Keto Pumpkin Bread | Diabetes Strong

By electricdiet / October 27, 2020


For a rich fall treat, try this keto pumpkin bread topped with cream cheese frosting and chopped nuts! Enjoy it with coffee, for an afternoon snack, or as a mouth-watering dessert.

Keto pumpkin bread loaf on a white platter with one slice in the foreground

Pumpkin has become a staple of fall cooking. From the infamous Pumpkin Spices Latte to pumpkin cookies and everything in between, you’ll find this winter squash everywhere as soon as there’s a chill in the air.

And if you follow a low carb or keto way of eating, that doesn’t mean you have to miss out on delicious pumpkin baked goods. In fact, this keto pumpkin bread is a great way to celebrate the season!

With the right blend of low carb ingredients, some pumpkin purée, and a little cream cheese frosting to top it all off, this loaf becomes the perfect autumn treat. It’s so soft, so delicious, and so easy to make.

How to make keto pumpkin bread

This recipe is as simple as mixing together the ingredients to make the batter, baking, then mixing up and adding the frosting.

Pumpkin loaf ingredients in separate ramekins, as seen from above

Step 1: Preheat the oven to 360°F (180°C) and line a 4×8 inch loaf pan with parchment paper. Spray with cooking spray.

Step 2: In a large mixing bowl, add the almond flour, coconut flour, flax meal, psyllium husk, cinnamon, nutmeg, cloves, baking powder, and salt.

Dry ingredients in a glass bowl, unmixed

Step 3: Whisk together the dry ingredients.

Dry ingredients in a glass bowl with a whisk

Step 4: In a separate bowl, whisk together the pumpkin purée, eggs, cream cheese, butter, stevia, and white vinegar until smooth.

Step 5: Add the wet ingredients to the dry ingredients and mix until you have a thick batter.

Batter in a glass bowl with a whisk

Step 6: Pour the batter into the prepared loaf pan.

Unbaked pumpkin bread batter in the loaf pan

Step 7: Place in the oven to bake. Check the loaf after 30 minutes and if the top is getting too dark, cover the loaf with foil for the rest of the cooking time.

Step 8: Bake the loaf for 50 minutes or until a toothpick inserted in the center comes out clean, then remove from the oven and allow to cool in the loaf pan for about 10 minutes.

Step 9: Remove the loaf from the pan and set it on a wire rack to cool completely.

Pumpkin loaf on a cooling rack

Step 10: In a small bowl, combine the cream cheese, powdered stevia, and vanilla extract to make the frosting.

Unglazed loaf of pumpkin bread next to glaze and chopped nuts

Step 11: Once the loaf is completely cooled, spread the frosting on top and garnish with chopped nuts.

Whole loaf of pumpkin bread on a serving tray next to a bowl of chopped nuts, as seen from above

Just cut the loaf into 12 slices, and then your delicious low-carb pumpkin bread will be ready to enjoy!

Pumpkin loaf on a serving tray with one slice in the foreground

Is pumpkin good for keto?

Pumpkin can be enjoyed in moderation on a low carb diet. Thankfully, a little goes a long way, so it’s easy to watch your portions.

For example, this recipe calls for 1 cup of pumpkin purée, which contains about 8 grams of carbohydrates and 0.6 grams of fiber. That means there’s 7.4 net carbs in the whole loaf!

Divide that up into 12 slices, and you have yourself a perfectly keto-friendly treat.

Just make sure that you’re using pumpkin purée, NOT pumpkin pie filling! Pumpkin purée should be just pumpkin with nothing added, whereas pumpkin pie filling can be packed with sugar and fillers.

Storage

If you have leftover pumpkin bread, simply store the rest in an airtight container in the refrigerator.

It will stay fresh for up to 5 days, so you can enjoy this delicious pumpkin treat all throughout the week!

Pumpkin loaf on a serving tray next to a plate with a slice and a bowl for chopped nuts

Other diabetic-friendly pumpkin recipes

Looking for a few more delicious recipes that include pumpkin? There are plenty of ways to enjoy this wonderful ingredient!

Here are two of my favorite options that I know you’ll love:

You can also read this roundup I created of the best keto fat bomb recipes for even more great low carb inspiration!

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

Recipe Card

Keto Pumpkin Bread

For a rich fall treat, try this Keto pumpkin bread topped with cream cheese frosting and chopped nuts! Enjoy it with coffee, for an afternoon snack, or as a mouth-watering dessert.

Prep Time:10 minutes

Cook Time:50 minutes

Cooling Time:45 minutes

Total Time:1 hour 45 minutes

Servings:12

Loaf of pumpkin bread with slice

Instructions

  • Preheat the oven to 360°F (180°C) and line a 4×8 inch loaf pan with parchment paper. Spray with cooking spray.

  • In a large mixing bowl, add the almond flour, coconut flour, flax meal, psyllium husk, cinnamon, nutmeg, cloves, baking powder, and salt.

  • Whisk together the dry ingredients.

  • In a separate bowl, whisk together the pumpkin purée, eggs, cream cheese, butter, stevia, and white vinegar until smooth.

  • Add the wet ingredients to the dry ingredients and mix until you have a thick batter.

  • Pour the batter in to the prepared loaf pan.

  • Place in the oven to bake. Check the loaf after 30 minutes and if the top is getting too dark, cover the loaf with foil for the rest of the cooking time.

  • Bake the loaf for 50 minutes or until a toothpick inserted in the center comes out clean, then remove from the oven and allow to cool in the loaf pan for about 10 minutes.

  • Remove the loaf from pan and set it on a wire rack to cool completely.

  • In a small bowl, combine the cream cheese, powdered stevia, and vanilla extract to make the frosting.

  • Once the loaf is completely cooled, spread the frosting on top and garnish with chopped nuts.

Recipe Notes

This recipe is for 12 servings. If you cut the loaf into 12 slices, each slice will be 1 serving.
Make sure you’re using canned pumpkin purée, not pumpkin pie filling! 
Leftovers can be stored in an airtight container in the refrigerator for up to 5 days.

Nutrition Info Per Serving

Nutrition Facts

Keto Pumpkin Bread

Amount Per Serving (1 slice)

Calories 197
Calories from Fat 148

% Daily Value*

Fat 16.4g25%

Saturated Fat 8.3g42%

Trans Fat 0g

Polyunsaturated Fat 1g

Monounsaturated Fat 3.4g

Cholesterol 94.5mg32%

Sodium 314.2mg13%

Potassium 72mg2%

Carbohydrates 7.3g2%

Fiber 3.7g15%

Sugar 2.1g2%

Protein 5.2g10%

Net carbs 3.6g

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

Course: Breakfast, Dessert, Snack

Cuisine: American

Keyword: gluten-free, keto bread, keto pumpkin bread, pumpkin bread



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When I Dip, You Dip, We Dip – Corn Dip!

By electricdiet / October 25, 2020


Weekend Entertaining with Corn Dip

There seems to always be a reason to have friends hang out – a weekend grill-out, rooting on your team, or just good old-fashioned game night. Whatever the gathering – this Corn Dip from Holly Clegg’s trim&TERRIFIC KITCHEN 101: Secrets to Cooking Confidence cookbook, is the perfect appetizer to whip together. This dip is the epitome of fresh + convenience = homemade: you probably have these easy pantry-friendly ingredients already stocked. When entertaining, make it easy on yourself by making ahead of time, refrigerate and reheat in the microwave until heated through. Diabetic-friendly, and definitely a people-pleaser, this creamy dip with a kick is a sure-fire hit!

Corn Dip
A quick and easy, economical dip that’s creamy, crunchy, and spicy in each bite.

    Servings10 (1/4 cup) servings

    Ingredients

    • 1tablespoon


      olive oil

    • 2cups


      frozen cornthawed

    • 1/2cup


      chopped onion

    • 1/3cup


      chopped red bell pepper

    • 1/2cup


      chopped green onions

    • 2tablespoons


      chopped jalapenofound in jar

    • 2tablespoons


      light mayonnaise

    • 1/3cup


      nonfat sour cream

    • 2/3cup


      shredded reduced-fat sharp Cheddar cheese



    • salt and pepper to taste

    Instructions
    1. In large nonstick skillet, heat oil and add corn cooking over medium heat, stirring until golden brown, about 5 minutes.

    2. Add onion and pepper, sauté until tender, 3-4 minutes.

    3. Add green onions, jalapeño, mayonnaise, sour cream and cheese, stirring until heated and bubbly; cheese is melted. Season to taste.

    Recipe Notes

    Calories 89, Calories from Fat 39%, Fat 4g, Saturated Fat 1g, Cholesterol 7mg, Sodium 108mg, Carbohydrates 11g, Dietary Fiber 1g, Total Sugars 3g, Protein 4g, Dietary Exchanges: 1/2 starch, 1 fat

    Terrific Tip: If making ahead of time, refrigerate and reheat in microwave until heated.

    Serve Up Your Favorite Dips

    3 Tier Oval Bowl Set3 Tier Oval Bowl Set3 Tier Oval Bowl SetChip and Dip Serving Bowl SetChip and Dip Serving Bowl SetChip and Dip Serving Bowl SetMud Pie Circa Chip N Dip Set, WhiteMud Pie Circa Chip N Dip Set, WhiteMud Pie Circa Chip N Dip Set, White

    Cajun Red Beans rice Red beans recipe Mardi Gras Theme party

    Dig Into Louisiana Red Bean Dip

    Whether you are watching football at home or tuning in to the latest reality tv show, dig into Louisiana Red Bean Dip recipe. The most delicious way to start your menu! A dip that has the savory flavors reminiscent of Cajun red beans rice dish celebrating the festivities of Mardi Gras. Plus, this speedy appetizer, Red Beans recipe is full of heart healthy fiber.

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    tailgating recipes

    You will love Holly’s 25 Favorite Football Tailgating Recipes available for only $1.99! Included are dips, pick-ups, hearty food and the best of Holly’s sweet treats.  Best of all, it comes with a SHOPPING LIST so all the work is done for you from your menu to your grocery run.

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    The post When I Dip, You Dip, We Dip – Corn Dip! appeared first on The Healthy Cooking Blog.



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    LDL Cholesterol and Dysglycemia: an Intriguing Physiological Relationship

    By electricdiet / October 22, 2020


    It is an unexpected but pleasant surprise when new clinical relationships are identified, and one of the most interesting is the inverse association between LDL cholesterol (LDLc) and type 2 diabetes (T2D) risk. Evidence from both randomized clinical trials and genetic studies indicates that regulation of plasma lipids and glycemic control is more closely linked than previously assumed, yet in a counterintuitive, one could even say paradoxical, manner. Meta-analyses of randomized clinical trials have found that drugs designed to reduce LDLc, in addition to their hypolipidemic and cardioprotective effects, appear to also modestly increase T2D risk (1,2). Furthermore, naturally occurring genetic variation in molecular targets of LDLc-lowering therapy, such as genetic variants in or near HMGCR, NCP1L1, and PCSK9 genes, have been found to be associated with impaired insulin sensitivity and new-onset T2D, particularly among people with impaired fasting glucose levels (36). Further supporting that this is a fundamental biologic relationship, individuals with familial hypercholesterolemia, a dominantly inherited disease characterized by high plasma levels of LDLc due to genetic mutations in LDLR or APOB genes, appear to have a lower prevalence of diabetes than unaffected relatives (7). However, not all genetic variants that raise LDLc have similar effects on glycemic control (8). This suggests that the mechanism by which LDLc is reduced might have relevant implications for glycemic deterioration and reveal potential important mechanisms for diabetogenesis in general.

    As reported in this issue of Diabetes, Klimentidis et al. (9) conducted a study to examine the phenotypic and genotypic relationships between LDLc and T2D (Fig. 1). Using data from the UK Biobank (n = 431,167), they confirmed findings from previous reports that LDLc is inversely associated with T2D prevalence, in this case with an odds ratio of 0.41 [95% CI 0.39, 0.43] per each mmol/L increase in LDLc, which they term an “opposite direction of effect.” Although the magnitude of their observed association was higher than in other studies considering T2D incidence as opposed to T2D prevalence (1,2,10,11), these findings remained similar in several sensitivity analyses taking into account potential bias such as incomplete case ascertainment or the presence of a collider. The phenotypic associations also showed a paradoxical relationship of increased LDLc with increased HbA1c, which may be an indication of the difficulties of studying the relationship between these complex phenotypes in a cross-sectional study.

