Cinnamon Rolls with Biscuits Make Easy Cinnamon Rolls Recipe with Kids

By electricdiet / June 16, 2020


Cinnamon Rolls with Biscuits – Easy Cinnamon Rolls Recipe Fun For Kids

Cinnamon Rolls with biscuits make favorite easy cinnamon rolls recipe. In fact, you probably have these four ingredients for Easy Cinnamon Rolls from KITCHEN 101 cookbook already in your panty to easily make cinnamon roll biscuits. You can whip up this simple cinnamon roll breakfast and the kids love to help make them.  Watch and you will see if you involve the kids, usually they will quickly eat it.

Cinnamon Rolls with Biscuits Best Easy Cinnamon Rolls Recipe!

You might have an easy cinnamon rolls recipe but you will really like these cinnamon rolls with biscuits.  First, you can be creative with the recipe and add nuts, dried fruit or whatever extra ingredients you like.  Also, believe it or not, this is a diabetic recipe.  In KITCHEN 101, all recipes have nutritional information and have a “D” to highlight all diabetic recipes.  There are different size biscuit cans but the analysis is based on the 10-count can of biscuits.  Also, look for whole wheat biscuits to use.

Easy Cinnamon Rolls
Keep canned biscuits handy to make this fast and fabulous treat for breakfast or a snack.

    Servings10 rolls

    Ingredients

    • 1


      can refrigerated biscuits or whole wheat biscuits10-biscuit

    • 2tablespoons


      butter

    • 1tablespoon


      sugar

    • 1teaspoon


      ground cinnamon

    • 1/4cup


      chopped pecansoptional

    Instructions
    1. Preheat oven 425°F. Coat 15x10x1-inch baking sheet with nonstick cooking spray.

    2. Flatten each biscuit with your hand or rolling pin. Spread each biscuit with butter.

    3. In small bowl, combine sugar and cinnamon together. Sprinkle cinnamon mixture on top of butter; sprinkle with pecans, if desired.

    4. Roll up each biscuit like a cigar and form a circle by putting the ends together. Bake 8-10 minutes or until golden brown.

    Recipe Notes

    Calories 76, Calories from Fat 35%, Fat 3g, Saturated Fat 1g, Cholesterol 6mg, Sodium 210mg, Carbohydrates 11g, Dietary Fiber 0g, Total Sugars 3g, Protein 1g, Dietary Exchanges: 1 starch, 1/2 fat

    Terrific Tip: These are freezer-friendly so keep in freezer to have to pop out for a quick snack.

    Nutrition Nugget: Light, low-fat meals, especially breakfast foods seem to be the best tolerated of all foods while you are having chemotherapy.

    Another Fun Biscuit Recipe Bunny Biscuits

    Another fun kid biscuit recipe is for Holly’s popular Bunny Biscuits. Don’t save them only for Easter. They are fun and so good to eat year round!

    Get All of Holly’s Healthy Easy Cookbooks

    The post Cinnamon Rolls with Biscuits Make Easy Cinnamon Rolls Recipe with Kids appeared first on The Healthy Cooking Blog.



    Sell Unused Diabetic Strips Today!

    Hyperuricemia Predisposes to the Onset of Diabetes via Promoting Pancreatic β-Cell Death in Uricase-Deficient Male Mice

    By electricdiet / June 14, 2020


    Introduction

    Hyperuricemia (HU), a metabolic condition characterized by elevated serum urate (SU), is a primary cause of gout. Epidemiological studies have demonstrated the rapid increasing prevalence of HU worldwide in the last decades (13). Although some recent studies have highlighted the connection between SU and glucose homeostasis and indicated that each 1 mg/dL increase in SU was accompanied by a 17% increase in the risk of type 2 diabetes (4,5), there is no conclusion of a causal relationship between HU and diabetes. The Mendelian randomization study by Sluijs et al. (6) did not support a causal effect of circulating urate on diabetes onset and thus concluded that urate-lowering therapies (ULTs) may therefore not be beneficial in reducing diabetes risk. Therefore, whether or how SU is involved in disrupting glucose homeostasis remains elusive. Moreover, the investigation of the possible relationship between urate and glucose metabolism has been hindered by the lack of an appropriate animal model.

    The etiology of diabetes is multifactorial, including insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. Adequate insulin secretion from pancreatic β-cells is necessary to maintain blood glucose homeostasis. Notably, the apoptosis and the subsequent loss of function of pancreatic β-cells is the major contributor to the progression of diabetes. Although the relationship between HU and diabetes has been implicated, the molecular underpinnings of diabetic β-cell apoptosis promoted by HU remain poorly understood. One piece of evidences from Jia et al. (7) demonstrated that soluble urate can directly cause β-cell death and dysfunction by activation of the nuclear factor-κB–inducible nitric oxide synthase–nitric oxide (NF-κB–iNOS–NO) signal axis in vitro. Thus, understanding the mechanisms of β-cell survival in basal or stress conditions associated with HU is imperative for creating new strategies to prevent and manage diabetes, especially for HU individuals.

    Uricase (Uox) expressed in rodents can further degrade uric acid into allantoin (8), which has hindered the establishment of suitable rodent models for HU (9). We previously established a spontaneous HU mouse model with Uox gene deficiency that is characterized by long-term stable SU levels (7–9 mg/dL) (10). This animal model lays a foundation for further glycometabolism investigations. In the current study, we address the following questions: 1) whether the Uox-KO mouse develops spontaneous glucose abnormities (such as insulin resistance, compromised β-cell functions) and even diabetes, 2) whether HU imposes stresses on glucose phenotypes or pancreatic β-cells with additional high-fat diet (HFD) and/or streptozotocin (STZ) stimulation in the Uox gene-deficiency mouse model, and 3) how urate works in mouse isolated islets if we have evidence that urate attacks pancreatic β-cells.

    Research Design and Methods

    Animals

    Uox knockout (KO) mice and their wild-type (WT) counterparts (C57BL/6J background) were generated as previously described (10). Briefly, a region of 28 base pairs in exon 3 of the Uox gene was deleted using the transcription activator-like effector nuclease technique. The animals were maintained in a temperature-controlled room (22°C), with humidity at 55%, and on a 12-h light-dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) under specific pathogen-free conditions. After a 2-week acclimation period, 8-week-old mice were randomly assigned to two groups (1:1) and fed with an HFD (45% total fat, 35% protein, and 20% carbohydrate) or a regular chow diet ad libitum with free access to water for 20 weeks.

    To provoke the potential role of urate in animals, we fed mice the HFD (45% fat, 35% protein, and 20% carbohydrate) for 1 week and then injected them with multiple low-dose STZ (MLD-STZ, 40 mg/kg body wt i.p.) daily for 5 consecutive days. Specifically, STZ was freshly dissolved in 0.1 mol/L citrate buffer (pH 4.5). For comparison, mice were administered STZ and fed a normal diet. Random-fed (9:00–10:00 a.m.) blood glucose levels were determined by glucometer (ACCU-CHEK Inform; Roche Pharmaceuticals). To evaluate the effect of ULT, pegloticase (also known as Krystexxa or Puricase), purchased from Horizon Pharma, was administrated to mice by tail vein injection at a dose of 0.5 mg/kg, with the first injection on the first day of the MLD-STZ intervention, and the second injection was on the 10th day of the MLD-STZ intervention.

    Male mice were used for all studies shown, and the age of mice is indicated in the figures. This study was approved by the Affiliated Hospital of Qingdao University Animal Research Ethics Committee.

    Blood Biochemistry

    Mice were fasted overnight before serum biochemical tests. Blood was collected from the outer canthus the next morning. SU levels were measured immediately from the serum of anesthetized breathing mice (11), using an automatic biochemical analyzer (Toshiba, Tokyo, Japan). Serum creatinine levels and lipid profiles, including total cholesterol (TC), triglycerides (TG), and HDL and LDL cholesterol were assessed by the automatic biochemical analyzer (Toshiba) as well. Blood glucose levels were monitored by tail bleeding with a glucometer (ACCU-CHEK Inform). Diabetes was defined as a random blood glucose of ≥16.7 mmol/L (12).

    Glucose Tolerance Test, Insulin Tolerance Test, and Glucose-Stimulated Insulin Secretion

    Mice were fasted for 8 h before a glucose tolerance test (GTT) or insulin tolerance test (ITT) and then injected with d-glucose at 2 g/kg body wt i.p. or 1 unit/kg insulin (Humulin R; Eli Lilly). Blood was collected at predetermined times (0, 15, 30, 60, and 120 min) after the glucose injection, and the blood glucose levels were determined using a glucometer (ACCU-CHEK Inform).

