easy to forget that complications of diabetes can appear in this part of the body. High blood sugars, in particular, can lead to skin conditions that develop slowly.

      Itchy skin is one of those conditions.

      In this article, we’ll look at how diabetes can cause itchy skin and available treatment options.

      How diabetes can cause itchy skin

      Diabetes-related skin conditions are typically the result of persistently high blood sugar levels. The American Diabetes Association explains that persistently high blood sugar levels will:

      • Feed and fuel the growth of fungus
      • Damage and destroy the cells of your skin
      • Reduce the flow of oxygen required for maintenance and healing of skin
      • Severely increase the risk of infection in minor cuts, blisters, scrapes

      When it comes to itchy skin and diabetes, there are three causes that result from high blood sugar levels:

      • Growth of fungus
      • Severe dry skin
      • Reduced blood circulation
      • Diabetic neuropathy

      Let’s take a closer look at these four sources of itchiness.

      Fungal Infections

      You’ve probably heard of yeast infections in regards to a women’s genital health issue, but people with diabetes can develop yeast infections in a variety of other locations on their bodies.

      The itchiness associated with the growth of fungus can often feel like an extreme burning along with extreme itchiness.

      Referred to as “Candida albicans,” this fungal infection is fueled and fed by the excess sugar in your blood. Depending on the location of the body, it can also be referred to as:

      • Vaginal yeast infection
      • Jock itch
      • Athlete’s foot
      • Ringworm

      Locations of your body that can be affected by fungal infections include:

      • Moist folds in the skin (between rolls of body fat, for example)
      • Underneath the lower portion of your breasts
      • Around your nails
      • Between your fingers and toes
      • In the corners of your mouth
      • Under the foreskin in uncircumcised men
      • In and around the vagina in women
      • In your armpits
      • In and around your groin area

      There are a few things all of these locations have in common: they are generally moist and less exposed to fresh air.

      When your blood sugars are running higher, that excess glucose in your bloodstream becomes the breeding ground for the growth of itchy, relentless fungus.

      Treating fungal infections in people with diabetes

      A fungal infection is not something that will simply go away on its own. You’ll likely find the itchiness and burning so unbearable, you’ll be desperate for treatment.

      Here are three guidelines for managing a fungal infection:

      Improve your blood sugar levels

      Using medication to treat the area affected by fungus growth will only do so much if your blood sugars are still persistently high. You need to address your blood sugars. If they continue to be high, you will likely continue to face fungus issues.

      Unless your high blood sugars were a temporary issue you had to endure (because of illness, for example, or taking a steroid for an injury), you should call your diabetes healthcare team immediately to adjust your medications and reduce your blood sugars as quickly as possible.

      Get the right medication for the right type of fungus

      You may need to consult a dermatologist or your local pharmacist to be sure you’re using the right anti-fungal treatment option for the type of fungus you’re dealing with.

      Treatments for yeast infections are fairly obvious (Vagisil, for example, is a popular over-the-counter option), but more severe yeast infections may require a visit to your gynecologist and a prescription-strength treatment option.

      There are topical ointments, capsules to swallow, suppositories, lotions — the list goes on and on — so you need to make sure you’re using the right treatment for the right type of fungal infection.

      (And again, part of this medication process should include adjusting your diabetes meds so your blood sugars don’t continue to induce fungal infections.)

      Improve your self-care skin habits

      For fungal infections developing under your breasts or within folds of skin, this is a big sign that your bathing and self-care habits need an upgrade. Overall, the goal is to reduce or prevent moisture from sitting on your skin for long periods of time.

      For instance, you could:

      • Change your underwear and/or socks halfway through the day to reduce moisture.
      • Thoroughly wash and/or dry your body off after exercising.
      • For persistent sweating after exercise, consider taking a quick cool shower to reduce your body temperature and reduce your post-workout sweating.
      • Wash your hands thoroughly several times a day.
      • If you wear a “daily” panty-liner, be sure to change it more often.
      • Wear a body-suit that prevents moisture from accumulating between your thighs and crotch area.
      • Wear a bra or torso garment that thoroughly lifts the breast off your abdomen and separates the two areas with fabric.
      • If you wear diapers, change your diaper more often — even if it is clean.
      • Losing weight will help reduce the number of areas where moisture can accumulate.

      Severe dry skin

      Dry skin may sound like no big deal, but it can become severe enough that it creates a great deal of uncomfortable or painful itching and burning.

      It is possible that your dry skin may have nothing to do with your diabetes — especially if your blood sugars are in a generally healthy range. However, if you do struggle with persistently high blood sugar levels, they are likely causing or worsening your dry skin.  

      Treating severe dry skin

      Treating severe dry skin in people with diabetes can be a combination of simple and complicated.