    Figure 1
    Figure 1

    Overview of main study findings. In a cross-sectional study within the UK Biobank (n = 431,167), Klimentidis et al. (9) reported that LDLc is inversely associated with T2D prevalence (odds ratio 0.41 [95% CI 0.39, 0.43] per each mmol/L increase in LDLc). Using genetic data from UK Biobank and DIAGRAM (n = 898,130), Klimentidis et al. identified 44 genomic regions, with opposite associations between LDLc and T2D (31 of them then replicated) enriched for genes associated with NAFLD. Their findings suggest that the diabetogenic effect of lipid-lowering medications is in part mediated by increased liver fat content.

    To identify genetic variants with opposite effects on LDLc and T2D prevalence, Klimentidis et al. conducted a cleverly designed two-stage genome-wide association study. They first identified genetic variants associated with LDLc in UK Biobank. Then, to confirm those dual LDLc-T2D variant associations, they used T2D summary statistics data from the Diabetes Genetics Replication and Meta-Analysis (DIAGRAM) consortium (n = 898,130). This led to the initial identification of 44 genomic regions with opposite associations between LDLc and T2D, and 31 of them then replicated in the independent data sets for their association with LDLc. A number of the genomic regions identified by Klimentidis et al. were previously known or suspected to be inversely associated with circulating LDLc and T2D (HMGCR, NPC1L1, APOE), but the authors also identified 14 genomic regions without evidence of previous association with LDLc or T2D. Thus, first of all, this is a novel way to identify new LDLc and T2D loci. Computational characterization of identified genomic regions suggests that genetic variants that have an opposite effect on LDLc and T2D are enriched for genes associated with nonalcoholic fatty liver disease (NAFLD). Of particular interest is the observation that for some of the identified genomic regions, the same exact variant that has the joint effect on LDLc and T2D is the variant associated with increased NAFLD (i.e., GCKR, PNPLA3, PPP1R3B, or TM6SF2), highlighting the relevance of liver metabolism on plasma lipids and glycemic control.

    There are some limitations to the study. First, this version of UK Biobank is a cross-sectional study, and it will be of interest to repeat these analyses when prospective data become available. For example, it is possible that T2D cases are more likely to be newly diagnosed patients not yet under lipid-lowering therapy, those with intolerance to lipid-lowering medications, people misreporting lipid-lowering medications, or older T2D individuals not requiring lipid-lowering medications. Propensity score analyses were implemented to account for this potential bias, but confounding could still exist (12). Second, this is not a traditional joint-phenotype genetic study in which the same participants have the phenotype of interest. By including data for LDLc from participants within the UK Biobank, and a separate data set to investigate whether these genetic variants associated with LDLc have a divergent effect on T2D, it is possible that differences in genome-wide association study characteristics may introduce some noise. Third, while mapping variants to genes is difficult, and only some of these loci exhibited colocalization of the association signals, it is still the case that we often infer that the closest gene is the most likely causal gene. Emerging data indicate that this is not always the case (13).

    Overall, findings from Klimentidis et al. provide a new perspective on the debate regarding the intriguing physiological relationship between lipids and dysglycemia and put liver metabolism in the spotlight. Animal and human physiological studies have found that fat accumulation in the liver leads to hepatic insulin resistance and that direct and indirect mechanisms exist to control insulin’s regulation of hepatic glucose and fat production (14,15). The observation that T2D signals identified for their dual association with low LDLc are mainly insulin-resistance loci, as opposite to the growing number of T2D loci primarily associated with insulin secretion, is well aligned with previous physiological data linking insulin resistance with hepatic fatty acid uptake and adipose tissue dysfunction. In this context, lipid-lowering strategies promoting adipose tissue expandability might have relevant implications to reduce glycemic deterioration associated with reducing LDLc, as it has been recently demonstrated for both gain- and loss-of-function variants in the LPL gene and T2D risk (16). Evidence from the current study may foster new lines of investigation to gain insights, not only into the underlying mechanisms responsible for the diabetogenic effect of LDLc-lowering medications, but into the etiology of T2D itself. Such knowledge will hopefully be used to inform public health and individual strategies to leverage more personalized and efficient approaches to manage dyslipidemia among people with impaired fasting glucose levels.

    Article Information

    Funding. J.M. is supported by European Commission Horizon 2020 program grant H2020-MSCA-IF-2015-703787 and National Institutes of Health (NIH) grant P30 DK040561. J.I.R. is supported in part by NIH National Center for Advancing Translational Sciences (NCATS) UCLA CTSI Grant Number UL1TR001881 and National Institute of Diabetes and Digestive and Kidney Diseases Diabetes Research Center grant DK063491 to the Southern California Diabetes Endocrinology Research Center. J.I.R. is also supported in part by NIH grant R01-HL151855–01.

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



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    Cold Pea Soup with Crab and Mint

    By electricdiet / October 20, 2020


    This vibrant cold pea soup with crab and mint is so rich and delicious! Best of all, there’s no cooking required — simply allow it to chill for about an hour.

    Cold Pea Soup with Crab and Mint in  a glass

    As soon as the weather starts to warm up, I always crave chilled soups. They’re perfect with a crispy salad on a hot day, especially if you’ve been working outside.

    This cold pea soup with crab and mint is such a refreshing and flavorful way to cool down! Best of all, there’s no cooking required, so you don’t even have to heat up your kitchen to whip up a batch.

    Simply blend the ingredients, allow them to chill, then add the lump crab garnish. It’s so easy and so delicious. Plus I just love the vibrant flavor!

    How to make cold pea soup with crab and mint

    This refreshing soup comes together in 6 simple steps.

    Step 1: Combine the peas, broth, and salt in a blender. Purée until smooth.

    Step 2: With the motor running, add the olive oil in a slow, steady stream.

    Step 3: Stop the motor and add the lemon juice. Quickly pulse once or twice to blend.

    Step 4: Chill in the refrigerator for 1 hour.

    Step 5: In a small bowl, combine the chili paste and mint. Gently stir in the crab, keeping the big lumps intact.

    Step 6: Pour the soup into small bowls or cups. Garnish with the crab mixture and serve immediately.

    This wonderful chilled soup may become your warm-weather staple!

    Tips for making your perfect soup

    Need to thaw the frozen peas quickly? Just put them in a strainer and run cold water over them for a few minutes, using your fingers to break up the clumps. They’ll be ready in no time.

    When it comes to choosing your Asian-style chili paste, keep in mind that some varieties are spicier than others. You could also use chili sauce or even cocktail sauce, but be sure to look for a brand that’s low in sodium and sugar.

    Want a thicker soup? Just use less broth.

    If you’re watching sodium, consider skipping the kosher salt and using your own homemade chicken bone broth. This will reduce the amount of sodium by about half.

    Storage

    Want to prep this recipe ahead of time? Simply whip up the ingredients in the blender, then store in the refrigerator for up to 24 hours. You can even store the soup right in the blender container!

    Once you’re ready to enjoy, blend the soup again for a few seconds, then proceed with the recipe as usual.

    Pea soup in individual serving glasses topped with crab garnish

    Other refreshing soup recipes

    I don’t think there’s ever a bad time to enjoy soup. Hot or cold, refreshing or hearty, there is so much variety to choose from! Here are a few of my favorite soup recipes I know you’ll love:

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

    Recipe Card

    Pea soup topped with crab garnish

    Cold Pea Soup with Crab and Mint

    This vibrant cold pea soup with crab and mint is so rich and delicious! Best of all, there’s no cooking required — simply allow it to chill for about an hour.

    Prep Time:10 minutes

    Chill Time:1 hour

    Total Time:1 hour 10 minutes

    Author:Shelby Kinnaird

    Servings:4

    Instructions

    • Combine the peas, broth, and salt in a blender. Purée until smooth.

    • With the motor running, add the olive oil in a slow, steady stream.

    • Stop the motor and add the lemon juice. Quickly pulse once or twice to blend.

    • Chill in the refrigerator for 1 hour.

    • In a small bowl, combine the chili paste and mint. Gently stir in the crab, keeping the big lumps intact.

    • Pour the soup into small bowls or cups. Garnish with the crab mixture and serve immediately.

    Recipe Notes

    This recipe is for 4 servings of soup.
    For a thicker soup, use less broth.
    Watching sodium? Use your own homemade salt-free broth and skip the added kosher salt to cut the amount of sodium almost by half.
    To prep this recipe ahead of time, whip up the ingredients in the blender, then store in the refrigerator for up to 24 hours. When you’re ready to enjoy, blend the soup again for a few seconds, then proceed with the recipe as usual.

    Nutrition Info Per Serving

    Nutrition Facts

    Cold Pea Soup with Crab and Mint

    Amount Per Serving (1 cup)

    Calories 196
    Calories from Fat 124

    % Daily Value*

    Fat 13.8g21%

    Saturated Fat 1.8g11%

    Trans Fat 0g

    Polyunsaturated Fat 7g

    Monounsaturated Fat 5g

    Cholesterol 30mg10%

    Sodium 313.1mg14%

    Potassium 259.7mg7%

    Carbohydrates 11.6g4%

    Fiber 3g13%

    Sugar 4.6g5%

    Protein 8.1g16%

    Vitamin A 1500IU30%

    Vitamin C 21.5mg26%

    Calcium 90mg9%

    Iron 1.6mg9%

    Net carbs 8.6g

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

    Course: Soup

    Cuisine: American

    Diet: Diabetic

    Keyword: chilled pea soup, cold pea soup, pea soup



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    Buffalo Beef Burger Salad with Marzetti® Tastefully Dressed™ Buttermilk Romano Ranch Dressing 

    By electricdiet / October 18, 2020





    Buffalo Beef Burger Salad with Marzetti® Tastefully Dressed™ Buttermilk Romano Ranch Dressing  – My Bizzy Kitchen


























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    Keto Meatloaf with Tomato Glace

    By electricdiet / October 16, 2020


    This easy Keto meatloaf is great for feeding a crowd or meal-prepping for the week! The recipe swaps out the bread crumbs for a tasty dish that’s low in carbs.

    Keto Meatloaf with tomato topping and fresh thyme garnish on a white cutting board

    Meatloaf is a staple under the “quick and easy dinner” category. It’s a great way to feed a crowd or meal-prep for the week!

    Traditional meatloaf uses breadcrumbs to hold the loaf together, which adds a lot of unnecessary carbs. So to make this Keto meatloaf, we’ll simply swap out the bread crumbs for low-carb ingredients.

    The next time you’re looking for a classic comfort food that you can feel good about eating, definitely give this tasty low carb meatloaf a try.

    How to make Keto meatloaf

    This recipe is incredibly simple to make. All you have to do is mix the ingredients and bake!

    Ingredients in separate ramekins on a wooden cutting board, as seen from above

    Step 1: Preheat your oven to 350°F (180°C) and grease a loaf pan well.

    Step 2: In a pan over medium heat, sauté the onion until translucent. Add the garlic and cook for 30 seconds until fragrant, then remove from heat.

    Step 3: In a large mixing bowl, add the sautéed onion and garlic along with the ground beef, liquid smoke, flax seed meal, eggs, Worcestershire sauce, tomato paste, dried oregano, salt, and pepper.

    You can leave out the liquid smoke if you don’t have any, but it does add a great smoky flavor.

    Ingredients unmixed in a glass bowl with a wooden spoon

    Step 4: Mix the ingredients well, but be careful not to over-mix so the meatloaf will still have some texture. If the mixture seems too wet, you can add up to 2 tablespoons of almond flour as needed.

    Ingredients mixed in a glass bowl with a wooden spoon, as seen from above

    Step 5: Transfer the mixture to the loaf pan.

    Raw meatloaf ingredients pressed into a baking dish, as seen from above

    Step 6: Bake the meatloaf for 40 minutes.

    Ingredients for tomato glaze in separate ramekins

    Step 7: In a small bowl, add all of the ingredients for the glaze and mix until smooth.

    Tomato glaze mixed in a small ramekin with a wooden spoon

    Step 8: Spread the tomato glaze over the meatloaf.

    Tomato topping added to the meatloaf

    Step 9: Return the meatloaf to the oven and bake for another 30 minutes until completely cooked through.

    Step 10: Remove the meatloaf from the oven and allow it to stand for 15 minutes.

    Once it’s ready, garnish with fresh thyme, cut into 14 servings, and enjoy!

    Finished meatloaf on a white serving tray garnished with fresh thyme, as seen from above

    What to serve with low carb meatloaf

    Looking for a few great sides to serve with your low carb meatloaf? Good news: there are so many options to choose from!