    Glucose-stimulated insulin secretion (GSIS) testing in vivo and in vitro was performed. Briefly, mice were fasted for 8 h and injected with 2 g/kg d-glucose i.p., and serum insulin levels were tested at 0, 15, 30, and 60 min for in vivo GSIS. For in vitro GSIS, isolated islets were purified and harvested by handpicking under a stereomicroscope as described below. The islets (10 per well) were seeded in 24-well plates and then cultured in complete RPMI 1640 (Gibco, Life Technologies) with 10% FBS (HyClone; GE Healthcare, Little Chalfont, U.K.) in 5% CO2 at 37°C overnight. After incubation for 1 h in glucose-free Krebs buffer (115 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 20 mmol/L NaHCO3, 16 mmol/L HEPES, 2.56 mmol/L CaCl2, and 0.2% BSA), the islets were treated for 1 h in Krebs buffer with low (3.3 mmol/L) or high (16.7 mmol/L) concentrations of glucose. After treatment, the supernatants were obtained for determination of insulin concentration with an ultrasensitive ELISA kit (ALPCO Diagnostics, Salem, NH).

    Pathology Analysis

    Mice were sacrificed to extract their pancreas for the pathology analysis. The pancreas was immediately dissected, weighed, and fixed in 10% formalin on ice for 30 min, followed by paraffin embedding of 5-μm serial sections. Tissue serial sections were stained by hematoxylin-eosin (HE) and separately incubated with anti-insulin (1:200) (ab7842; Abcam) rabbit polyclonal antibodies for immunohistochemistry. Primary islet apoptosis was analyzed by the terminal deoxynucleotidyl TUNEL technique according to the manufacturer’s instructions (catalog no. 11684795910; Roche). The samples were stained with DAPI to visualize total cells. Pancreatic sections were also immunostained with anti-insulin antibody to identify β-cells. β-Cell fractional area was determined by quantifying the percentage of insulin-positive pancreas area as a total of the full pancreas area for each section, followed by averaging of six sections per mouse. Images were analyzed using the Pannoramic Digital Slide Scanner. The insulin-positive areas were analyzed by Image-Pro Plus 6 software. β-Cell mass was calculated by multiplying the β-cell fractional area with the initial pancreatic wet weight.

    The kidneys were removed, fixed in 10% formalin, embedded in paraffin, and then cut into 4-μm sections. The sections were used for HE, periodic acid Schiff–methenamine (PASM) staining, and periodic acid Schiff (PAS) staining. The percentage of tubule injuries was assessed by scoring six renal cortical tubule sections in randomly selected fields for each group. The glomerulosclerosis index (GSI) was calculated as [(1 × N1) + (2 × N2) + (3 × N3) + (4 × N4)]/(N0 + N1 + N2 + N3 + N4), where Nx was the number of glomeruli with each given score (0 for normal glomeruli, 1 for up to 25% involvement, 2 for up to 50% involvement, 3 for up to 75% involvement, and 4 for >75% sclerosis). The average GSI was analyzed based on three given PAS staining sections for each group. Renal crystal sections were obtained from absolute ethanol-fixed kidneys to detect urate crystals under polarized light. The urate crystal areas in each group were calculated by the Image-Pro Plus 6.0 system.

    Islets Isolation

    Pancreatic islets were isolated from mice with digestive enzyme (13,14). Briefly, a mouse was euthanized with CO2, followed by cervical dislocation, and then placed supine under a stereomicroscope, with the abdomen cleaned with 75% ethanol. A laparotomy was performed by cutting the skin and the muscular tissue of the thorax with a V-incision from the pubic region up to the diaphragm to expose the abdominal cavity. The common bile duct close to the duodenum was ligated for the retrograde puncture of the common bile duct, followed by a slow perfusion of 3 mL prechilled collagenase IV (catalog no. C5138S; Sigma-Aldrich) at 0.5 mg/mL concentration (dissolved in Hanks’ balanced salt solution) to fully expand the pancreatic body and tail. The pancreas was then excised and digested at 37°C for 11 min. Islets were purified in Histopaque-1077 (catalog no. 10771; Sigma-Aldrich) by vortexing gently for several seconds.

    Microarrays

    RNA Isolation and Quantification

    Isolated islets from mice were prepared for the following RNA extraction. Briefly, total RNA was extracted and purified using RNeasy Micro Kit (catalog no. 74004; QIAGEN), following the manufacturer’s instructions, and checked for a registrant identification number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent Technologies). Only those samples with a 260 nm–to–280 nm ratio between 1.8 and 2.1, a 28S-to-18S ratio within 1.5 and 2.0, and gel electrophoresis that showed clear 28S and 18S bands and a weak 5S strip were further processed.

    Library Preparation

    Total RNA samples (100–1,000 ng) were polyA enriched, reverse-transcribed into double-stranded cDNA, and then labeled by Low Input Quick Amp Labeling Kit (catalog no. 5190-2305; One-Color; Agilent Technologies) following the manufacturer’s instructions. Each slide was hybridized with 600 ng Cy3-labeled cRNA using the Gene Expression Hybridization Kit (catalog no. 5188-5242; Agilent Technologies) in a hybridization oven. Microarray was performed on Agilent platform using Agilent SurePrint G3 Mouse Gene Expression Microarray 8 × 60K chips (Agilent Technologies).

    Data Analysis

    Data were extracted with Feature Extraction 10.7 software (Agilent Technologies). We used a cutoff of normalized array values (log2-transformed values >2.0 or <0.5) for islet tissue transcripts. Raw data were normalized by quantile algorithm. Limma and agilp packages were loaded in R 3.4.1 software.

    Statistical Analysis

    All experimental statistical analyses were performed using GraphPad Prism 8 software (GraphPad). For two-group comparisons, the Student t test was used. For multiple comparisons, one-way ANOVA, followed by a Dunnett test, was used to compare each group versus a vehicle-treated group. Data are presented as mean ± SEM. Differences with P < 0.05 were considered statistically significant.

    Results

    Uox-KO Mice Develop Glucose Intolerance but Not Diabetes

    In our previous study, we successfully established the mouse model with Uox gene (which is mainly expressed in liver) (Supplementary Fig. 1) deficiency to mimic human HU (10). Shown in Fig. 1A, SU levels in Uox-KO male mice were dramatically increased compared with WT mice (P < 0.001) and stabilized at elevated levels (7–9 mg/dL) from 8 to 56 weeks of age. No difference in body weight was observed between age-matched Uox-KO and WT mice (Fig. 1B). HU did not alter fasting blood glucose or plasma insulin levels even with aging (from 8 to 56 weeks) (Fig. 1C and D). As for lipid profiles, no difference was shown in TC or HDL and LDL cholesterol between Uox-KO males and their WT counterparts, whereas TG were significantly lower in Uox-KO mice compared with WT mice at each age point (P < 0.001 at 8 weeks, 24 weeks, and 56 weeks) (Fig. 1E–H).

    Figure 1
    Figure 1

    General characteristics of Uox-KO male mice and WT counterparts stratified by age. A: SU levels were measured at the age of 8, 24, and 56 weeks in KO and WT mice (n = 9). B: Body weights were evaluated accordingly (n = 8). Fasting blood glucose (C) and fasting plasma insulin (D) were detected (n = 5–8). Lipid profiles, including TC (E), TG (F), LDL cholesterol (G), and HDL cholesterol (H) were determined in 8-, 24‐, and 56-week-old mice (n = 6–8). ***P < 0.001. Data are expressed as mean ± SEM.

    GTT and ITT were performed in mice to further assess the impact of HU on glucose homeostasis. In basal condition, Uox-KO males showed a significant impairment in glucose tolerance, indicated by remarkable elevated blood glucose concentrations at 15 and 30 min after 2 g/kg glucose administration at 8 and 24 weeks of age (Fig. 2A and B) and at 30 min when they were 56 weeks old (Fig. 2C). ITT revealed no insulin resistance in Uox-KO males, even with prolonged HU stress (Fig. 2D–F). These data show Uox-KO male mice do not develop diabetes spontaneously though with aging.

    Figure 2
    Figure 2

    HU impairs glucose tolerance in Uox-KO male mice. GTT were performed in Uox-KO mice and WT controls with a 2 g/kg glucose i.p. injection after 8 h fasting at the age of 8 weeks (A), 24 weeks (B), and 56 weeks (C), separately (n = 6). DF: Insulin (1 unit/kg) was administrated i.p. after 8 h fasting for ITT in 8-, 24‐, and 56-week-old mice (n = 6). *P < 0.05, **P < 0.01. Data are expressed as mean ± SEM.

    HU Does Not Impair Insulin Sensitivity in HFD-Fed Mice

    We fed Uox-KO male mice and the control WT littermates with the HFD or the normal chow diet, starting at 8 weeks of age, and continued for 20 weeks. Both genotypes displayed comparable SU levels and circulating lipids (including TC and HDL and LDL cholesterol) before and after the experimental feeding (Table 1). The HFD elevated TC, TG, and LDL cholesterol and decreased HDL cholesterol in both genotypes (Table 1). After 20 weeks of exposure to the HFD, the WT mice showed increased body weight compared with the Uox-KO mice (Table 1). Although plasma insulin levels displayed a significant elevation in HFD-fed WT mice compared with chow diet–fed mice, the HFD-fed Uox-KO mice showed a dramatic reduction in plasma insulin levels compared with the HFD-fed WT mice (Table 1). This result indicates a failure of compensatory insulin production by pancreatic β-cells in Uox-KO mice.