      Improve your blood sugar levels

      Above all else, it’s critical to work with your healthcare team to lower your blood sugar levels. When your blood sugar levels are persistently high, your blood flow is limited and your skin isn’t getting the healthy nutrients and oxygen it needs to thrive and heal each day.

      Improving your overall blood sugars is not an easy task. It will require dedication, discipline, patience, and determination!

      Improve your skin-care routine

      The easier part is making simple changes your skin-care routine:

      • Reduce how often you take showers or baths, especially in the dryer winter months.
      • Avoid taking overly hot showers which can further damage and dry out your skin.
      • Use a “mild” soap — and look closely at the ingredients in your soap.
      • Avoid soap and body products containing fragrances, colors, and other commercial additives that don’t serve the wellbeing of your skin.
      • Apply a moisturizer after every bath or shower — and perhaps a second time during the day in the dryer winter months.
      • Do your research: are the products you’re using to wash and moisturize your skin as natural as possible? When it comes to quality in the ingredients of skin products, less is more!

      If your itching and dry skin escalate to a degree that isn’t soothed by lotions and leaves you feeling remarkably uncomfortable, contact your healthcare team immediately for a referral to a dermatologist!

      Poor blood circulation: stasis dermatitis or varicose eczema

      Stasis dermatitis (also known as “varicose eczema” or “venous eczema” or “gravitational eczema”) is a condition that can result from poor blood circulation.

      For people with diabetes, poor blood circulation is a known complication, particularly in those with persistently high blood sugar levels.

      What is stasis dermatitis?

      For stasis dermatitis, the valves within your veins become weaker due to the lack of blood flow. Eventually, blood can actually leak from your veins into the muscle, fat, and skin tissue in your legs.

      Stasis dermatitis usually develops in the lower part of your body — your legs, feet, and eventually your calves. It can develop in other parts of your body, like your hands, arms, and face, too.

      A complete list of symptoms for stasis dermatitis includes:

      • Severe itchiness
      • Swelling that increases towards the end of your day, and decreases while you sleep
      • Very visible varicose veins (thick purple and red lines on your skin)
      • Crusty, red skin
      • Dry, cracked skin
      • An overall heaviness or soreness in the affected area, especially after standing or walking for a longer period of time
      • Painful to touch, in more severe degrees of the condition

      The condition usually gets worse if ignored, and you may notice those areas of skin become thicker and harder over time and become more visibly red and irritated.

      Treating stasis dermatitis

      The National Eczema Organization (NEA) stresses that stasis dermatitis will not improve until the underlying cause of the condition is addressed.

      In people with diabetes, the biggest culprit is persistently high blood sugars.

      Improve your blood sugar levels

      This means your first action towards treatment is to contact your diabetes healthcare team and ask for the help you need to adjust your medications, nutrition, and other health-related habits to improve your blood sugar levels.

      Remember, even a small change in medication dosages can have a big impact on blood sugar levels! Don’t hesitate to ask for help in making these changes. Even if you aren’t ready to make changes in how you eat or exercise, you have plenty of options to lower your blood sugar levels as quickly as possible.

      Use a topical steroid or other prescribed topical treatments

      A topical steroid can bring quick relief for many cases of stasis dermatitis and would be prescribed by your doctor or a dermatologist.

      “Sometimes covering the steroid with a wet or dry wrap or an Unna boot can greatly assist in severe cases,” explains the NEO. “An Unna boot is a type of gauze bandage with healing medications in it and provides compression to help with fluid build up.”

      For cases where topical steroids aren’t appropriate — or if they’ve already been used for a length of time — your doctor can proscribe other topical medications like tacrolimus or pimecrolimus.

      Wear pressure stockings

      The NEO also recommends wearing pressure stockings to improve blood flow and help prevent further ruptures and leakages in your veins.

      Have ruptured or leaking veins surgically repaired

      For more severe cases of ruptured and leaking veins, surgery may be necessary — but surgery isn’t necessarily an option for everyone.

      Diabetic neuropathy

      Diabetic neuropathy is a serious condition that can develop when high blood glucose levels cause damage to nerve fibers, particularly those in the feet and hands.

      Some of the earliest signs of neuropathy include itchiness, burning, and tingling in the affected areas, so it’s important to contact your doctor if you are experiencing any of these symptoms.

      To learn more about neuropathy, read Diabetic Neuropathy: Symptoms & Treatment Options.

      What you can do to avoid diabetes-related itching

      At the end of the day, all of these skin conditions can be avoided with reasonable blood sugar management. You don’t have to be perfect — everyone experiences high and low blood sugars.

      The American Diabetes Association recommends aiming for an A1c at or below 7.0 percent to reduce your risk of developing any and all diabetes complications.



      Sell Unused Diabetic Strips Today!

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