    For a hearty serving of vegetables, just whip up this Keto broccoli casserole. It’s packed with healthy greens and rich, cheesy goodness.

    Want to go the traditional meat-and-potatoes route? Roasted cauliflower mash is a great way to satisfy your craving for mashed potatoes, but without the unwanted carbs.

    You could also make a batch of low carb cornbread for a truly hearty and satisfying dinner. The cornbread will stay fresh for up to 5 days, so it makes a great snack or side throughout the week!

    Meatloaf with one slice cut

    Storage

    Traditional meatloaf has always been a great way to guarantee delicious leftovers, and this Keto version is no different. It’s one of my favorite ways to meal prep!

    Any extra slices of meatloaf should be stored in an airtight container in the refrigerator. They will stay fresh for 3-4 days.

    Close-up of meatloaf, garnished with fresh thyme

    Other Keto dinner recipes

    Looking for more low carb dinner inspiration? There are so many delicious meals to help you stay in ketosis! Here are a few of my favorites that I know you’ll love:

    You can also check out this Ketogenic Meal Plan for even more keto inspiration!

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

    Recipe Card

    Keto Meatloaf

    Easy Keto meatloaf is a delicious dish that’s great for feeding a crowd or meal-prepping for the week! This recipe swaps out the bread crumbs for a tasty dish that’s low in carbs.

    Prep Time:10 minutes

    Cook Time:1 hour

    Cooling Time:10 minutes

    Total Time:1 hour 20 minutes

    Servings:14

    Keto meatloaf garnished with thyme

    Instructions

    • Preheat your oven to 350°F (180°C) and grease a loaf pan well.

    • In a pan over medium heat, sauté the onion until translucent. Add the garlic and cook for 30 seconds until fragrant, then remove from heat.

    • In a large mixing bowl, add the sautéed onion and garlic along with the ground beef, liquid smoke, flax seed meal, eggs, Worcestershire sauce, tomato paste, dried oregano, salt, and pepper.

    • Mix the ingredients well, but be careful not to over-mix so the meatloaf will still have some texture. If the mixture seems too wet, you can add up to 2 tablespoons of almond flour as needed.

    • Transfer the mixture to the loaf pan.

    • Bake the meatloaf for 40 minutes.

    • In a small bowl, add all of the ingredients for the glaze and mix until smooth.

    • Spread the tomato glaze over the meatloaf.

    • Return the meatloaf to the oven and bake for another 30 minutes until completely cooked through.

    • Remove the meatloaf from the oven and allow it to stand for 15 minutes.

    Recipe Notes

    This recipe is for 14 servings of meatloaf. If you cut the loaf into 14 slices, each slice will be 1 serving.
    Leftovers can be stored in an airtight container in the refrigerator for 3-4 days.
    You can leave out the liquid smoke if you don’t have any. The meatloaf will still taste great!

    Nutrition Info Per Serving

    Nutrition Facts

    Keto Meatloaf

    Amount Per Serving (1 slice)

    Calories 171
    Calories from Fat 86

    % Daily Value*

    Fat 9.6g15%

    Saturated Fat 2.9g15%

    Trans Fat 0g

    Polyunsaturated Fat 0.9g

    Monounsaturated Fat 0.3g

    Cholesterol 66.9mg22%

    Sodium 479.3mg20%

    Potassium 184.2mg5%

    Carbohydrates 5.7g2%

    Fiber 2.4g10%

    Sugar 2.3g3%

    Protein 15.7g31%

    Net carbs 3.3g

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

    Course: Main Course

    Cuisine: American

    Keyword: keto, Keto meatloaf, Low-carb Banana Bread, meatloaf



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    Oh So Satisfying Best Chicken Tortilla Soup Recipe

    By electricdiet / October 14, 2020


    Crowd Pleasing Chicken Tortilla Soup

    Best Chicken Tortilla Soup recipe from Holly Clegg’s Gulf Coast Favorites cookbook is a great cool weather soup when you have extras to feed and when your goal is eating healthy. A cross between an easy White Chicken Chili and Tortilla Soup, this chunky chicken tortilla soup recipe is filled with beans, corn, and southwestern flair. The best of both worlds!  This recipe is a thicker, chunkier soup with corn and beans. When serving the soup,  serve with condiments.  Top this healthy chicken tortilla soup recipe with cheese, avocados, and tortilla strips.  Chunky, hearty and oh soooo good!  When you make it, we bet there won’t be one drop of this delicious soup left.

    Best Chicken Tortilla Soup
    Leftover chicken (Rotisserie chicken), southwestern seasonings and canned broth quickly turn into a mouth-watering one-pot meal.

      Servings13 (1 cup) servings

      Ingredients

      • 46-8-inch


        flour tortillascut into 1/4-inch strips

      • 1cup


        chopped onion

      • 1teaspoon


        minced garlic

      • 1 1/2pounds


        boneless, skinless chicken breastscut into strips (4 cups cooked)

      • 1(28-ounce) can


        chopped tomatoes with juice

      • 6cups


        fat-free chicken broth

      • 1(4-ounce) can


        chopped green chiliesdrained

      • 1(16-ounce) package


        frozen corn

      • 1(15 1/2-ounce) can


        Great Northern or navy white beansdrained and rinsed

      • 2tablespoons


        lime juice

      • 1 1/2teaspoons


        ground cumin

      • 1tablespoon


        chili powder

      • 1/2cup


        chopped green onions

      • 1cup


        shredded reduced-fat Mexican-blend or Cheddar cheese

      • 1


        small avocadopeeled and diced

      Instructions
      1. Preheat oven 350°F. Place tortilla strips on baking sheet and bake 10-15 minutes or until crisp. Set aside.


      2. In nonstick pot coated with nonstick cooking spray, sauté onion and garlic over medium heat until tender, about 7 minutes.


      3. Add chicken and continue cooking until chicken is done, about 5-7 minutes. Add tomatoes with juice, broth, green chilies, corn, beans, lime juice, cumin, and chili powder. Bring to boil, reduce heat and simmer about 10-15 minutes.


      4. Serve soup topped with tortilla strips, green onions, cheese, and avocado.

      Recipe Notes

      Per Serving: Calories 209 Calories from Fat 17% Fat 4g, Saturated Fat 2g, Cholesterol 37mg Sodium 571mg Carbohydrates 24g Dietary Fiber 5g Total Sugars 4g, Protein 20g, Dietary Exchanges:  1 1/2 starch,  2 lean meat

      Chicken Tortilla Soup

      Amazing Diabetic Delicious Soup

      Yes, this delicious Chicken Tortilla Soup recipe makes an easy diabetic chicken tortilla soup.  All Holly Clegg’s recipes include nutritional information and this recipe is even DIABETIC! It also has 5 grams fiber making it a high fiber food option and we always need to include more fiber into our diet.  This is a one-meal dish and works great in a slow cooker or take a short cut with rotisserie chicken! Lots of people love a chicken tortilla slow cooker soup plus it’s great to serve the soup in!

      Oh So Satisfying Cool Weather Soup

      This Chicken Tortilla Soup is one of Holly Clegg’s favorites from her Louisiana kitchen! With her Gulf Coast Favorites cookbook  you get all of her healthy Cajun recipes! You’ll find all of your favorite soup recipes and many more meals like most popular Chicken and Sausage Gumbo with a healthy, delicious and EASY roux!

      Best of all, these easy Cajun recipes you can make wherever you are and they are also healthy Cajun recipes. Who said Louisiana cooking wasn’t good for you?!

      Serve Best Chicken Tortilla Soup Recipe in White Soup Bowl

      You will really like these plain soup bowls! If you don’t have soup bowls, these bowls make a great option. They are the perfect size, dishwasher safe and with colder weather around the corner, you’ll find you will be pulling out these bowls all the time.

      They go with whatever dishes you have and this easy Chicken Tortilla Soup is a one-meal dish-even hearty enough to fill up those big eaters and so delicious!

      Chicken Tortilla Soup Recipes Perfect For Tailgating in Your Home = HOMEGATING!

      You will love serving this best Chicken Tortilla Soup recipe for company coming over to watch games or gatherings.  You can even keep the soup warm in a slow cooker and serve with mugs.  Have the condiments in a bowl surrounding the soup and let everyone serve themselves.  Who doesn’t love Chicken Tortilla Soup?!  The ingredient list might look long but the recipe is easy.  If you can open cans you can cook this soup!

      Team Holly’s New Favorite Pot Holders 

      Once you use these silicon pot holders, they will be your one and only kitchen pot holders for several reasons. They are easy to use and best of all, they never get dirty. Cloth pot holders end up so filthy so these colorful clean heat resistant pot holders are inexpensive and the best!

      Simplify Weekly Meal Planning with Holly Clegg’s Diabetic Meal Plan Downloadable

      Can you eat delicious food that is also good for you? Of course! Diabetic friendly meals definitely do not have to be boring and tasteless. This Diabetic Meal Plan & Recipes Downloadable is your easy go-to guide to meal planning diabetic meals the whole family will love. This comprehensive guide includes 13 weekly recipes, from dinners, lunch, snacks and dessert.

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      The post Oh So Satisfying Best Chicken Tortilla Soup Recipe appeared first on The Healthy Cooking Blog.



      Sell Unused Diabetic Strips Today!

      Maintaining Myocardial Glucose Utilization in Diabetic Cardiomyopathy Accelerates Mitochondrial Dysfunction

      By electricdiet / October 12, 2020


      Abstract

      Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

      Introduction

      Of the numerous complications associated with diabetes, cardiovascular diseases remain the major cause of death. Both insulin-deficient (type 1) and insulin-resistant (type 2) diabetes are accompanied by a complex milieu of systemic changes including hyperlipidemia and hyperglycemia. It has long been known that despite increased availability of multiple myocardial substrates, in uncontrolled diabetes the heart shows increased fatty acid (FA) utilization and reduced glucose utilization (1). These metabolic changes acting in concert with mitochondrial dysfunction, oxidative stress, and aberrant intracellular signaling are believed to contribute to ventricular dysfunction even in the absence of coronary artery disease and hypertension (2).

      Typically, the healthy heart shows flexibility in its ability to utilize ketone bodies, lactate, FA, and glucose to meet its constant energy demands. These substrates are utilized in a concentration-dependent manner, with a preference for FA. The relative balance between glucose and FA utilization is maintained via allosteric regulation by intermediates of glucose and FA metabolism (3). In diabetes, impaired insulin signaling increases circulating free FA as a result of increased lipolysis (4), which contributes to increased myocardial FA utilization. The mechanisms linking increased FA utilization, lipotoxicity, mitochondrial dysfunction, and diabetic cardiomyopathy are well studied (5). In contrast, many mechanisms including reduced cardiac GLUT4 protein levels contribute to impaired myocardial glucose utilization in diabetes (6), but the contribution of GLUT4 remains incompletely defined. Furthermore, the contribution of glucose toxicity in diabetic cardiomyopathy is less well understood, particularly in light of the relative reduction in myocardial glucose utilization in diabetes. Support for a causative role of increased glucose has been suggested by the emerging relationship between certain glucose-lowering therapies and reduction of heart failure in diabetes (7). Specifically, evidence linking glucose lowering by sodium–glucose cotransporter 2 inhibitors and the reduction of heart failure has reignited interest in the relationship between myocardial energetics and glucose availability in modulating heart failure risk in diabetes (810). Thus, increased understanding of mechanisms by which glucose may directly contribute to myocardial dysfunction and mitochondrial impairment, characteristic of diabetic cardiomyopathy, may further inform the complex interaction between myocardial metabolism and heart failure, particularly in the context of diabetes.