    Table 1

    Body weight and serum biochemical profiles of Uox-KO and WT mice after 20 weeks on an HFD or normal chow diet

    We also performed GTT and quantified the area under the curve (AUC) that integrates the values from 0 to 120 min of GTT. After 20 weeks of being fed with the chow diet or HFD, blood glucose in WT and Uox-KO male mice was restored to normal concentrations at 120 min after the glucose challenge (Fig. 3A), despite that a higher peak at the 30-min point was observed in HFD-fed Uox-KO mice. However, after 20 weeks of being fed the HFD, both genotypes exhibited a retarded glucose clearance compared with mice fed the normal chow diet (Fig. 3A). Remarkably, the blood glucose levels in HFD-fed Uox-KO mice were sustained at a dramatically higher level over the whole course of GTT compared with HFD-fed WT controls (Fig. 3A), resulting in a significant increase in the AUC of the GTT (Fig. 3B) and indicative of severe glucose intolerance.

    Figure 3
    Figure 3

    HU strengthens glucose intolerance with HFD stimuli. A: GTTs were used for chow diet– and HFD-fed 8-week-old Uox-KO male mice and WT controls (n = 8). B: AUC integrated from the period of 0–120 min was calculated based on the GTT curves. ITTs were monitored (C), and corresponding AUCs were determined (8-week-old mice, n = 8) (D). E: Plasma insulin levels were measured with plasma collected at 0, 15, 30, and 60 min after glucose injection (2 g/kg i.p.) in 8-week-old Uox-KO mice and WT mice (n = 6 in each group). F: Supernatant insulin levels were determined after in vitro GSIS with isolated pancreatic β-cells (n = 6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as mean ± SEM.

    The development of glucose dysmetabolism is ultimately attributable to impaired insulin action or insufficient insulin production, or both. To clarify whether insulin resistance accounts for the glycol-metabolic disorders in HFD-fed Uox-KO mice, we used an ITT. Compared with the chow diet, the disappearance of glucose after the insulin challenge was comparable by the HFD (Fig. 3C), displaying no insulin resistance in diet-induced Uox-KO male mice. The AUC of the ITT was comparable between HFD-fed WT and Uox-KO mice as well (Fig. 3D), suggesting an equivalent degree of insulin resistance in the two genotypes.

    Islet function was determined by in vivo and in vitro GSIS. Similar to the results of Uox-KO mice islet function in our previous study (10), HFD-fed Uox-KO mice secreted a significantly lower amount of insulin at 15 and 30 min (Fig. 3E), consistent with the GTT results above. Although isolated islets derived from HFD-fed Uox-KO mice secreted a similar amount of insulin as HFD-fed WT islets at low glucose concentration (3.3 mmol/L), insulin secretion was decreased by 22.6% in HFD-fed Uox-KO islets at a high glucose concentration (16.7 mmol/L), as shown in Fig. 3F. Collectively, these data suggest that HU impairs GSIS in HFD-fed Uox-deficient males, contributing to increased glucose intolerance.

    HU Induces Diabetes With External MLD-STZ Stimulation

    Given HU compromised β-cell functions and impaired glucose tolerance, we suppose urate worsens hyperglycemia in an established model of diabetes induced by HFD + MLD-STZ (40 mg/kg/day for 5 days). Pegloticase, an approved recombinant porcine-like uricase drug, was indicated for the treatment of HU mice. As indicated by Fig. 4A, the random glucose levels in STZ-induced diabetic Uox-KO (STZ-KO) mice elevated markedly on day 7, and then severe hyperglycemia developed, which over time resulted in uncontrolled diabetes (day 0 vs. day 7, P < 0.01; day 0 vs. day 8–20, P < 0.001), whereas the STZ-induced diabetic WT (STZ-WT) mice showed stable glucose levels on the first 8 days (P > 0.05) and were significantly elevated afterward (P < 0.01). Consistent with the previous reports, a low dose of STZ enhanced random glucose, and 23.1% of the mice developed diabetes. However, 87.5% of the STZ-KO mice exhibited average random blood glucose levels >16.7 mmol/L on day 11 and afterward (Fig. 4B). ULT lowered the diabetes incidence, although without a significant difference, and ultimately reached 75.0% in STZ-KO mice (Fig. 4B). Furthermore, ULT delayed the diabetes onset from day 7 to day 10 (Fig. 4B). Therefore, MLD-STZ stimuli could accelerate the development of diabetes, and ULT can only partially reverse this deterioration in STZ-KO males.

    Figure 4
    Figure 4

    HU induces diabetes with external MLD-STZ stimulation. A: Random blood glucose levels were monitored daily after administration of MLD-STZ (40 mg/kg/day for 5 days) for 20 consecutive days (8-week-old mice, n = 13, 8, and 8 for STZ-WT mice, STZ-KO mice, and pegloticase therapy ULT-KO mice separately, respectively). B: The diabetes incidence in 8-week-old mice was calculated by percentage (n = 13, 8, and 8 in each group, respectively). Diabetes was defined as random blood glucose ≥16.7 mmol/L. P trend value is presented as *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as mean ± SEM.

    HU-Related Diabetes Is Manifested by Increased β-Cell Apoptosis

    For determination of whether impaired glucose intolerance was caused by a reduction in β-cell number and islet mass, we extracted mice pancreas tissues for further analysis. No apparent histological lesions, stained with HE or with antibodies against insulin, were detected in the pancreatic islets of the Uox-KO males and controls (Fig. 5A and B). Pancreatic sections were coimmunostained with anti-insulin antibody to visualize β-cells. Compared with the control groups, pancreatic insulin content was lower in STZ-KO mice than in control mice (Fig. 5A and B). Accordingly, relative β-cell mass, determined by insulin immunoreactivity, was 62% lower in STZ-KO mice (Fig. 5A and B). To determine whether cell death contributes to reduced β-cell mass in STZ-KO mice, we measured islet apoptosis by TUNEL staining. The number of TUNEL-positive β-cells was higher in STZ-KO mice than in control mice (Fig. 5C and D), tending to a 42% increase (Fig. 5C and D). This finding suggests HU promotes not only the loss of β-cell mass but also apoptosis of the pancreatic β-cell in STZ-KO mice. These morphological alterations are consistent with the changes in plasma insulin levels, indicating that β-cell apoptosis chiefly contributes to the onset of diabetes in STZ-KO males.

    Figure 5
    Figure 5

    HU causes pancreatic β-cell death under MLD-STZ stimuli. A: Pancreatic sections were histologically stained for HE and immunohistochemically (IHC) stained for insulin, respectively (8-week-old mice, n = 6). B: Total areas of pancreatic tissues and insulin-positive cells were traced manually and determined by counting 10 islets or more in 6 sections per mouse (8-week-old mice, n = 6) in each group. β-Cell mass was analyzed, and results are shown in multiplying the β-cell ratio (insulin-positive areas–to–total area) with the initial pancreatic wet weight. C: Insulin-positive cell apoptosis was analyzed by the terminal deoxynucleotidyl TUNEL (8-week-old mice, n = 6). D: Double-positive ratio of insulin and TUNEL was measured in each group (8-week-old mice, n = 6). Scale bars = 50 μm. **P < 0.01, ***P < 0.001. Data are expressed as mean ± SEM.

    We next asked whether ULT would be able to reverse the pancreatic β-cell apoptosis with MLD-STZ in HU mice. Shown in Fig. 6A, the β-cell masses were significantly smaller in STZ-KO males than in STZ-WT controls (P < 0.001). However, the ULT did not reverse the loss of β-cell mass (STZ-KO vs. ULT-KO, P > 0.05) (Fig. 6A). MLD-STZ exaggerated the islet β-cell death compared between STZ-KO and STZ-WT mice, indicated by TUNEL staining (Fig. 6B). Consistently, the number of TUNEL-positive β-cells was significantly higher in STZ-KO mice than in their controls (Fig. 6C). However, no significant decreases in TUNEL-positive β-cell signals (Fig. 6B) or analysis numbers (STZ-KO vs. ULT-KO, P > 0.05) (Fig. 6C) were detected after 21 days of ULT in STZ-induced Uox-KO mice. Thus, our findings did not support the benefits of ULT on β-cell vitality.

    Figure 6
    Figure 6

    ULT reversed few pancreatic β-cell deaths stimulated by MLD-STZ. Mice were sacrificed on the 21st day after the first MLD-STZ (40 mg/kg/day for 5 days) injection. A: β-Cell mass was calculated by multiplying the β-cell ratio (insulin-positive areas–to–total area) with the initial pancreatic wet weight (11-week-old mice, n = 3). B: Insulin-positive cell apoptosis was analyzed by the terminal deoxynucleotidyl TUNEL (11-week-old mice, n = 3). C: Double-positive ratio of insulin and TUNEL was measured in each group (11-week-old mice, n = 3). Scale bars = 50 μm. **P < 0.01, ***P < 0.001. Data are expressed as mean ± SEM.

    ULT Ameliorates Tubulointerstitial Injury in Diabetes

    To evaluate renal changes after STZ stimulation and ULT intervention, we did kidney functional and histological experiments in both genotypes. SU levels were significantly higher in STZ-induced Uox-KO male mice than in their WT controls (P < 0.001) (Fig. 7A). The same results were shown in serum creatinine levels, which are indicators of renal function (P < 0.001) (Fig. 7B). ULT increased renal function in STZ-KO males, displaying significant decreases in SU and creatinine levels (P < 0.001) (Fig. 7A and B).

    Figure 7
    Figure 7

    Renal function and pathological changes were evaluated in male 11-week-old mice. A: SU levels were measured at the end of experiments (n = 13 for WT and n = 8 for Uox-KO mice in each group). B: Serum creatinine levels, indicators of renal function, were determined by the automatic biochemical analyzer (n = 13 for WT and n = 8 for Uox-KO mice in each group). C: Renal pathohistology was detected by HE staining, PASM staining, and PAS staining (n = 6). Deposits of renal urate crystals were investigated under polarized light (n = 3). D: Quantification of tubulointerstitial injury. Quantitative analysis for tubulointerstitial injury was assessed by scoring six renal cortical tubule sections (original magnification ×200, HE) in randomly selected fields for each group (n = 6). E: The GSI was calculated to evaluate diabetic nephropathy based on three given PAS staining sections for each group (n = 6). F: Urate crystal areas were calculated by the Image-Pro Plus 6.0 system with arbitrary units (n = 3). Scale bars = 50 μm in HE (original magnification ×200), PASM, PAS, and polarized light sections. Scale bars = 100 μm in HE (original magnification ×400). *P < 0.05, ***P < 0.001. Data are expressed as mean ± SEM.

    STZ-KO mice showed dilated Bowman spaces and tubules and collapsed and necrotic nephrons by pathological analysis (Fig. 7C). The tubular damage was significantly severe, with tubular dilation, detachment of tubular epithelial cells, and condensation of tubular nuclei appearance in STZ-KO mice (Fig. 7C). ULT prevented the development of these lesions indicated by decreased percentage of tubular damage in ULT-KO mice (Fig. 7D). PASM staining exhibited obvious glomerular mesangial hyperplasia, increased glomerular matrix, and thickened glomerular basement membrane in diabetic STZ-KO mice compared with their WT controls (Fig. 7C). PAS staining documented glomerular mesangial expansion and glomerular sclerosis, early features of diabetic nephropathy, in STZ-KO mice compared with STZ-WT mice (Fig. 7C). The GSI, calculated by PAS staining sections, was increased in STZ-KO mice compared with STZ-WT mice (P < 0.05) (Fig. 7E). However, ULT did not prevent the glomerular injury in STZ-induced HU mice as quantified by GSI (Fig. 7E). HU STZ-KO mice showed significant renal urate crystal deposits (P > 0.05) (Fig. 7C). Crystals were dissolved by ULT in STZ-KO mice with decreased urate crystal areas (P < 0.001) (Fig. 7F). The histological analysis presented that ULT exhibited a significant therapeutic effect of HU-crystal–associated kidney injury and tubulointerstitial injury manifestation in diabetic nephropathy.

    Differentially Expressed Genes in HU and/or Diabetic Mice

    We then wondered whether differentially expressed genes (DEGs) in islets of the HU and/or diabetic mice would explain the molecular mechanism of urate on impaired glucose metabolism. Microarray data were represented by heat maps (Fig. 8A and B) in subgroup comparisons of Uox-KO mice versus Uox-WT mice and STZ-KO mice versus STZ-WT mice. We selected the genes based on adjusted P < 0.05 and absolute fold change >2. In Uox-KO versus Uox-WT groups, 850 of 2,018 genes were upregulated and 1,168 of 2,018 genes were downregulated (Fig. 8C). Whereas in STZ-KO versus STZ-WT groups, 29 of 171 genes were upregulated and 142 of 171 genes were downregulated (Fig. 8D). When compared between Uox-KO versus Uox-WT and STZ-KO versus STZ-WT groups together, one gene (Stk17β) was shared in the upregulated DEGs and five genes (Fut4-ps1, Erich3, 1700027H10Rik, Kcnh2, and Klhl32) in the downregulated gene set (Fig. 8C and D). These shared genes were the urate primacy functioning genes. It is notable that the shared upregulated gene, Stk17β, plays a key role in a wide variety of cell death signaling pathways.

    Figure 8
    Figure 8

    Gene set enrichment analysis comparing isolated islets from HU and/or diabetic mice. Heat maps from mouse isolated islets are shown. Each colored box represents the normalized expression level of a given gene in a particular experimental condition of WT vs. KO (A) and STZ-WT vs. STZ-KO (B). Red denotes upregulation and blue denotes downregulation according to the color scale. Shared upregulated (C) and downregulated (D) DEG numbers were enriched in WT vs. KO and in STZ-WT vs. STZ-KO groups. One gene (Stk17β) was shared in the upregulated gene set and five genes (Fut4-ps1, Erich3, 1700027H10Rik, Kcnh2, and Klhl32) were shared in the downregulated gene set, with adjusted P < 0.05 and absolute fold change >2 (8-week-old WT and KO mice and 11-week-old STZ-WT and STZ-KO mice, n = 3 in each group).

    Discussion

    Whether HU is a causal or noncausal factor for diabetes remains controversial. Although numerous clinical studies have showed that HU predicts the development of diabetes and that the ULT could reduce the diabetes incidence or fasting glucose levels accordingly, the causal relationship between HU and diabetes and related mechanisms remains elusive. The current study firstly demonstrated that HU augmented the existing glycometabolism abnormality induced by MLD-STZ and induced diabetes by promoting not only the loss of β-cell mass but also pancreatic β-cell apoptosis in STZ-induced Uox-KO male mice. Although HU increased the incidence of diabetes when accompanied with STZ stimuli, ULT can only ameliorate the incidence by a small proportion without significant statistical differences. In addition, our transcriptomic results indicated that Stk17β is a possible target gene in HU-induced β-cell apoptosis.

    Multiple lines of evidence have shown the association between HU and diabetes. A community-based study in the U.S. demonstrated the risk of diabetes incidence increased 18% with every 1 mg/dL SU elevation, and the association remained significant after adjustment for fasting glucose and insulin levels (15). The diabetes incidence was 19% for SU ≤7 mg/dL patients, 23% for SU 7 mg/dL to ≤9 mg/dL, and 27% for SU >9 mg/dL in an 80-month follow-up investigation that included 1,923 U.S. veterans (15). This study also indicated ∼8.7% of all new cases of diabetes were statistically attributed to HU (15). The age- and sex-adjusted hazard ratio for diabetes was 2.83 for the fourth quartile of SU in subjects from the Rotterdam study (16). Although epidemiology studies showed that HU predicts the development of diabetes, no causality was found in the Mendelian randomization study (6).

    Thus, for further phenotype and mechanism explorations, a major obstruction is the lack of an appropriate animal model given that Uox in most mammals, including rodents, is functional. Here, we were able to use Uox-KO mice to study the potential role of urate in glucose metabolism. Traditional establishment strategies, such as potassium oxonate (a chemical inhibitor of uricase) i.p. injection (17), would interfere with the effects of HU per se as the additional exogenous intervention. An advantage here is that this mouse model is a suitable HU model because of its consistent and stable SU elevations to simulate a human-specific biological background of elevated urate (10).

    Next, we further stimulated this spontaneous HU mouse model with the HFD and/or MLD-STZ. A phenotypic heterogeneity in glycometabolism existed between sexes in the Uox-KO model, displaying severe glucose intolerance and more sensitivity to STZ in Uox-KO males than in females in a previous study (10). To avoid confounding factors due to sex or sex hormone differences, only male mice were used in the current study. Further investigations in female mice would help to delineate a full picture of the effect of HU on glucose metabolism.

    Our data show significant elevated blood glucose concentrations at 15 and 30 min of GTT under basal conditions and in response to high-fat feeding (Figs. 2 and 3). Moreover, the plasma insulin drop indicated the compromise of β-cell function is responsible for glucose intolerance. The link of HU and insulin resistance has been extensively reported and discussed (18,19). Spontaneous HU represents one of the metabolic syndromes resulting from the interactions between genetic and environmental factors, including dietary and behavioral factors, which also contribute to insulin resistance in studies (20). Thus, genetically modified HU mouse models lack major complicated genetic or nongenetic risk factors, and HU indeed did not induce insulin resistance, which is normally obesity related. We then fed Uox-KO mice and WT controls the HFD or normal chow diet. The results showed HFD-fed Uox-KO mice displayed a dramatic reduction in plasma insulin levels compared with HFD-fed WT mice, which indicated a failure of compensatory insulin production by β-cells in the Uox-KO mice. Glycol-metabolism evaluations, including GTT, ITT, and GSIS, hint at severe glucose intolerance, with unchanged insulin sensitivity in HFD-induced Uox-KO mice (Fig. 3). To be noted, neither the fasting glucose nor fasting insulin changed in the Uox-KO mice, even with aging (Fig. 1). In this study, the lack of being able to see a urate-to-insulin resistance relationship is based on the use of a high-fat rather than a high-sugar diet. A similar conclusion was drawn by Kelly and colleagues (21) that systemic HU, while clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causal. Mendelian randomization studies that focused on genetic variants associated with urate, thereby removing confounding factors such as obesity, showed elevated urate could not independently predict diabetes development (6,22), which also supports our study findings. However, in models of sugar-/fructose-induced insulin resistance or in models where liver xanthine oxidase activity is increased, then one does see urate-dependent insulin resistance (23,24), which is likely mediated by gluconeogenesis (25). Further investigations using sugar-/fructose-fed Uox-KO mice are needed to detect the relationship between urate and insulin resistance.