      Slc2a4 (a.k.a. GLUT4) is the major mediator of myocardial glucose uptake in the contracting heart (11). Overexpression of GLUT4 in skeletal muscle, adipose tissue, and heart prevents the metabolic abnormalities characteristic of diabetic cardiomyopathy; however, it has not been clear whether these benefits are directly attributable to GLUT4 in cardiomyocytes or improved systemic metabolic homeostasis (12). Increased expression from birth of the related family member Slc2a1 (i.e., GLUT1) protected against pressure overload–induced heart failure (13). We have more recently reported that inducible GLUT1 overexpression in the context of pressure overload hypertrophy prevented mitochondrial dysfunction without rescuing contractile dysfunction (14). Conversely, loss of GLUT4 expression predisposes the heart to failure in response to either chronic pressure overload or intermittent exercise training (15). Together, these and other studies strongly support the concept that glucose utilization and GLUT expression may play an important role in the myocardial adaptation to hemodynamic stress. In contrast, constitutive increases in glucose uptake might exacerbate ventricular dysfunction in response to diet-induced obesity (16). However, it remains to be determined whether maintenance of normal rates of myocardial glucose uptake and utilization in the context of diabetes could be cardioprotective. Under conditions of sustained glucose exposure in cultured cardiomyocytes, impaired mitochondrial function has been correlated with the protein posttranslational modification of O-GlcNAcylation that may contribute to decreased mitochondrial complex activity (17,18). In addition to direct modification of mitochondrial proteins, glucose also regulates gene expression by modifying transcription factors and modulating pathways that induce epigenetic changes (19). These data support that under certain circumstances restoring glucose delivery in the context of high extracellular glucose may actually impair cellular function. Thus, increased understanding of mechanisms by which glucose may directly contribute to altered gene expression and differential protein modifications will inform mechanisms linking myocardial mitochondrial impairment to the complex interaction between myocardial metabolism and heart failure in the context of diabetes.

      In the current study, we use an inducible cardiac-specific, myc-tagged GLUT4 overexpression mouse model (mG4H) that normalizes myocardial glucose uptake in the context of diabetes. This model also enabled us to define glucose-specific changes in gene expression, mitochondrial protein content, and mitochondrial bioenergetics. We observed that restoration of glucose delivery by maintaining or increasing GLUT4 expression in the context of diabetes enhances glycolytic function but exacerbates the decline in glucose oxidation, prevents the increase in FA oxidation, and accelerates the decline in mitochondrial oxidative phosphorylation (OXPHOS) capacity. These changes were associated with posttranslational modifications (i.e., O-GlcNAcylation) of mitochondrial proteins and transcription factors that contribute to changes in gene expression that would predict mitochondrial impairment. Thus, the decline in cardiac glucose utilization in the context of diabetes may play an adaptive role in reducing glucotoxicity, and circumventing this could worsen outcomes.

      Research Design and Methods

      Generation of Inducible Cardiomyocyte-Specific GLUT4 Overexpression in Mouse

      Mice were studied in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Utah and University of Alabama at Birmingham. To generate the inducible mycGLUT4 transgene, a c-myc epitope–tagged Rattus norvegicus GLUT4 cDNA (20) was subcloned into the pTRE2 vector (Clontech Laboratories) that contains a promoter region under regulation of tetracycline response elements and injected into oocytes from FVB/NJ mice. Offspring with germ line transmission were crossed with mice expressing αMHC-tON (21), for inducible cardiomyocyte expression. For all studies, 8- to 10-week-old double transgenic mice (mG4H) were the experimental group with αMHC-tON as controls (Con). Both male and female mice were used for a subset of studies, e.g., echocardiography and isolated working heart. As no differences between the sexes were seen, data were combined. For all other studies, male mice were used. The mice were housed at 22°C on a 12 h–12 h light-dark cycle with ad libitum access to food (8656; Harlan Teklad) and water. The transgene was induced by injection with 100 µg doxycycline (DOX) (500 µg/mL in 0.9% NaCl) (Sigma-Aldrich) and switched to 1 g/kg DOX-containing chow (F5820; Bio-Serv).

      Cell Culture, Promoter Reporter Construction, and Luciferase Assays

      Cell Culture

      C2C12 Mus musculus muscle myoblasts were purchased from ATCC (Manassas, VA). Low passage cells (<10) were maintained at 37°C under 5% CO2 in DMEM containing 4.5 g/L glucose supplemented with 10% FBS. When cells were confluent, cell medium was changed to DMEM supplemented with 2% horse serum for myotube differentiation.

      Adenoviral Expression Vectors

      Adenoviral green fluorescent protein (GFP) and O-GlcNAcase (OGA) have previously been described (22).

      Mammalian Expression Vectors

      pCMV-Sp1 and empty control plasmids were provided by Dr. Jonathan M. Horowitz, and their construction has previously been described (23).

      Reporter Constructs

      The mouse Ndufa9 gene promoter was generated by PCR amplification of FVB/NJ genomic DNA followed by cloning into the pGL4.1 as previously described (24). All promoter deletions had the same 3′-primer: 5′-GAGAGCTCCAAATATCCCTTCTCTGC-3′, which has a SacI-cloning site 994 bp downstream from the transcription start site (TSS). The −2 kb, “full-length,” promoter reporter 5′-primer, 5′ACTGGCCGGTACCACAGGAAG-3′, has a KpnI-cloning site 1,855 bp upstream from the TSS. The deletions had KpnI sites for the −0.5-kb fragment 504 bp, 5′-AGGTACCGTGAACACTAA-3′, and the −0.3-kb fragment 326 bp, 5′-AGGTACCCTGCAGAGCT-3′, upstream from the TSS, respectively.

      Site-Directed Mutagenesis

      Site-directed mutagenesis of the candidate Sp1 response elements (REs) was performed using the QuikChangeII Kit (Agilent, Santa Clara, CA) following the manufacturer’s protocol with the following modifications. Candidate Sp1-REs were mutated as shown in Fig. 5A. The sites were mutated with the following primers: Sp1-RE1, 5′-CAGTGACTAGATGTGTGGGAGCTCGGGTGATACTCAGAGGGGA-3′/5′-TCCCCTCTGAGTATCACCCGAGCTCCCACACATCTAGTCACTG-3′; Sp1-RE2, 5′-GCGTGCGAGGGGCTTAGAGCTCGGGCGCCTCTGCAT-3′/5′-ATGCAGAGGCGCCCGAGCTCTAAGCCCCTCGCACGC-3′; and Sp1-RE3 (M3) 5′-CAGGGGAAAGGGAGCTCGGACGGCAGGAC-3′/5′-GTCCTGCCGTCCGAGCTCCCTTTCCCCTG-3′.

      Luciferase Assays

      The constructs described above and shown in Fig. 5A were used for promoter reporter luciferase assays as previously described (24). C2C12 cells were plated at a density of 140,000 cells/mL in 12-well plates and transfected using Lipofectamine 2000 (Invitrogen, Waltham, MA) as previously described (24). Firefly luciferase reporter plasmids were cotransfected with CMV promoter–driven Renilla luciferase to control for transfection efficiency and the appropriate mammalian expression vector (e.g., pCMV-Sp1) or its corresponding empty vector. For luciferase reporter assays, cells were harvested and analyzed using Dual-Glo (Promega, Madison, WI) and were read according to the manufacturer’s protocols.

      Electron Microscopy

      Tissue samples were fixed and processed for histology and electron microscopy as previously described (25). Mitochondrial volume density was determined as the mitochondrion-containing fraction of a 32-by-32 grid placed on pictures of ×3,500 magnification (n = 3 hearts and 2–3 pictures/heart). Mitochondrial number was determined in identical-size pictures of ×18,000 magnification (n = 3 hearts and 2–3 pictures/heart).

      Histological Analysis

      Fresh hearts were fixed by immersion in 10% buffered formalin and analyzed as previously described (15). Tissue was embedded in paraffin, sectioned, and stained by the manufacturer’s protocol with Masson trichrome for visualization of fibrotic tissue on 5–10 images per group. Light microscopy was performed using a Leica DMRB inverted microscope (Leica Microsystems, Wetzlar, Germany) and captured using XnView software (XnSoft, Reims, France).

      Protein Analysis by Immunoprecipitation and Western Blotting

      Whole-cell, mitochondria-enriched, or nuclear-enriched protein extracts were resolved by SDS-PAGE and following transfer to polyvinylidene fluoride membranes were probed with antibodies as defined below. Detection and quantification were performed by measuring fluorescently labeled secondary antibodies using the Odyssey Infrared Imaging System and accompanying software (version 3.0; LI-COR Biosciences, Lincoln, NE). Immunoprecipitations were performed as follows. Nuclear fractions were isolated from left ventricular tissue, and protein was quantified. A total of 5% of the lysate was saved for input. Anti–O-GlcNAc antibody (5 μg) (RL2, ab2735; Abcam, Cambridge, U.K.) was conjugated with magnetic beads (cat. no. 10004D, Dynabeads; Thermo Fisher Scientific, Waltham, MA) in PBS with 0.02% Tween-20 at room temperature for 90 min. Protein lysate (1 mg) was diluted with 1 mL radioimmunoprecipitation assay buffer and added to the antibody-beads conjugate and incubated overnight at 4°C. Beads were washed five times with radioimmunoprecipitation assay buffer, and protein was eluted with Tris-Glycine SDS sample buffer (cat. no. LC2676; Invitrogen, Carlsbad, CA) containing NuPAGE sample reducing agent (NP009; Invitrogen) and blotted (immunoblot) with the anti-Sp1 antibody (07-645; EMD Millipore, Burlington, MA). Other antibodies used were for 4-aminobutyrate aminotransferase (ABAT) (ab108259; Abcam), complex IV (CIV) subunit COX4l1 (A21348; Invitrogen), GAPDH (CS-2118; Cell Signaling Technology, Danvers, MA), GlcNAc (CTD110.6, MMS-248R; Covance, Princeton, NJ), GlcNAc (RL2, ab2735; Abcam), GLUT4 (ab654; Abcam), Myc (sc-13922; Santa Cruz Biotechnology, Santa Cruz, CA), complex I (CI) subunit NDUFA9 (459100; Invitrogen), Sp1 (07-645; EMD Millipore), complex II (CII) subunit, SDHB (A21345; Invitrogen), complex III (CIII) subunit UQCRC1 (ab110252; Abcam), and VDAC (PA1-954A; Affinity BioReagents, Golden, CO).

      In Vivo Cardiac Contractile Function

      Echocardiography

      Echocardiography was performed in a subset of the mice prior to respiration studies as previously described (26). Mice were anesthetized with isoflurane, first at an induction dose of 2%, and then transferred to a warmed platform with a nose cone at 1%–1.5% maintenance isoflurane and imaged in the left lateral decubitus position with a linear 13-MHz probe (Vivid V echocardiograph; GE Healthcare). Cardiac dimensions and function were calculated from these digital images as previously described (27). Diastolic function was assessed with the VisualSonics Vevo 2100 Imaging System (Toronto, Canada) with the mouse under isoflurane anesthesia using the same protocol as above. Spectral Doppler was used to determine transmitral early (E) and atrial (A) wave peak velocities with the ratio of E to A (E/A) calculated. Peak early (E′) annular velocities were recorded in tissue Doppler mode.

      Left Ventricular Catheterization

      Left ventricular catheterization was performed on an overlapping subset of animals, invasive left ventricular hemodynamic measurements were performed with a temperature-calibrated 1.4-Fr micromanometer-tipped catheter (Millar Instruments, Houston, TX) inserted through the right carotid artery in anesthetized mice and analyzed as previously described (27).

      Microarray and Bioinformatics Analysis

      For transcriptomic array-based analysis, left ventricular tissue was harvested from transgenic mG4H or Con mice following 4 weeks of treatment as outlined above (n = 6). RNA isolation was performed as described below. Extracted RNA was analyzed to ensure RNA quality, with RNA integrity number >7. Gene expression intensity was quantified using the Mouse GE 4x44K v2 Microarray (Agilent) by the Health Sciences Center (HSC) Core at the University of Utah as previously described (25). Significant changes in gene expression were then analyzed for functional and network gene set enrichment analysis, along with curated literature-supported candidate upstream regulators, using Ingenuity Pathway Analysis (QIAGEN, Hilden, Germany) unless otherwise specified.

      Mitochondrial Purification

      Hearts were freshly excised; the atria were separated from the ventricles, and the right ventricle was dissected from the left ventricle. Mitochondria were isolated from the left ventricle by differential centrifugation as previously described (28).

      OXPHOS Complex Activity Assays

      Activities of individual complexes of the respiratory chain were determined spectrophotometrically, corrected to mitochondrial protein levels, and presented as percent of vehicle (Veh)-treated Con. CI activity, NADH dehydrogenase, was measured as reduction of 2,6-dichloroindophenol by electrons from decylubiquinol following oxidation of NADH by CI (29); CII activity, succinate dehydrogenase, CIII activity, coenzyme Q–cytochrome c reductase, and CIV activity, cytochrome c oxidase, were measured as previously described (30).