    In the current study, we report that Uox deficiency predisposes mice to the onset of diabetes, which results from a loss of β-cell mass under conditions of the HFD accompanied by MLD-STZ. The study has provided both functional and mechanistic data showing the proapoptotic effect of HU in β-cells. This is the first in vivo investigation suggesting a critical role of HU in acceleration of the progression from impaired glucose tolerance into diabetes via the action of promoting β-cell death. Increasing incidence of diabetes is also accompanied by decreased islet area and β-cell mass (Figs. 4 and 5), which corroborates that this glucose intolerance induced by HU is primarily caused by β-cell death. Multiple factors and signaling mechanisms have been demonstrated to influence β-cell compensation, of which β-cell apoptosis has emerged as a key event causing decompensation of β-cells and the development of diabetes (26). Insulin deficiency has been demonstrated to play a causative role in the development of diabetes from both animal and human studies (26). In line with this, Uox-KO mice induced by MLD-STZ develop diabetes, accompanied by hypoinsulinemia and increased β-cell apoptosis. The diabetic phenotype of Uox-KO mice not only indicates that loss of functional β-cell mass is a key cause of the disease but also reveals a critical role of HU that is required for maintaining β-cell survival in the STZ-induced condition. However, it is worth noting that no differences were found in β-cell mass and insulin production between Uox-KO mice and WT controls fed the normal chow diet, suggesting that HU is not required for islet development and is dispensable under nonstressed physiological conditions.

    The benefits of ULT are still inconclusive. Randomized trials have reported that ULT by allopurinol improves insulin resistance in asymptomatic HU individuals (27,28), and similar improvement in insulin resistance has been observed with the use of benzbromarone (29). However, in a gout population, the incidence of diabetes was lower in urate-lowering drug (ULD) users than in nonusers (30). In vivo study showed oral administration of ULD dose-dependently reduced the blood glucose level and improved glucose tolerance and insulin resistance in db/db mice (31). Whether asymptomatic HU should be treated remains controversial, as Sluijs et al. (6) concluded that SU is not causally linked to diabetes and that ULTs may therefore not be beneficial in lowering diabetes risk. A long-held notion in diabetes is that macrophages within the islet produce reactive oxygen species and proinflammatory cytokines, creating a β-cell cytotoxic environment (32). Pegloticase, a recombinant uricase, exerts its urate-lowering role by degrading urate with a mild oxidative stress. Lowering SU may also reduce oxidative stress because urate, although an antioxidant in noncellular systems, is a pro-oxidant in cellular systems. The present in vivo ULTs exhibited only a partial reverse function on reducing diabetes incidence (Fig. 4) and could not ameliorate β-cell apoptosis (Fig. 6) or glomerular lesions in diabetes (Fig. 7). Substantial short-term ULT did not have a direct protective effect on β-cell apoptosis or antidiabetic potential, suggesting that, on its own, this might not be an effective strategy for restoring β-cell function in STZ-induced HU mice. Longer-term ULT needs to be done in future work to solidify the conclusion.

    One of the interesting findings in this study is that ULT improved tubulointerstitial injury but not glomerular lesions in STZ-KO mice. Gilbert and Cooper (33) suggested that tubulointerstitial injury is a major feature of diabetic nephropathy and that its development may reflect influences that are common to other forms of renal disease and also those that are unique to diabetes. An in vivo study showed HU plays a pathogenic role in the mild tubulointerstitial injury associated with diabetic nephropathy but not glomerular damage in diabetic mice (34). Other factors, such as high glucose or oxidative stress, could be responsible for the diabetic glomerular lesion because high glucose is well known to be one of the major stimuli to accelerate extracellular matrix deposition in diabetic glomeruli (35).

    Contrary to studies that have previously reported a positive causal association with progression of chronic kidney diseases (CKDs) (36), urate concentration was not causally related to the development of diabetic nephropathy in a Mendelian randomization study by Ahola et al. (37). This Mendelian randomization analysis suggested that the SU concentration does not have any causal effect on diabetic kidney complications but is rather a downstream marker of the kidney damage (37). Moreover, as one Mendelian randomization study suggests that SU is a causal risk factor for CKD in the general population (38), it might be that SU plays a role only in the processes leading to nondiabetic renal disease rather than in pure diabetic nephropathy. Furthermore, the authors of a post hoc analysis in diabetic nephropathy with mean follow-up of 3 years concluded that urate was weakly associated with decline in the glomerular filtration rate (GFR) in patients with type 1 diabetes with overt nephropathy (39). Evidence showed urate may facilitate the development and progression of CKD in people with diabetes (4042). However, the benefits of urate-lowering are still putative and inconclusive. A post hoc analysis of the Febuxostat Open-Label Clinical Trial of Urate-Lowering Efficacy and Safety Study (FOCUS) with 116 HU patients treated with febuxostat for 5 years found an inverse correlation between urate and estimated (e)GFR and projected an improvement in eGFR of 1 mL/min/1.73 m2 for every 1 mg/dL decrease in SU (43). In a randomized clinical trial, the authors found a significant association between higher urate and lower GFR (P = 0.017), while this association was absent in the allopurinol treatment (P = 0.61) (44).

    However, the renal status in the above studies was assessed with the eGFR and/or urinary albumin excretion rate, which manifested the glomerular not the tubular function. Despite the current availability of conclusive trial data, Bartáková et al. (45) have recommended 10–15% lower SU cutoff values for people with diabetes to confer protection against kidney disease. In an animal study, Kosugi et al. (34) found that db/db mice developed HU and glomerular mesangial expansion (an early feature of diabetic nephropathy). Allopurinol treatment significantly lowered urate levels and reduced tubulointerstitial injury but without amelioration of glomerular lesions in diabetes (34). These results were consistent with our reported data. However, the mixed conclusion of whether lowering SU benefits diabetic nephropathy from Mendelian randomization and clinical and animal studies needs to be carefully explained.

    The underlying mechanism of how urate interferes with the function of pancreatic β-cells has not been well elucidated. Roncal-Jimenez et al. (46) showed that sugar-induced HU likely played a role in the development of diabetes through an effect that included a direct urate effect on islet insulin secretion. Previous studies demonstrated β-cell toxicity of soluble urate via the NF-κB–iNOS–NO signal axis (7). Our research is the first study to elucidate the molecular changes occurring specifically in HU and/or diabetic islets by microarray. The transcriptomic analysis of isolated islets indicates major alterations in shared DEGs with up- and downregulation that might contribute to the accelerated progression of urate-induced cell death. Death-associated protein kinase–related apoptosis-inducing kinase-2 (Drak2), also known as Stk17β, is a serine/threonine kinase that executes its role by apoptosis-related pathways. Stk17β rapidly induces apoptosis in mouse islet β-cells by inflammatory cytokines (47) and free fatty acids (48). Stk17β overexpression, along with the cytokine assaults, has been demonstrated to lead to aggravated apoptosis of β-cells (47), which was consistent with our transcriptomic results (Fig. 8). Mao et al. (47) suggested the inflammatory cytokine/Stk17β/p70S6 kinase pathway seemed to be critical in islet apoptosis. They also demonstrated Stk17β acted through caspase-9 in apoptosis (47). Together with our transcriptomic profiles, these lines of evidence indicate that Stk17β is a potential gene in urate-mediated islet apoptosis of STZ-induced diabetes.

    Particularly, reduced levels of TG and unchanged TC levels in Uox-KO mice may partially explain the absence of an insulin-resistance phenotype given TG are an independent risk factor for the future development of insulin resistance (49). The decreased TG levels are attributable to sex hormones per se, since it has been reported that testosterone enhances TG in rodents (50) while our HU mice had lower testosterone (data not shown).

    Limitations of this study also need to be pointed out. To meet the animal ethic, the mice were euthanized with CO2, followed by cervical dislocation before islet isolation. CO2, as an additional stress on the islets, may magnify gene expression differences between KO and WT islets. Although Uox-KO mice displayed high SU levels comparable to those observed in adult humans, their germline disruption of Uox produced embryonic or postnatal developmental effects such as nephropathy. We also acknowledge this potential caveat that the mice developed kidney disease and urate crystalluria spontaneously, as we previously described (10). To separate whether the impaired glucose tolerance is due to the impaired renal function or to the urate, mice with a conditional and inducible disruption of Uox (e.g., tamoxifen-responsive cre mice) may be used to obviate the concern.