      Preparation of Isolated Cardiomyocytes and Glucose Uptake/GLUT4 Translocation Assays

      Cardiac myocytes were isolated as previously described (31). Perfused hearts were digested with type I collagenase, and once the heart became translucent and soft, it was minced and myocytes were dissociated by sequential washing in buffer. Cells were pelleted by centrifugation and used for glucose transport assays. Studies commenced after waiting for 90 min to allow cardiomyocytes to attach. Cells were washed and then treated with or without insulin, 2-deoxyglucose (2DG), and 1 μCi/μL [3H]2DG (NEN Life Science Products, Boston, MA), and glucose uptake was determined as previously described (14). To measure GLUT4 translocation by exofacial exposure of myc epitope in nonpermeabilized cardiomyocytes, we performed immunofluorescence assay as previously described (32).

      Respiration and ATP Measurements in Saponin-Permeabilized Cardiac Fibers

      The respiratory rates of saponin-permeabilized fibers were determined using a fiber-optic oxygen sensor (Ocean Optics, Largo, FL) as previously described (33). Studies were performed with three independent substrates: 1) glutamate/malate, 2) pyruvate/malate, or 3) palmitoylcarnitine/malate. Respiratory rates are defined as follows: V0, permeabilized fibers in the presence of substrate; VADP, maximally stimulated respiration following addition of ADP; and VOligo, oligomycin uncoupled, following addition of oligomycin, which inhibits ATP synthase. ATP generation was determined with the ENLITEN ATP Assay System (Promega). ATP/O was calculated as the ratio of ATP synthesis rates and VADP.

      RNA Isolation and Quantification

      For each group, six individual male mice with at least one sibling pair in another group were harvested between 6:00 a.m. and 7:00 a.m., right ventricle was dissected away, and left ventricle was immediately placed in RNA, later followed by isolation, using the RNeasy Fibrous Tissue Mini Kit (QIAGEN), purification, cDNA synthesis, and labeling. Either microarray quantification (as described above) or quantitative PCR (qPCR) was performed as previously described (25). Primer pairs were designed based on GenBank accession numbers with default settings. Dissociation curves followed by agarose gel size confirmation were analyzed for all primer pairs to ensure single product amplification. Oligos for qPCR analysis were used as follows: for transforming growth factor β1 (Tfgb1) NM_011577.2 forward 5′-CGGAATACAGGGCTTTCGAT-3′, reverse 5′-TTCATGTCATGGATGGTGCC-3′; collagen type I α1 (Col1a1) NM_007743.3 forward 5′-TACAGTGGATTGCAGGGTCT-3′, reverse 5′-TCTACCATCTTTGCCAACGG-3′; transgelin (i.e., SM22α, Tagln) NM_011526.5 forward 5′-GCGACTAGTGGAGTGGATTG-3′, reverse 5′-GATCCCTCAGGATACAGGCT-3′; actin α2 (αSMA, Acta2) NM_007392.3 forward 5′-CCGCCATGTATGTGGCTATT-3′, reverse 5′-AGATAGGCACGTTGTGAGTC-3′; elastin (Eln) NM_012751.1 forward 5′-GCAGAGGAGCAAAAGCTTATTTC-3′, reverse 5′-CCAGCACAGCCAAGACATTGT-3′; ribosomal protein lateral stalk subunit P0 (36B4, Rplp0) NM_007475.5 forward 5′-ACCTCCTTCTTCCGGCTTT-3′, reverse 5′-CTCCAGTCTTTATCAGCTGC-3′; 4-aminobutyrate aminotransferase (Abat) NM_001170978.1 forward 5′-CCAGGAAGCCTTACCGGAT-3′, reverse 5′-TTGTTGAGCAGGTCTTCCCG-3′; and peptidylprolyl isomerase (Cphn, Ppia) NM_008907.2 forward 5′-AGCACTGGAGAGAAAGGATTTGG-3′, reverse 5′-TCTTCTTGCTGGTCTTGCCATT-3′.

      Streptozotocin-Induced Diabetes

      Mice were made diabetic using the low-dose streptozotocin (STZ) protocol as outlined by the Animal Models of Diabetic Complications Consortium. After 4–6 h of fasting, mice were injected with a stock solution of STZ (7.5 mg/mL) at 55 mg/kg body wt or an equivalent volume of sodium citrate buffer (0.1 mol/L, pH 4.5) for five consecutive days. On day 7 and weekly thereafter, blood glucose was monitored using a standard Contour glucometer (Ascensia, Basel, Switzerland). Mice were considered diabetic if their blood glucose was >200 mg/dL.

      Substrate Metabolism and Contractile Function in Isolated Working Hearts

      Cardiac substrate metabolism was measured in hearts isolated from Con and mG4H mice 4 weeks after STZ administration and 2 weeks of DOX treatment. Hearts were prepared and perfused using protocols previously described (15). For determination of metabolism, rates of glycolysis for exogenous glucose, glucose oxidation, and palmitate oxidation were measured over a 60-min period in working hearts, and aortic pressures and cardiac performance including coronary flow were monitored by collection of coronary sinus effluent, with the caveat that Thebesian vein drainage may be included in the measurement, as we have previously described (34).

      Tissue and Serum Metabolite Levels

      Blood was obtained from the submandibular facial vein following Goldenrod Animal Lancet (MEDIpoint, Inc., Mineola, NY) puncture. Serum was used for measurement of free FA (FFA) and triglyceride (TG) levels. Circulating TG and FFA concentrations were determined using kits (Wako Diagnostics, Mountain View, CA). Blood glucose was measured from tail vein puncture using the Contour glucometer. Tissue glycogen levels were measured in extract from liver, gastrocnemius, or heart as previously described (35). Results are presented as glucose released from glycogen corrected to tissue weight.

      Two-Dimensional Electrophoresis Analysis and Identification of Glycosylated Mitochondrial Proteins

      Two-dimensional PAGE (2D-PAGE) was performed as previously described with the following modifications (36). In brief, following protein separation, gels were stained with Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kit (P21875; Thermo Fisher Scientific) following the manufacturer’s protocol. Quantitative differences in the protein patterns between the Con-Veh group and the three treatment groups (i.e., Con-STZ, mG4H-Veh, and mG4H-STZ) were analyzed. Spots that significantly changed in these groups were selected using the statistical module of the MELANIE III software package.

      In-Gel Digestion and Mass Spectrometry

      Gel spots were destained and subjected to trypsin (20 ng/μL; Promega) digest. Further extraction of peptides from the gel material was performed twice, and these solutions were collected and combined. The supernatant solutions of extracted peptides were combined and dried in a vacuum centrifuge. The samples were then subjected to liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis by the HSC Core at the University of Utah. Specifically, peptides were analyzed using a nano–LC-MS/MS system comprised of a nano-LC pump (Eksigent, AB SCIEX, Framingham, MA) and an LTQ-FT mass spectrometer (Thermo Electron Corporation, Waltham, MA). LTQ-FT MS raw data files were processed to peak lists with BioWorks Browser 3.2 software (Thermo Electron Corporation). Resulting DTA files from each data acquisition file were merged, and the data file was searched for identified proteins against the National Center for Biotechnology Information (NCBI) database, mouse taxonomy subdatabase, using Mascot search engine (version 2.2.1; Matrix Science). Identified peptides were accepted only when the Mascot ion score value exceeded 20.

      Quantification and Statistical Analysis

      Data are presented as means ± SEM. Sample number (n) indicates the number of independent biological samples (individual mice or wells of cells) in each experiment. Unpaired Student t test was used to analyze comparisons between two groups. Data sets with more than two groups were analyzed by two-way ANOVA, and significance was assessed using Tukey post hoc analysis. Statistical calculations were performed using the JMP Pro 9.0 software package (SAS Institute, Cary, NC) or GraphPad Prism 8 software (GraphPad, San Diego, CA), and significance was set at P < 0.05. For array-based gene expression analysis, statistical significance was assessed with a Benjamini-Hochberg P value adjustment.

      Data and Resource Availability

      All data from this article are available from the corresponding authors upon request. The gene expression data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO) (37) and are accessible through GEO Series accession number GSE123975 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE123975).

      Results

      Development of Inducible Cardiomyocyte-Specific GLUT4 Transgenic Mice

      To modulate glucose uptake in the adult mouse heart, we generated double transgenic mice with DOX-inducible cardiac-specific expression of an myc-tagged GLUT4 transgene (mG4H). Western blot analysis revealed a fivefold increase in GLUT4 protein following 2 weeks of transgene induction (Fig. 1A). Increases in GLUT4 protein levels were observed as early as 2 days after introduction of DOX, reaching maximum levels at about 1 week (Supplementary Fig. 1A). Transgene induction was not observed in single transgenic animals, in double transgenic animals without DOX, or in other tissues examined (Fig. 1A, Supplementary Fig. 1A and B, and data not shown).

      Figure 1
      Figure 1

      mG4H mice exhibit inducible cardiomyocyte-specific expression of the mycGLUT4 transgene that enhances glucose (glc) uptake and storage in the heart without influencing systemic metabolic homeostasis or cellular structure. A: Western blot analysis of whole cell extracts for Myc-tag or GLUT4 protein from biventricular heart tissue in the hearts of single transgenic (Con) and double transgenic (mG4H) mice in the absence or presence of DOX. d, days (additional conditions and tissues described in Supplementary Fig. 1A and B). B: IHC for Myc tag of primary adult cardiomyocytes isolated from mG4H mice 2 weeks following DOX induction in the absence or presence of insulin. C: Basal and insulin-stimulated 2DG uptake in primary adult cardiomyocytes isolated from Con and mG4H mice following 2 weeks of DOX treatment (n = 3–4). Ins, 0.1 nmol/L insulin. D: Heart glycogen content of Con and mG4H mice following 2 weeks of DOX treatment (n = 5), with additional tissues in Supplementary Fig. 1C and D. E: Development of hyperglycemia following STZ treatment compared with Veh-injected mice. Serum FFAs (F) (n = 5–7) and TG (G) (n = 5–7). Body weight (H) (n = 11–14) and heart weight (HW)–to–tibia length (TL) ratio (I) (n = 5–9) at 4 weeks following Veh or STZ treatment. J: Western blot analysis as in A, with STZ treatment (n = 3). Histological examination of cardiac tissue using trichrome staining (K) and qPCR for RNA levels of fibrotic markers (n = 3–7) (L). Additional contractile function data associated with these different conditions is listed in Supplementary Tables 1 and 2. All quantitative data are means ± SEM. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. Con-Veh.

      Enhanced Glucose Uptake and Storage Following GLUT4 Transgene Induction

      To assess transgene function, we isolated cardiac myocytes from mG4H mice following 2 weeks of DOX treatment. We performed immunofluorescence histochemistry (IHC) for detection of the exofacial myc-tag epitope of the transgene in nonpermeabilized cells. There was patchy GLUT4 protein on the sarcolemma of cardiomyocytes under basal conditions, and recruitment to the sarcolemma was markedly increased in response to insulin treatment (Fig. 1B). Using similarly prepared cells from both littermate Con and mG4H mice, we measured [3H]2DG uptake. 2DG uptake was increased 3.2-fold by transgene induction alone, which was similar to insulin-treated cells isolated from Con mice (Fig. 1C). Insulin further increased 2DG uptake by another 2.2-fold above the increased baseline uptake in cells isolated from mG4H mice (Fig. 1C). Compared with hearts of Con mice, mG4H hearts contained ∼2.6-fold more glucose stored as glycogen (Fig. 1D) but not in other tissues examined including liver and skeletal muscle (Supplementary Fig. 1C and D).

      Development of Insulin-Deficient Diabetes Is Not Altered by Cardiac GLUT4 Transgene Induction

      To determine the role of restoring glucose delivery to the heart under diabetic conditions, we used the low-dose STZ model of insulin-deficient diabetes. By 1 week after the initial STZ injection, all treated mice were hyperglycemic (blood glucose >200 mg/dL) compared with Veh-injected Con. At 2 weeks after the initial STZ or Veh injection, all mice were switched to DOX-containing chow to induce GLUT4 transgene expression in mG4H mice and followed an additional 2 weeks. At this 4-week time point, all STZ-treated mice had blood glucose >500 mg/dL compared with values <150 mg/dL in Veh-injected mice (Fig. 1E). Blood was collected, mice were euthanized, weights were collected, and tissue was snap-frozen for molecular analysis at this 4-week time point. Consistent with the development of diabetes, STZ-treated mice were also hyperlipidemic as shown by significantly elevated serum FFA (Fig. 1F) and TG (Fig. 1G) concentrations. As expected, STZ-induced diabetes resulted in lower body weight (Fig. 1H) and a lower heart weight–to–tibia length ratio (Fig. 1I). Cardiomyocyte-specific overexpression of GLUT4 had no effect on these parameters. Furthermore, Western blot analysis revealed that STZ treatment in turn had no significant effect on DOX-induced GLUT4 overexpression (Fig. 1J). Interestingly, GLUT4 overexpression had relatively little effect on cardiac histology (Fig. 1K) or fibrotic marker gene expression (Fig. 1L).