    In conclusion, the current study demonstrates that urate per se is insufficient to induce diabetes, while it impairs glucose tolerance. For the first time, our research using a constitutive HU model corroborates that high levels of urate predispose mice to diabetes by disrupting β-cell function. Diabetes incidence, β-cell death, or glomerular lesions in diabetes were not reversed by ULT with significance; however, ULT displayed a therapeutic effect on HU-crystal–associated kidney injury and tubulointerstitial damage in diabetic nephropathy. Transcriptomic profiling suggested Stk17β is a urate primacy gene in β-cell apoptosis. We believe that our microarray data specifically from isolated islets of HU mice can serve as a valuable resource for investigators in the field for further exploration of specific genes involved in HU with glycol-metabolic dysfunction.

    Article Information

    Acknowledgments. The authors thank pathologists Shihong Shao, Xiangyan Zhang, and Feng Hou (Department of Pathology, the Affiliated Hospital of Qingdao University) for their professional technical support.

    Funding. This study was supported by research project grants from the National Key Research and Development Program (2016YFC0903400), the National Science Foundation of China (31900413, 81520108007, and 81770869), the Shandong Province Key Research and Development Program (2018CXGC1207), and the Shandong Province Natural Science Foundation (ZR2018ZC1053).

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

    Author Contributions. J.L., Y.H., L.C., Z.L., X.L., H.Z., H.L., W.S., A.J., and Y.W. performed the experiments. J.L., Y.H., H.Y., and C.L. designed the study. J.L., Y.H., X.X., H.Y., and C.L. analyzed and interpreted the data. All authors approved the final version to be published. J.L. and C.L. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.



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    Diabetic Foodie: What’s Next? – Diabetic Foodie

    By electricdiet / June 12, 2020


    Change is good. At least that’s what I keep telling myself.

    Last fall, I attended the DiabetesSisters PODS Leadership conference. Shawn Shepheard of The Leadership Advantage led a session about creating our vision for the future. We each wrote down our biggest opportunities and what challenges might keep them from becoming reality. It was the kind of quality time you rarely get the luxury of spending on yourself. The experience forced me to think long and hard about Diabetic Foodie’s future.

    After the conference, I reached out to Shawn and asked if he could help me work on a 2020 business plan for Diabetic Foodie. I felt it needed some new energy. He agreed and we had a call that was life-changing.

    The best time for new beginnings is now

    Discovering My Unique Abilities

    Shawn suggested I spend some time thinking about the things I’m passionate about that I love to do. He said people who are “successful” and “happy” spend more than 80% of their time using these unique abilities. Through a series of exercises, I determined I was at about 17%. Yikes.

    Next, I tried to figure out how I could mold Diabetic Foodie into something that allowed me to use my unique abilities most of the time. To make that work, I would need to hire a bunch of people to do the things I didn’t want to do, and I would have to manage them all. Management is something I’m fairly good at, but have no passion for. It’s not one of my unique abilities.

    I taught a couple of classes at my local Lifelong Learning Institute and really enjoyed it. Around the same time, I had a conversation with my friend from DiabetesSisters, Christel Oerum of Diabetes Strong, about my work with Shawn. Christel mentioned she was interested in starting a food blog.

    What’s Next for Shelby?

    By January I was making plans to offer my first in-person cooking class. I had located a commercial kitchen to use and scheduled an Instant Pot class for March 21. Nine people signed up. My lifelong best friend passed away on March 7 and her memorial service was planned for March 21, so I rescheduled my cooking class for May 16. Almost immediately stay-at-home orders were issued for my state (Virginia) through June 10. Both the memorial service and the class were postponed indefinitely.

    In business-speak, I pivoted. I offered my Instant Pot class online via Zoom. Cameras, lighting, and the configuration of my kitchen posed challenges, but it was kind of fun trying to figure it all out. And, thank goodness, my audience was very forgiving!

    As staying home became the “new normal,” I started hearing from people who lived alone. Their social outlet, eating in restaurants with friends and family, had been taken away. They were looking for connection. I realized that while we couldn’t share food together, we could share a food experience. We could cook together online and have fun conversation along the way. Cook & Chat with Shelby was born.

    Sometimes you just have to let go and see what happens

    My ultimate dream is to combine cooking classes with travel experiences, but the pandemic won’t allow that to happen for a while. The good news is that I have plenty of time to work out the details!

    If you want to keep up with what I’ve got cooking (so to speak), please reach out via my website (currently a work in progress) or on Instagram or Twitter. I’d also love it if you’d sign up for the Cook & Chat with Shelby mailing list.

    I want to Cook and Chat

     

    What’s Next for Diabetic Foodie?

    What does this mean for Diabetic Foodie? Remember that conversation I had with Christel? We both realized that Diabetes Strong taking over the reins of Diabetic Foodie made perfect sense for both of us. To that end, as of June 1, 2020, I’ll be leaving Diabetic Foodie in the oh-so-capable hands of the team at Diabetes Strong. You can expect great things in the future.

    Please welcome the new owners of Diabetic Foodie and show them all of the love you’ve thrown my way over the last 10 years.

    I’m so grateful for all of you and everything you have taught me. Until we meet again…
    Shelby





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    Now and Then – My Bizzy Kitchen

    By electricdiet / June 10, 2020


    This past weekend my step-son and his wife were in town from Texas.  It was ironic that this same weekend on my TimeHop was a picture of Joe and Hannah – Joe was a sophomore in high school and Hannah was graduating 8th grade.

    FIFTEEN YEARS!  It’s hard to believe that much time has gone by.  And how things have changed.  Both are married now.  Both are on their own.  Both make me proud to be their Momma.

    We had our first BBQ at Hannah and Jacob’s house a mere four days after they moved in.  I know.  I had no other expectation because you all know Hannah is an organizing freak.  In fact, I went to her house at lunch to drop something off today, and she had already rearranged how she organized her kitchen cabinets within six days of moving in.

    They have a delightful back patio.

    Hannah and I went to Walmart on Saturday to pick up stuff for the BBQ and I kept finding stuff to throw in the basket.  I had put away some money knowing that she would need stuff after they moved in that they hadn’t thought of – like a pizza cutter (totally necessary!) garbage can for the kitchen etc.  When we stumbled upon the tiki torches, I just started putting them in our basket without even asking her.

    I stopped myself in my tracks.  I looked at her and said “Hannah, this is your house with Jacob, just tell me to shut the hell up if I overstep my boundaries.”  To which Hannah replied “I love you, and yes, we definitely need tiki torches!”

    Joe – it was so great to see you.  I know what ever career path you take next will be amazing.

    And Liz, so happy to see you and that you were able to spend so much time with your family this last week.

    It’s still a bit surreal that Hannah and Jacob aren’t here anymore.  Who knew a pandemic would give them the drive to move out.  I know it’s something they have wanted to do for a long time, but having been burned on their last attempt, and then getting outbid on other places, it was hard not to have low expectations.

    But they are . . . home.  Today when I walked in you would have thought they lived there for years.  I am so happy for all my kids, and yes, their spouses are my kids too. 😀

    Come back tomorrow because I made THE BEST PORK WONTONS I’ve ever made.  No.  Like seriously.  And they are WW friendly and delicious!

    Until next time. . . be well.

     



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    How to Treat a Low Blood Sugar (Without Eating Everything in Sight)

    By electricdiet / June 8, 2020


    We’ve all been in the position of having a bad low blood sugar, where all you want to do is eat everything in sight. There’s no reasoning with yourself. It’s like a demon has hijacked your self-control!

    It makes perfect sense–your body just wants to get your blood sugar back up to a safe level, and it’s doing its best to ensure that the low is corrected.

    But eating your weight in carbs is just going to result in high blood sugar, and then you are dealing with the opposite problem. What’s a person with diabetes to do?

    Woman eating glucose tabs

    First of all, fix the low. Get your blood glucose level above 70 mg/dl (4 mmol/L).

    It’s best to use a dextrose-based treatment, like glucose tablets or gel, to get the BG up to a safe level. Using something high glycemic index is important when you are very low and need to quickly raise the blood glucose, especially if you have a lot of insulin on board.

    Be careful to treat with the right amount of carb, as opposed to over-treating.

    • If you weigh less than 60 lbs, a gram of carb will raise your BG about 6 points.
    • If you weigh 100-160 lbs, a gram of carb should raise your BG about 4 points.
    • If you weigh 160-220 lbs, a gram of carb might raise you 3 points.

    And give the carbohydrates 15-20 minutes to raise the BG. Do a follow-up fingerstick because there is a lag time with CGM sensors, and the BG you get with a fingerstick will be a more accurate reflection of your response to your treatment. If you are still low, repeat the treatment.

    I remember a low where I munched my way through the pantry, and I neglected to count my carbs as I was doing it. I had absolutely no idea how much to bolus for! Boy, did I learn my lesson.

    I never did that again – I’ve always made sure to carb count and take insulin to cover whatever I eat that’s in excess of what I needed to do to control the low blood glucose levels. Sometimes experience is the best teacher.

    I was driving when I had the worst low I’ve ever had. Thank God I had glucose gel in the center console and glucose tablets on my key chain. I was sucking down the gel as I was pulling off the road.

    Ordinarily, I would have wanted to eat (and eat and eat) after that. But I had to sit in my car and wait for my BG to come up, so food wasn’t readily available. And do you know what? By the time I got home, the desire to eat had passed! I was very surprised, but it did. That was a good lesson for me, too.

    If your blood glucose is rising and getting higher than you’d like after you’ve treated your low, you can choose to exercise and get yourself away from your tempting kitchen.

    Take a little walk to use up some of that food you ate for the low BG and avoid the need to take corrective insulin to bring it down again – vicious cycle, no?

    Another strategy to avoid overeating after a low? Enlist the help of the people around you. Sometimes we all need a little support. Who knows, they might just be willing to sit with you while you wait for the low to come up or they may go on that walk with you.

    Suggested next posts: How To Treat Low Blood Sugar At Night and What a Low Blood Sugar Feels Like



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    Healthy Enchiladas Recipe-Easy Cookbook Highlights Diabetic Recipes

    By electricdiet / June 6, 2020


    Healthy Enchiladas Recipe Is Also Favorite Beef Diabetic Enchilada Recipe!

    Holly’s healthy enchiladas recipe is a favorite enchiladas.  This diabetic enchilada recipe made with beef makes an amazing and easy southwestern dish. There is no magical diabetes diet.  You need to practice straightforward healthy lifestyle changes, moderate sugar, fat, and portion control. Holly’s cookbook, KITCHEN 101: Secrets to Cooking Confidence, takes the thinking out of healthy diabetic meal choices.  This book focuses on delicious easy healthy recipes. Every recipe includes nutrition facts and diabetic exchanges.  Also, the symbol D’ highlights diabetic recipes and these dishes fit into the American Diabetes Association standards.

    Easy Beef Enchiladas
    Ridiculously easy and absolutely delicious dinner pronto!

      Servings10 enchiladas

      Ingredients

      • 1pound


        ground sirloin

      • 2teaspoons


        chili powder

      • 1cup


        salsa

      • 1cup


        corn

      • 1cup


        packed fresh baby spinach

      • 1 1/2cups


        shredded reduced-fat Mexican blend cheese

      • 10


        flour tortillasroom temperature, 6–8-inch

      • 1 1/2cups


        enchilada saucefound in can

      • 1


        bunch green onionschopped

      Instructions
      1. Preheat oven 350°F. Coat 13 x 9 x 2-inch baking dish with nonstick cooking spray.

      2. In large nonstick skillet, cook meat 6–8 minutes or until meat is done and drain any excess fat.

      3. Add chili powder, salsa, corn, and spinach; continue cooking about 5 minutes. Remove from heat, set aside.

      4. Spoon about 1/4 cup meat mixture and 1 tablespoon cheese onto a tortilla. Roll and place seam side down in prepared baking dish. Repeat with remaining tortillas.

      5. Pour enchilada sauce evenly over filled tortillas in baking dish and sprinkle with any remaining cheese and green onions. Bake, covered with foil for 20 minutes or until thoroughly heated.

      Recipe Notes

      Per Serving: Calories 237, Calories from Fat 24%, Fat 6g, Saturated Fat 3g, Cholesterol 35mg, Sodium 739mg, Carbohydrates 28g, Dietary Fiber 2g, Total Sugars 3g, Protein 17g, Dietary Exchanges: 1 1/2 starch, 1 vegetable, 2 lean meat

      Diabetic Recipes Highlighted in KITCHEN 101

      Holly’s grandmother had diabetes and she would watch her measure out her boring food.  That image stuck with Holly so she started writing cookbooks and always included the diabetic exchanges.  In KITCHEN 101, diabetic recipes are indicated with a “D” throughout the book so people living with diabetes can understand you can still enjoy your favorite food without making too many changes in food selection.  It’s the first mainstream cookbook that indicates diabetic recipes!

      Holly’s Easy Healthy Enchiladas Recipe Fit into Diabetic Diet

      The proof is in the recipes.  You will LOVE the simplicity and flavor of these Easy Beef Enchiladas.  They have only 2 Weight Watcher Pointsplus.  Holly’s goal is to infiltrate diabetic-friendly recipes throughout a mainstream cookbook to prove that everyone can enjoy the same food.

      • 64% of adults in the United States either are overweight or obese.
      • Projected 44 million to have diabetes in next 20 years.
      • Americans eat out 4 times a week.

      Holly’s KITCHEN 101 Cookbook to the Rescue for Easy Healthy Recipes!

      Who  has time to cook?  To the rescue- Holly’s KITCHEN 101. In this cookbook she highlights easy diabetic recipes with the ADA guidelines with a “D” for easy reference.  Best of all, you don’t have to change what you eat but just how you prepare the food.  Holly’s arthritis, cancer and KITCHEN 101 cookbooks all have a “D” to indicate diabetic living recipes. You won’t believe how amazing these easy diabetic recipes are!  The proof is in the tasting.

      KITCHEN 101 is a time saver cookbook and includes your favorite comfort food healthier! Rotisserie chicken is the secret in Quick Chicken Lasagna and turns lasagna into a quick recipe. This cookbook simplifies cooking and perfect for the busy person or new cook.

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

      diabetic meal plan

      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.

      Diabetic Enchiladas a favorite southwestern dish in the book!

      Holly created her books to guide families to embrace healthy cooking.  These recipes are for people with or without medical issues. Diabetic-friendly recipes are indicated with a “D” in KITCHEN 101.  Try sampling the easy healthy recipes from KITCHEN 101 on the healthy food blog. Make this recipe gluten-free with corn tortillas.  Have fun in the kitchen cooking healthy and remember there isn’t a magical diabetes diet.  It is the healthiest way to eat!  The best easy mainstream diabetic cookbook is KITCHEN 101 

      Get All Holly’s Healthy Easy Cookbooks

      The post Healthy Enchiladas Recipe-Easy Cookbook Highlights Diabetic Recipes appeared first on The Healthy Cooking Blog.



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      Potential Protection Against Type 2 Diabetes in Obesity Through Lower CD36 Expression and Improved Exocytosis in β-Cells

      By electricdiet / June 4, 2020


      Abstract

      Obesity is a risk factor for type 2 diabetes (T2D); however, not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from donors with T2D and non-T2D (ND), especially obese donors (BMI ≥30 kg/m2). Islets from obese donors with T2D had reduced insulin secretion, decreased β-cell exocytosis, and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis, and reduced granule docking. This was accompanied by reduced expression of the exocytotic proteins SNAP25, STXBP1, and VAMP2, likely because CD36 induced downregulation of the insulin receptor substrate (IRS) proteins, suppressed the insulin-signaling phosphatidylinositol 3-kinase/AKT pathway, and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human β-cell line EndoC-βH1 increased IRS1 and exocytotic protein levels, improved granule docking, and enhanced insulin secretion. Our results demonstrate that β-cells from obese donors with T2D have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit β-cell function in T2D associated with obesity.

      Introduction

      Hyperglycemia caused by insufficient insulin action characterizes type 2 diabetes (T2D). Insulin resistance and defective insulin secretion are the two major pathogenic factors of the disease, and both are strongly associated with lifestyle and genetic components (1,2). Obesity is one of the strong risk factors for the development of T2D. In obesity, lipid accumulation is common not only in adipose tissue but also in ectopic tissues such as the liver and skeletal muscle. The intracellular lipid accumulation in ectopic tissues leads to impaired insulin signaling and promotes systemic insulin resistance (3). However, not all obese individuals develop T2D because pancreatic β-cells can adjust, to a certain extent, for an increasing demand of insulin. Pancreatic β-cell dysfunction is central in the failure to adjust for the increased insulin resistance. Indeed, reduced first-phase insulin response can, at least in some individuals, be observed already before the development of T2D (4). These findings suggest that those who cannot adapt to the extra demand by increased insulin secretion are prone to T2D.

      Like insulin target tissues, the insulin-producing β-cells have been shown to be damaged by excessive lipid accumulation, a concept known as β-cell lipotoxicity (5). In this condition, accumulated lipids, specifically triacylglycerol, cause cellular stress, dysfunction, and death of the β-cell. In fact, increased accumulation of lipid droplets is observed with increased BMI in human β-cells (6). A number of in vitro studies have identified mechanisms involved in impaired insulin secretion by chronic fatty acid (FA) elevation (7,8), including those affecting exocytosis (9). Moreover, insulin signaling in β-cells is essential not only for growth but also for proper regulation of the cellular function (1012). Hence, together these findings indicate that insulin resistance and defective insulin secretion are likely to share common etiologies in terms of lipid accumulation. Both endogenous FA synthesis and FA uptake are considered causally important for increased lipid accumulation in β-cells (13,14).

      We show in this study that, among human donors with obesity, insulin secretion capacity of pancreatic islets and β-cell exocytosis were significantly lower in donors with T2D than in non-T2D (ND). We compared expression levels of the FA transporters in their islets to address the role of facilitated FA uptake for the defective insulin secretion. We further explored in detail the signaling pathway involved in CD36-modulated insulin secretion in β-cells using INS-1 cells carrying a Tet-on system for CD36 overexpression. Finally, we tested the therapeutic potential of a CD36-neutralizing antibody to improve β-cell function in human EndoC-βH1 cells.