      Induction of GLUT4 Transgene Following Short-term Hyperglycemia Did Not Alter In Vivo Systolic Cardiac Function or Exacerbate Diastolic Dysfunction

      Systolic function was assessed by echocardiography (Fig. 2A). The primary difference observed was reduced cardiac output in STZ-treated animals irrespective of genotype, with a trend toward lower cardiac output in Veh-treated mG4H mice and no differences in ejection fraction (Fig. 2B and Supplementary Table 1). The change in cardiac output may reflect the smaller heart size and body weight. Invasive left ventricular catheterization revealed equivalent degrees of systolic dysfunction in STZ-treated Con and mG4H mice (Supplementary Table 2). Two independent echocardiographic measurements were used to estimate diastolic function: E/A ratio and the E/E′ ratio. GLUT4 induction was characterized by decreased A-wave amplitude leading to an increase in the E/A ratio that was independent of STZ treatment (Fig. 2C and D). In Con animals, the mean E/A ratio was higher following STZ treatment, but overlapping values in a subset of Veh-treated Con animals precluded statistical significance. Tissue Doppler imaging showed reduced E′-wave amplitude in both STZ-treated groups leading to comparable elevations in E/E′ ratios. Although the mean E/E′ ratio was higher in Veh-treated mG4H mice, values were partially overlapping in Veh-treated Con animals, precluding statistical significance (Fig. 2E and F). With results taken together, it appears that short-term STZ-induced diabetes does not impair systolic dysfunction in vivo, but evidence for some degree of diastolic dysfunction is apparent, and depending on the measurement used, transgene induction might also induce diastolic dysfunction. However, there is little evidence of synergistic exacerbation by transgene induction and diabetes.

      Figure 2
      Figure 2

      Echocardiographic analysis of in vivo cardiac dimensions and function in diabetic mice with short-term overexpression of GLUT4. Representative M-mode images (A) show preserved systolic function following STZ treatment or GLUT4 transgene induction. B: Quantification of ejection fraction from M-mode; additional parameters are reported in Supplementary Table 1. Transmitral Doppler flow reveals diastolic dysfunction in mG4H mice manifested by increased E/A ratio (n = 5–11); representative images (C) and quantification (D). Tissue Doppler imaging also identified an elevated E/E′ ratio in STZ-treated diabetic mice independent of genotype (n = 5–11); representative images (E) and quantification (F). Quantitative data are means ± SEM. *P < 0.05 or **P < 0.01 vs. Con-Veh.

      Substrate Metabolism and Ex Vivo Function in Diabetes Are Altered by GLUT4 Transgene Induction

      Cardiac glucose utilization by either glycolysis or oxidation was increased in perfused isolated working heart (IWH), following GLUT4 overexpression in mG4H (Fig. 3A and B). As expected, glucose utilization was decreased in hearts of Con-STZ mice. Diabetes-induced repression of glycolysis was reversed with GLUT4 overexpression (Fig. 3A), but impairment in glucose oxidation was exacerbated (Fig. 3B). The expected compensatory increase in FA utilization, as measured by palmitate oxidation in IWH, was observed in hearts of Con-STZ mice (Fig. 3C). Consistent with mitochondrial oxidative impairment as suggested by reduced glucose oxidation, palmitate oxidation failed to increase in hearts of mG4H-STZ mice (Fig. 3C). There were subtle, but statistically significant, increases in basal ex vivo contractile function in hearts of mG4H-Veh mice including aortic flow, cardiac output, and cardiac power, which were reversed in diabetic animals (Fig. 3DF and Supplementary Table 3).

      Figure 3
      Figure 3

      Cardiac function and substrate metabolism in IWH is altered in diabetic mice with short-term overexpression of GLUT4. Glycolysis (A) (n = 6–10), glucose (glc) oxidation (B) (n = 6–10), and palmitate oxidation (C) (n = 5–13) in IWH from mice 4 weeks after STZ treatment and DOX-induced transgene expression for 2 weeks. DOX was administered 2 weeks after STZ treatment (heart weight [HW]). Ex vivo contractile function of data pooled from both glucose and palmitate metabolism studies for aortic flow (D), cardiac output (E), and cardiac power (F). Additional contractile function data associated with these perfusions are shown in Supplementary Table 3. Quantitative data are means ± SEM. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. Con-Veh.

      Mitochondrial Oxygen Consumption and Respiratory Coupling

      Mitochondrial respiratory function was further assessed using saponin-permeabilized cardiac fibers from hearts of Con and mG4H mice in the presence or absence of diabetes. Basal respiration (V0) was significantly reduced only in the mG4H-STZ group with glutamate or pyruvate as substrates (Fig. 4A and B). Maximal glutamate-supported ADP-stimulated mitochondrial oxygen consumption (VADP) was equivalently suppressed by diabetes in Con and mG4H mice and in animals in the mG4H-STZ group. In contrast, VADP was suppressed in pyruvate- and palmitoylcarnitine-treated samples only in the mG4H-STZ group (Fig. 4AC). ATP synthesis was suppressed in the mG4H-STZ group with either pyruvate or palmitoylcarnitine as substrates (Fig. 4E and F) but was unchanged with glutamate (Fig. 4D). Interestingly, the only change in ATP/O ratio was in the mG4H-Veh treatment group incubated with palmitoylcarnitine (Fig. 4GI). There were no statistically significant changes from GLUT4 overexpression (mG4H) in mitochondrial number, and a modest, but significant, increase in mitochondrial volume was observed in both STZ groups (Fig. 4JL).

      Figure 4
      Figure 4

      Mitochondrial respiratory parameters and ATP synthesis rates are decreased by induction of GLUT4 following short-term hyperglycemia with minor changes in mitochondrial number and structure. Mitochondrial respiration in saponin-permeabilized cardiac fibers examining V0, VADP, and VOligo oxygen utilization with glutamate/malate (GM) (A) (n = 7–9), pyruvate/malate (PM) (B) (n = 6–9), or palmitoylcarnitine/malate (PC) (C) (n = 4–6) as substrates (fiber weight [FW]). ATP synthesis rates (DF) and ATP/O ratios (GI) in comparably treated fibers from the same mice as in AC. J: Representative electron micrographs of cardiac tissue; left, low magnification, and right, high magnification of different heart sections. K: Mitochondrial number per high-power field (hpf) (n = 3 hearts). L: Mitochondrial volume density as a percentage (n = 3 hearts). Quantitative data are means ± SEM. *P < 0.05 or **P < 0.01 vs. Con-Veh.

      Mitochondrial Respiratory Complex Activity and O-GlcNAcylation of OXPHOS Proteins and Mitochondrial Enzymes

      For further elucidation of potential mechanisms for the mitochondrial respiratory defects observed, mitochondria were isolated from the four groups. In mG4H-STZ mice, there was a decrease in electron transport chain complex activity for all four electron transport chain complexes measured spectrophotometrically (Fig. 5AD). Diabetes alone reduced CII activity in Con mice (Fig. 5B). Analysis of mitochondrial OXPHOS protein content revealed a modest but significant decrease in CI protein NDUFA9 in mG4H-STZ mice (Fig. 5E and F). There were no significant changes in CII or CIII proteins, but a significant increase in CIV protein COX4l1 was observed in the mG4H-STZ group (Fig. 5GI). Various mechanisms have been evaluated to determine the role of glucose in regulating mitochondrial protein function, including changes in the posttranslational modification O-GlcNAcylation (38). Western blot analysis of isolated cardiac mitochondrial proteins (as in Fig. 5E) revealed a modest increase in protein O-GlcNAcylation in mG4H-Veh and a robust increase in STZ groups (Fig. 5J). To begin to identify O-GlcNAc–modified proteins, we first used a candidate approach and examined the previously identified NDUFA9 (17). There was a modest increase in protein O-GlcNAcylation of immunoprecipitated NDUFA9 by transgene induction and diabetes (Fig. 5K). We next performed targeted glycoproteomics to identify additional proteins with GlcNAc modifications. Specifically, we used isolated mitochondrial fractions (as in Fig. 5) and separated proteins by two-dimensional PAGE followed by Pro-Q Emerald 488 glycoprotein staining. Fourteen spots were then selected that showed similar staining in each of the three treatment groups relative to Con-Veh and were subjected to MS identification. All 14 proteins had one predominant peptide identified by Mascot database searches (Table 1). Furthermore, all proteins identified are known to be associated with mitochondria, and the majority have previously been shown to possess O-GlcNAcylation sites on proteins in the diabetic rat heart (39). The latter finding supports the conclusion that increasing glucose uptake alone (mG4H) may replicate certain changes in protein posttranslational regulation associated with the complex metabolic milieu that occurs in diabetes.

      Table 1

      MS-identified mitochondrial proteins with differential glycosylation in Veh- and STZ-treated Con and mG4H mice

      Figure 5
      Figure 5

      Induction of cardiac GLUT4 in diabetes decreases respiratory chain complex activities in parallel with O-GlcNAc modifications of mitochondrial proteins. Specific respiratory complex activities in mitochondria isolated from Con and mG4H mice treated as in Fig. 1E and beyond. NADH dehydrogenase CI activity (A) (n = 4), succinate dehydrogenase CII activity (B) (n = 4), coenzyme Q–cytochrome c reductase CIII activity (C) (n = 4), and cytochrome c oxidase CIV activity (D) (n = 3). E: Representative subunit protein levels from CI to CIV in isolated mitochondria corrected to VDAC. (CI, NDUFA9; CII, SDHB; CIII, UQCRC1; CIV, COX5I1.) FI: Quantification of data presented in E. J: Mitochondrial protein O-GlcNAcylation in isolated mitochondria. K: O-GlcNAcylation of NDUFA9 as assessed by immunoprecipitation (IP) followed by O-GlcNAc immunoblot (IB). Quantitative data are means ± SEM. *P < 0.05 or **P < 0.01 vs. Con-Veh.

      Expression Levels of the Mitochondrial CI Subunit Ndufa9 Is Directly Regulated by Glucose and O-GlcNAcylation

      To further explore the mechanisms by which glucose availability may regulate mitochondrial oxidative capacity, we used a candidate approach to define the molecular pathways by which glucose can alter gene expression. We reasoned that given the widespread changes in mitochondrial protein O-GlcNAcylation, parallel changes could be taking place in the nucleus. We focused initially on the transcription factor Sp1, a known target of regulation by O-GlcNAcylation (40), and determined whether its regulation of mitochondrial OXPHOS gene expression could be modulated by O-GlcNAcylation in the hearts of diabetic and mG4H mice. Transcript levels of the Ndufa9 subunit of OXPHOS CI were repressed in mG4H-STZ mice (Fig. 5F), suggesting a potential role for glucose-dependent repression of gene expression. Many regions of the Ndufa9 gene are evolutionarily conserved from human to mouse (Fig. 6A) and harbor candidate Sp1 REs. We cloned a large portion of the mouse Ndufa9 gene promoter (−2 to 1 kb) into a luciferase reporter and generated two deletion mutants that removed some of these candidate REs. Following transfection of the reporter plasmids into C2C12 myoblasts, cells were differentiated into myotubes. Promoter activity was assessed, revealing it was suppressed by high-glucose treatment (25 mmol/L) in the full-length and first deletion (−0.5 kb). However, this repression was lost upon further truncation (Fig. 6B). We then sequentially mutated each of the three candidate Sp1 REs in the glucose-responsive regions and found that only one site, between the −0.5 and −0.3 kb promoter fragments, was actually required for glucose-mediated transcriptional suppression (Fig. 6C and data not shown). Lastly, we used an Sp1 overexpression plasmid to demonstrate that Sp1 was sufficient to confer transcriptional suppression and further enhanced glucose-mediated repression of promoter activity (Fig. 6D).

      Figure 6
      Figure 6

      Evidence linking O-GlcNAcylation of Sp1 to the glucose-mediated regulation of Ndufa9 gene expression. A: Conservation of the mouse and human Ndufa9 gene promoter regions (https://ecrbrowser.dcode.org) and schema of luciferase (Luc) reporter constructs. Sp1 RE identified by Transcriptional Regulatory Element Database (TRED) (purple), exon 1 of Ndufa9 (black arrow), and TSS (e.g., −2 kb is 2,000 bp from the TSS). B: Luciferase reporter assay of Ndufa9 promoter deletion series in C2C12 myotubes (−2 kb, full-length reporter; −0.5 kb, 1.5-kb deletion; or −0.3 kb, minimal promoter [as in Fig. 5A]) mapping glucose-mediated transcriptional suppression (n = 3). C: Site-directed Sp1 response element mutation M3 in the full-length −2 kb Ndufa9 promoter luciferase reporter assay defining requirement of the site for glucose-mediated transcriptional suppression (n = 3). D: Cotransfection of Sp1 expression vector and luciferase reporter assay showing Sp1 supported glucose suppression of the full-length −2-kb Ndufa9 promoter in C2C12 myotubes (n = 3). E: Adenoviral overexpression of a GFP or OGA luciferase reporter assay supports an O-GlcNAcylation mechanism of glucose-mediated transcriptional suppression of the full-length −2-kb Ndufa9 promoter in C2C12 myotubes (n = 3). F: O-GlcNAcylation of Sp1 of nuclear proteins from hearts of mice treated as in Fig. 4 and assessed by immunoprecipitation (IP) and Sp1 immunoblot. IgG-negative Con was against a pooled sample containing an equal amount of lysate from each sample group. G: Quantification of Sp1 enriched over input following O-GlcNAcylation immunoprecipitation as in Fig. 5F (n = 3); P = 0.052 genotype effect by two-way ANOVA. Quantitative data are means ± SEM. *P < 0.05 or **P < 0.01 vs. promoter, 5.5 mmol/L glucose.

      To test the hypothesis that O-GlcNAcylation mediates the glucose-dependent transcriptional repression of Ndufa9 via Sp1, we repeated experiments following adenoviral overexpression of a control GFP or OGA (the enzyme that removes protein O-GlcNAcylation). OGA-mediated O-GlcNAc removal prevented glucose-mediated transcriptional repression, which was present when GFP alone was overexpressed (Fig. 6E). To determine whether a similar mechanism existed in the hearts of our mouse models, we performed protein immunoprecipitations from left ventricular nuclear lysates of mice treated as in Fig. 5, using an O-GlcNAc–specific antibody (RL2), and Sp1 was detected by using anti-Sp1 antibody (Fig. 6F). Diabetes induced a modest enrichment of cardiac Sp1 O-GlcNAcylation compared with nondiabetic mice that was enhanced in mG4H-STZ hearts (Fig. 6G). mG4H mice also showed increased glycosylation of cardiac Sp1, further supporting a glucose-specific role for O-GlcNAcylation of Sp1. Collectively, these results suggest that increased glucose availability may independently regulate Ndufa9 expression via protein O-GlcNAcylation.

      Identification of Glucose-Regulated Gene Expression Profile in the Heart

      To globally identify gene expression pathways impacted by increased myocardial cardiac glucose delivery versus STZ-induced diabetes, we used microarray-based gene expression analysis. Heatmap and hierarchical clustering of differentially expressed genes (DEGs) by STZ treatment (P < 0.01) revealed a distinct clustering by genotype within the Veh-treated mice, whereas samples failed to cluster by genotype within the STZ-treated group (Fig. 7A). To examine the genes that were regulated in the mG4H-STZ group, we used a volcano plot of mG4H-STZ DEGs (Fig. 7B). This approach revealed robust changes in numerous transcripts encoding enzymes that regulate intermediary metabolism, including pyruvate dehydrogenase kinase 4 (Pdk4) (2.9-fold increase, P = 1.6 × 10−5), acyl-CoA thioesterase 1 (Acot1) (4.4-fold increase, P = 4.2 × 10−7), and β-hydroxybutyrate dehydrogenase (Bdh1) (3.5-fold decrease, P = 1.3 × 10−5). The most differentially suppressed gene, Abat (4.8-fold decrease, P = 4.3 × 10−9), was further validated via qPCR (Fig. 7C) and Western blotting (Fig. 7D), which confirmed its repression in mG4H to the same extent as observed with Con-STZ. To further identify those genes whose regulation is mediated by glucose alone, we compared differential expression based on mG4H and/or STZ treatment via Venn diagram. This analysis identified 557 DEGs between pairwise differential expression analyses of mG4H-Veh, Con-STZ, and mG4H-STZ relative to Con-Veh (Fig. 7E and Supplementary Table 4). Three-dimensional scatterplot analysis revealed that the ketone metabolic enzyme Bdh1 was among the most suppressed (6.0-fold decrease in mG4H-STZ, P = 1.2 × 10−5; 3.7-fold decrease in mG4H, P = 7.9 × 10−7; and 6.1-fold decrease in STZ, P = 6.3 × 10−7), whereas Ucp2, Acot5, Acot1, and Pdk4 were among the most highly induced (Fig. 7F). Within the minority of DEGs inversely changed by mG4H alone, the gene encoding βMHC (Myh7) was the most differentially suppressed in mG4H (3.0-fold decrease, P = 0.004) but was induced in both Con-STZ (3.1-fold increase, P = 1.7 × 10−6) and mG4H-STZ (1.9-fold increase, P = 0.004) relative to Con-Veh. Pathway analysis of the overlapping and upregulated DEGs (Fig. 7E [yellow 359]) enriched for FA metabolic pathways (Fig. 7G and Supplementary Table 5), whereas downregulated overlapping DEGs (Fig. 7E [blue 168]) enriched for ketone body and amino acid metabolic pathways (Fig. 7G and Supplementary Table 5). Together, these findings support additional metabolic signaling connections between glucose delivery and regulation of cardiac metabolism.

      Figure 7
      Figure 7

      Analysis of glucose-dependent vs. diabetes-dependent gene expression patterns in the mouse heart. A: Heatmap and hierarchical clustering of DEGs from array-based analysis of left ventricles of mice following 4 weeks of low-dose STZ or Veh treatment. B: Volcano plot of mG4H-STZ relative to Con-Veh with the top 50 DEGs labeled. Differential expression of Abat in mG4H or Con mice treated with STZ or Veh by qPCR for RNA (C) and Western blot for protein levels (n = 6) (D). E: Venn diagram comparing gene expression in mG4H-Veh, Con-STZ, and mG4H-STZ relative to Con-Veh. Genes are listed in Supplementary Table 4. F: Coexpressed genes visualized on a three-dimensional scatterplot of fold change for mG4H-Veh vs. Con-Veh, Con-STZ vs. Con-Veh, and mG4H-STZ vs. Con-Veh. G: Gene set enrichment analysis of upregulated (yellow) and downregulated (blue) coexpressed DEGs. Genes listed in Supplementary Table 5. Quantitative data are means ± SEM. *P < 0.05 vs. Con-Veh. BW, body weight; P-val., P value.

      Discussion

      The objective of the current study was to test the hypothesis that restoring glucose delivery to the heart in the context of diabetes may ameliorate the pathophysiology of diabetic cardiomyopathy. Prior studies revealed that overexpression of GLUT4 in muscle, heart, and adipose tissue from birth prevented the characteristic cardiac metabolic changes and contractile dysfunction in mouse models of diabetes (12,41,42). We more recently reported that loss of cardiac GLUT4 increases susceptibility to heart failure in response to either physiologic or pathologic stress (15). Together, these studies support the hypothesis that maintaining GLUT4-mediated myocardial glucose utilization may be required to prevent diabetes-related ventricular dysfunction and to maintain function following a hemodynamic stress. However, our results indicate that GLUT4 restoration in the context of diabetes not only is unable to rescue either metabolic or contractile dysfunction but also may accelerate the development of diabetic cardiomyopathy. These observations support the perspective that myocardial insulin resistance, as defined by decreased glucose uptake, may provide cardioprotection by defending against fuel overload (43). The current study provides additional molecular insights into mechanisms linking glucotoxicity with mitochondrial dysfunction. Specifically, we observed that increasing myocardial glucose uptake promoted widespread O-GlcNAcylation of core mitochondrial OXPHOS subunits and metabolic enzymes and a representative transcription factor Sp1, which repressed expression of the CI subunit Ndufa9 in a glucose- and O-GlcNAc–dependent manner. Moreover, increased myocardial glucose alone induced a transcriptional program consistent with induction of FA metabolism and repression of ketone body and amino acid metabolism.

      Cardiac-specific overexpression of GLUT1 prevents contractile dysfunction and dilation following pressure overload (13), rescues cardiac dysfunction that is secondary to PPARα deficiency (44), and ameliorates ischemic injury associated with aging (45). In contrast, increased myocardial glucose uptake enhances cardiac dysfunction in diet-induced obesity (16), which supports the hypothesis that increasing myocardial glucose delivery in the context of nutrient excess becomes maladaptive.

      The short-term duration of hyperglycemia in this study was not expected to induce severe ventricular dysfunction. As such, the current study focused on early mechanisms that could contribute to diabetic cardiomyopathy. We therefore focused on potential mechanisms linking increased myocardial glucose utilization and altered myocardial mitochondrial metabolism. Although not exhaustive, two important modes of regulation were explored. The first was evidence of direct regulation of enzymatic function by posttranslational O-GlcNAcylation of mitochondrial proteins. We identified enzymes and mitochondrial proteins in which a link between O-GlcNAcylation and impaired catalytic activity was previously described in diabetes (17,39). The second was transcriptional regulation, in part, through O-GlcNAcylation of transcriptional regulators (46,47). It should be noted that additional mechanisms likely exist, such as an emerging role of metabolic regulation of epigenetics and its impact on gene expression (19).

      There has been resurgent interest in the relationship between cardiac ketone body utilization in heart failure (48,49). In fact, we found that one of the most heavily repressed genes by either GLUT4 expression or diabetes was Bdh1, a rate-limiting enzyme in ketone body oxidation. Of note, these changes linked to diabetes or glucotoxicity in rodent hearts are opposite to changes described in models of nondiabetic heart failure. Thus, the extent to which altered ketone metabolism might contribute to the mitochondrial and metabolic phenotypes of mG4H mice remains to be determined.

      The robust changes in the regulation of Abat are intriguing, and the relationship between this change and glucose-induced metabolic and mitochondrial impairment in response to glucotoxicity in the heart remains to be clarified. Abat encodes a metabolic enzyme that catabolizes γ-aminobutyric acid (GABA) for recycling into the tricarboxylic acid cycle as succinate. Prior studies have revealed significantly elevated levels of ABAT protein in failing human dilated cardiomyopathy (1.84-fold) and following trans-aortic constriction (1.95-fold) in the mouse (49). The impact of altered ABAT on mitochondrial substrate utilization and respiratory function remains to be determined, but its pattern of expression identifies another metabolic pathway that is differentially regulated in diabetic cardiomyopathy relative to heart failure arising from other causes.

      Despite its exciting and novel findings, it should be noted that the current study has some limitations that should be carefully considered. First, it has recently been shown that tetracyclines can alter mitochondrial function (50). We observed subtle effects of DOX in our pilot studies and chose to control for them by placing all mice on DOX. As such, our design should primarily reveal differences that can be attributed to increased glucose delivery in a background of DOX exposure. Second, this study focused on candidate proteins such as NDUFA9 and ABAT. We demonstrated that O-GlcNAcylation not only might directly regulate the function of NDUFA9 but also may regulate its expression via a similar modification of the Sp1 transcription factor. Although the regulation of gene expression by O-GlcNAcylation of transcription factors likely targets multiple pathways as suggested by our microarray pathway analysis, we have not definitively demonstrated that this specific mechanism mediates the global changes in gene expression that were observed. It is also unlikely that the modest change observed in NDUFA9 exclusively accounts for the mitochondrial oxidative impairment observed. Although germ line defects in NDUFA9 alone may lead to neonatal lethality (51), mice with cardiomyocyte-restricted loss of C1 (NDUFS4) are viable, exhibit preserved ventricular function in nonstressed hearts (52), and might even be protected from ischemia/reperfusion injury (53). Thus, it is likely that multiple pathways converge to limit mitochondrial oxidative capacity when glucose availability is increased. Furthermore, we chose to inducibly overexpress GLUT4, while prior studies have focused on constitutive GLUT1 and GLUT4 expression. Despite the known roles of insulin in regulating GLUT4 vesicle trafficking, and the notion that GLUT1 mediates basal levels of glucose uptake, it is now accepted that GLUT4 is the major contributor of basal glucose uptake in the beating heart, given its greater abundance and the role of myocardial contraction in regulating GLUT4 translocation to the sarcolemma. Moreover, given that GLUT4 is repressed in diabetic cardiomyopathy, we believe that increasing or maintaining GLUT4 content in the heart represents a reasonable approach for preserving myocardial glucose utilization in the context of diabetes. We used STZ-induced hyperglycemia for the current study. Although it is possible that other forms of diabetes may result in different findings, studies in GLUT1 transgenic mice following high-fat feeding–induced insulin resistance and diabetes suggest that maintaining glucose uptake in the context of increased FA delivery to the heart that occurs in type 1 and type 2 diabetes will be deleterious.

      In conclusion, the current study shows that in the presence of hyperglycemia, increasing myocardial glucose utilization accelerates the decline in glucose and FA oxidative capacity in IWH, reduces mitochondrial oxygen consumption in saponin-permeabilized cardiac fibers, and impacts mitochondrial OXPHOS activity. Our data suggest that the reduction in glucose uptake in the hyperglycemic state likely plays a protective role that limits glucotoxicity. Although increasing glucose delivery to the heart increased glucose uptake, storage, and utilization and was tolerated in the absence of metabolic stress, it clearly sensitized the heart to develop impaired mitochondrial function in the context of nutrient excess. Thus, in the presence of uncontrolled hyperglycemia, normalizing or increasing myocardial glucose utilization is likely to sensitize the heart to glucotoxicity, which will exacerbate mitochondrial dysfunction and worsen diabetic cardiomyopathy.

      Article Information

      Acknowledgments. HSC Core services from the University of Utah were provided for the following: microarray by Brian Dalley and Qian Yang, bioinformatics by Brett Milash, and mass spectrometry and proteomics by Chad Nelson and Krishna Parsawar. Electron microscopy images were provided by Elizabeth W. Weeks of EM Laboratories, Inc., Birmingham, AL.

      Funding. This work was supported by National Institutes of Health (NIH) grants R00 HL111322 and R01 HL133011 and JDRF Advanced Postdoctoral Fellowship 10-2009-267 to A.R.W. M.E.P. was supported by NIH grant F30 HL137240, O.K. was supported by NIH grant R01 GM108975, R.O.P. and M.K.B. were both supported by postdoctoral fellowships from the American Heart Association (AHA), and E.D.A. was supported by NIH grants R01 DK092065, R01 HL108379, and U01 HL087947 and is an established investigator of the AHA.

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

      Author Contributions. A.R.W. and E.D.A. secured funding, conceived the study, and wrote the manuscript. All authors reviewed and edited the manuscript. A.R.W. performed mouse studies, left ventricular catheterization and analysis, and promoter cloning and analysis, as well as multiple molecular studies (e.g., RNA quantification, protein quantification, metabolite analysis). J.C.S., M.K.B., L.W., and C.A.A. performed mouse studies, collected samples, and performed Western blots. J.C.S. performed and analyzed the fiber studies. C.M.-H. performed qPCR and echocardiography data analysis and reviewed all statistical analysis of the data as well as graphing of the data. M.E.P. performed microarray analysis and qPCR. O.K. performed and analyzed the respiratory complex activities. H.S. performed and analyzed the two-dimensional PAGE. R.O.P. performed 2DG uptake and the quantification of the mitochondria as appears in the electron micrographs. M.K.B. performed immunoprecipitation studies. J.T. performed IWH perfusion and analysis. A.C.-F. performed IHC. C.D.O., W.E.B., L.J.D., and S.E.L. performed and assisted in analysis of the echocardiography data. W.H.D. provided critical reagents for the O-GlcNAc studies. A.R.W. 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 October 27, 2019.
      • Accepted April 25, 2020.



      Sell Unused Diabetic Strips Today!

      Delicata Squash Pasta Sauce – My Bizzy Kitchen

      By electricdiet / October 10, 2020


      You guys – this delicata squash pasta sauce is money!  

      I saw my friend Courtney make a Pinch of Yum recipe using butternut squash, fried pancetta and sage.

      I had none of those ingredients!  

      this is a picture of roasted delicata squash cut in half on a cutting board

      But I did have delicata squash – duh!  If you’ve followed me for any length of time, you know my love of delicata squash is deep.

      What is Delicata Squash?

      I love that delicata squash is sometimes referred to as the lazy person’s squash, because the skin is edible and you don’t have to peel it like butternut squash.

      How do you roast it?

      If you have an air fryer, simply cut in half (seeds and all) and cook for 20 minutes at 400 degrees.  Once cooled, you can slice in half and easily remove the seeds.  I’ve used delicata squash to make delicata wonton soup, delicata hush puppies, and even delicata squash twice baked potatoes.

      Delicata Squash Pasta Sauce

      A creamy, slightly sweet pasta sauce that will soon be on your fall menu – so good!

      Prep Time5 mins

      Cook Time20 mins

      Servings: 6

      Calories: 59kcal

      • 1 large delicata squash roasted
      • 1 cup chicken broth
      • 1/2 cup unsweetened almond milk
      • 4 cloves garlic
      • 1/2 cup ricotta cheese
      • 2 tbsp whipped cream cheese
      • 1 tbsp Italian seasoning
      • 1/2 tsp crushed red pepper
      • Cut the delicata squash in half. Put in air fryer (or oven) at 400 degrees for 20 minutes, or until fork tender. Cool slightly, remove seeds.

      • In a blender, add the squash, chicken broth, almond milk, garlic, ricotta cheese, whipped cream cheese, Italian seasoning and crushed red pepper.

      • Because we still have the skin on the squash, blend the sauce for at least 3 minutes. There is sodium in the chicken broth, so I season to taste with salt and pepper at the end.

      This made 3 cups of sauce.  I used 1/2 cup of sauce to coat 1 cup of cooked pasta.
      It’s 1 point per 1/2 cup on all WW plans, or 3 points for one cup.

      Calories: 59kcal | Carbohydrates: 10g | Protein: 4g | Fat: 1g | Saturated Fat: 1g | Cholesterol: 4mg | Sodium: 240mg | Potassium: 331mg | Fiber: 2g | Sugar: 3g | Vitamin A: 1096IU | Vitamin C: 13mg | Calcium: 119mg | Iron: 1mg

      this is a photo of creamy delicata squash pasta sauce

      As soon as I made the pasta dish, I immediately thought of macaroni and cheese.  Yesterday for lunch I made a mini baked macaroni and cheese.  One cup cooked pasta, 1/2 cup delicata pasta sauce, 1 ounce good cheddar cheese.  Mixed that together and topped with a tablespoon of bread crumbs and baked in my air fryer at 400 for 10 minutes.

      Can you use dishes in an airfryer?

      Yep!  Think of your air fryer as a mini oven – whatever is oven safe is air fryer safe.

      this is dish with macaroni and cheese

      Pinky swear you can convince your family with either the pasta sauce or the mac n cheese.  Both are amazing and I am saving my delicata squash seeds to grow my own next spring.

      I think my daughter Hannah and son-in-law Jacob are happy to have their own place to live now.  If for nothing else not to be subjected to my love affair with delicata squash.  I am not sure I have convinced my twin sister either.  That’s okay – more for me!





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      Stuffed Celery with Cheese, Bacon, and Herbs (Low-Carb)

      By electricdiet / October 8, 2020


      These low-carb stuffed celery stalks are ready in minutes and are the perfect crunchy appetizers or snacks for summertime.

      6 stuffed celery stalks on a plate

      Stuffed celery is a fantastic keto snack! The combination of crunchy celery with a creamy filling is just perfection.

      You can stuff celery with so many flavors. For this recipe, I used classic ingredients like bacon pieces and fresh herbs, but you can really go wild here.

      How to make stuffed celery

      Step 1: Fry the bacon on a very hot pan until it’s crispy. Set aside on pieces of paper towel to drain and cool.

      Step 2: Clean the celery stalks, cut them in thirds, and set aside. Clean the fresh herbs and chop them into smaller pieces.

      ingredients for stuffed celery on a wooden board

      Tip: To make the celery sticks look nice and even, use a vegetable peeler and peel a layer off the bottom of the celery stalks so that they can stand upright on the plate.

      Step 3: Add the cream cheese to a food processor and process until whipped and creamy. Stop to scrape down the sides if necessary. 

      Step 4: Add in the cheddar cheese, fresh herbs, salt, pepper, and bacon bits and pulse until combined. Scrape down the sides as necessary.

      Ingredients in the food processor

      Tip: You can control the texture of the filling by blending it more or less. I prefer to have the mixture more on the chunky side so I only give it a few pulses. 

      Blended ingredients in food processor

      Step 5: Spoon the filling into the middle of the celery sticks, leaving an inch on the bottom free of filling and sprinkle with bacon pieces. This clear space makes it easier to handle the celery sticks.

      Stuffed celery with crispy bacon bits

      Filling variations

      You can make these stuffed celery sticks your own in so many ways. If you have a favorite flavor that you love, feel free to experiment and try out a whole bunch of ingredients to get that flavor just right.

      The rich cream cheese base works well with almost any flavor combination. You can try adding olives, pimento peppers, dill pickles, walnuts, and the list goes on. For some added kick, add a dash or two of hot sauce. 

      If you would like a lighter tasting filling, try substituting some cream cheese with a few tablespoons of mayonnaise. 

      Herbs and Spices

      You can use fresh or dry herbs in this recipe. When substituting with dry herbs, you need to use less as dry herbs are typically more potent than fresh herbs. You can replace a tablespoon of fresh herbs with one teaspoon of dry herbs. 

      In regards to spices, there are some spices you can use that compliment the stuffed celery well. Old Bay Seasoning, paprika (smoked or normal), and cumin are some to try out. Add according to taste, 1/4 to 1/2 teaspoon should do. 

      Stuffed celery sticks on a plate

      More healthy low-carb snack recipes

      Great tasting snacks are a great way to keep your blood sugar levels stable and the hunger at bay!

      Here are some of my favorite low-carb snacks to keep me going:

      You can also check out my roundup of diabetes-friendly dinner recipes for more healthy low-carb inspiration.

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

      Recipe Card

      Stuffed Celery with Cheese, Bacon, and Herbs

      Celery sticks stuffed with a creamy and delicious filling and topped with crispy bacon bits are sure to become your new favourite keto snack! 

      Prep Time:10 minutes

      Cook Time:0 minutes

      Total Time:10 minutes

      Servings:9

      Instructions

      • Fry the bacon on a very hot pan until it’s crispy. Set aside on pieces of paper towel to drain and cool.

      • Clean the celery stalks, cut them in thirds, and set aside. Clean the fresh herbs and chop them into smaller pieces.

      • Add the cream cheese to a food processor and process until whipped and creamy. Stop to scrape down the sides if necessary. 

      • Add in the cheddar cheese, fresh herbs, salt, pepper, and bacon bits and pulse until combined. Scrape down the sides as necessary.

      • Spoon the filling into the middle of the celery sticks, leaving an inch on the bottom free of filling and sprinkle with bacon pieces.

      Recipe Notes

      This recipe makes 9 servings. Each serving is 1/3 stuffed celery stalk
      You can use fresh or dry herbs in this recipe. You can replace a tablespoon of fresh herbs with one teaspoon of dry herbs. 
      You can control the texture of the filling by blending it more or less. I prefer to have the mixture more on the chunky side so I only give it a few pulses. 

      Nutrition Info Per Serving

      Nutrition Facts

      Stuffed Celery with Cheese, Bacon, and Herbs

      Amount Per Serving (1 stuffed celery stick)

      Calories 136
      Calories from Fat 113

      % Daily Value*

      Fat 12.6g19%

      Saturated Fat 7.5g38%

      Trans Fat 0g

      Polyunsaturated Fat 0.5g

      Monounsaturated Fat 2.8g

      Cholesterol 39.4mg13%

      Sodium 272.1mg11%

      Potassium 79.9mg2%

      Carbohydrates 2g1%

      Fiber 0.4g2%

      Sugar 1.4g2%

      Protein 4.2g8%

      Net carbs 1.6g

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

      Course: Appetizer

      Cuisine: American

      Keyword: low-carb stuffed celery



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