      Research Design and Methods

      Cell Line and Culture

      INS-1 cells carrying the Tet-on system for CD36 overexpression (15) were cultured with RPMI 1640 medium containing 11.1 mmol/L glucose, 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 50 mg/mL neomycin, 50 mg/mL hygromycin, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, and 50 μmol/L β-mercaptoethanol at 37°C in a humidified atmosphere with 5% CO2. To induce CD36 expression, cells were seeded in 24- or 48-well plates and cultured with or without 500 ng/mL doxycycline (Sigma-Aldrich, St. Louis, MO) for 72 h.

      EndoC-βH1 cells (16) were cultured in Matrigel/fibronectin-coated (100 μg/mL and 2 μg/mL, respectively) (Sigma-Aldrich) vessels with DMEM containing 5.6 mmol/L glucose, 2% BSA, 10 mmol/L nicotinamide, 50 μmol/L β-mercaptoethanol, 5.5 μg/mL transferrin, 6.7 ng/mL sodium selenite, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells were seeded in Matrigel/fibronectin-coated 48-well plates and cultured for 72 h with 2 μg/mL of a CD36-blocking antibody (FA6.125, catalog number 60084; STEMCELL Technologies, Vancouver, British Columbia, Canada) or an isotype control (MOPC-21, catalog number ab18443; Abcam, Cambridge, U.K.).

      Human Pancreatic Islets

      Human pancreatic islets were obtained from cadaver donors of European ancestry by the Nordic Network for Clinical Islet Transplantation. The islets were processed as previously described (17) and handpicked under stereomicroscope before use. To dissociate into single cells, the islets were incubated with 3 mmol/L EGTA in Hanks’ balanced salt solution containing 0.25% BSA for 15 min at 37°C followed by gentle pipetting. Donor characteristics for each experiment are shown in Supplementary Table 1. All procedures using human islets were approved by the ethical committees in Uppsala and Malmö/Lund, Sweden.

      Human Islet RNA-Sequencing Data Analysis

      Islet RNA-sequencing (RNA-seq) data were retrieved from the Gene Expression Omnibus with accession numbers GSE50398 and GSE108072 from the studies of Fadista et al. (18) and Gandasi et al. (19), respectively. The expression values are expressed as log2 counts per million after normalization and transformation using voom. Expression levels of selected genes were retrieved from 68 normal-body-weight (BMI <25 kg/m2) and 31 obese (BMI ≥30 kg/m2) donors.

      Insulin Secretion Assay

      For CD36 INS-1 cells, after being washed twice with 1 mL prewarmed secretion assay buffer (SAB) (1.16 mmol/L MgSO4, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 114 mmol/L NaCl, 2.5 mmol/L CaCl2, 25.5 mmol/L NaHCO3, 20 mmol/L HEPES, and 0.2% BSA, pH 7.2) with 2.8 mmol/L glucose, cells were preincubated in 0.5 mL SAB with 2.8 mmol/L glucose for 1 h. The cells were then stimulated for 1 h in 0.25 mL SAB with 2.8 mmol/L glucose or 16.7 mmol/L glucose or for 15 min in 0.25 mL SAB with 2.8 mmol/L glucose and 50 mmol/L KCl at 37°C. Secreted insulin levels were measured using Rat Insulin ELISA (Mercodia, Uppsala, Sweden), and the values were normalized to the protein content. Protein was extracted with 100 μL RIPA buffer (0.1% SDS, 150 nmol/L NaCl, 1% Triton X-100, and 50 mmol/L Tris-HCl, pH 8.0), and the content was analyzed using a BCA Protein Assay Kit (Thermo Fisher Scientific).

      For EndoC-βH1 cells, culture medium was changed to a medium containing 2.8 mmol/L glucose and further incubated for 18 h before the secretion assay. After two washes with 1 mL SAB containing 1 mmol/L glucose, cells were preincubated in 0.5 mL SAB with 1 mmol/L glucose for 2 h. The cells were then stimulated for 15 min with 0.25 mL SAB with 1 mmol/L glucose or 20 mmol/L glucose at 37°C. Secreted insulin levels were measured using Human Insulin ELISA (Mercodia), and the values were normalized to the protein content. Protein content was measured as above.

      For human islets, glucose-stimulated insulin secretion was examined using a dynamic glucose perifusion system to calculate the stimulation index as a quality control (20). To evaluate membrane depolarization-induced insulin secretion, batches of 12 islets were preincubated for 30 min in Krebs-Ringer bicarbonate buffer, pH 7.4, supplemented with 20 mmol/L HEPES, 0.1% BSA, and 1 mmol/L glucose and then stimulated for 1 h in Krebs-Ringer bicarbonate buffer with 70 mmol/L KCl and 1 mmol/L glucose.

      Insulin-Stimulation Assay

      Insulin-stimulation assay was performed on the confluent cells after 72 h of seeding. After washing with prewarmed PBS twice, the cells were preconditioned with the FBS-free culture medium containing 1 mmol/L glucose for 6 h to diminish an autocrine effect of insulin on the signaling pathway. The cells were then stimulated for 10 min in the conditioning medium without (0) or with 1 or 10 nmol/L human insulin (Actrapid Penfill; Novo Nordisk, Bagsværd, Denmark). After discarding the medium, the cells were frozen immediately in liquid nitrogen and lysed with one-time loading buffer, followed by sonication. The cell lysates were stored at −80°C until Western blot analysis.

      Subcellular Fractionation

      Subcellular fractionation of CD36 INS-1 cells was performed using a Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific). Purity of each fraction was verified by Western blot analysis.

      Western Blot Analysis

      Protein samples (7.5–10 μg protein/sample) were applied to SDS-PAGE using 4–15% TGX Stain-Free gels (Bio-Rad Laboratories, Hercules, CA). The gels were then activated with ultraviolet light for 1 min to visualize total protein on the blotted low-fluorescence polyvinylidene difluoride membrane using the Stain-Free technology (Bio-Rad Laboratories). Blotting was performed using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membrane blocking and incubation with primary antibodies were performed with 5% skim milk in Tris-buffered saline with Tween, except for phosphorylated proteins with 3% BSA in Tris-buffered saline with Tween. Primary antibodies and their dilutors are provided in Supplementary Table 2. Incubation with the primary antibodies was performed overnight at 4°C. Horseradish peroxidase–conjugated anti-rabbit (catalog number 170–6515; 1:2,000; Bio-Rad Laboratories), anti-mouse (catalog number P0447, 1:2,000; Agilent Technologies, Santa Clara, CA), and anti-goat (catalog number ab6741; 1:1,000; Abcam) Igs were used as secondary antibodies. Incubation with the secondary antibodies was performed for 1 h at room temperature. Clarity Western ECL Substrate (Abcam) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used for visualization of proteins with a ChemiDoc XRS+ System (Bio-Rad Laboratories). The signal intensity of each protein band was measured using Image Lab Software (version 5.2.1; Bio-Rad Laboratories) and normalized to that of the total protein bands in the lane, except for the phosphorylated protein band, which was normalized to the corresponding total protein band.

      Electrophysiology

      Whole-cell patch-clamp experiments were conducted on single human β-cells (cell capacitance >9 pF) or CD36 INS-1 cells as described previously (21). The recordings were performed using an EPC 10 patch-clamp amplifier with Patchmaster software (ver. 2–73; HEKA Elektronik, Lambrecht, Germany). Exocytosis was evoked by a train of 10 500-ms depolarizations from −70 to 0 mV applied at 1 Hz. Voltage-dependent currents were investigated using a current-voltage protocol in which the membrane was depolarized from −70 mV to voltages between −40 and +40 mV during 50 ms. The sustained current (I), measured during the latter 20 ms of the depolarizations, was considered as Ca2+ current.

      Immunocytochemistry

      Dissociated islet cells or CD36 INS-1 cells were fixed and stained as described elsewhere (22). Primary antibodies for CD36 (JC63.1, catalog number ab23680, 1:200; Abcam), insulin (catalog number 03–16049; 1:400; American Research Products, Inc., Waltham, MA), Na+K+ ATPase (catalog number ab76020; 1:200; Abcam), and FoxO1 (catalog number 2880; 1:200; Cell Signaling Technology) diluted in blocking buffer were treated for 2 h. After washing twice with PBS, the cells were exposed to fluorescent-labeled corresponding secondary antibodies (1:400) for 1 h, followed by postfixation with 3% paraformaldehyde in PBS. Immunofluorescence was observed with a confocal microscope (Meta 510 or LSM 880; Carl Zeiss, Oberkochen, Germany). Nuclear localization of FoxO1 was evaluated by the nuclear fluorescence intensity normalized to the whole-cell intensity. The nuclear region was defined by DAPI staining. The fluorescence intensity was analyzed with ZEN software (Carl Zeiss).

      Flow Cytometry

      Dissociated islet cells were stained with allophycocyanin (APC)–conjugated anti-CD36 (catalog number 562744; BD Biosciences) for 30 min in PBS with 0.5% BSA and 0.05% sodium azide. The cells were then fixed with 3.7% paraformaldehyde in PBS for 20 min and further stained intracellularly with Alexa Fluor 488–conjugated anti-insulin (catalog number IC1417A; R&D Systems) and phycoerythrin-conjugated anti-glucagon (catalog number 565860; BD Biosciences) for 30 min in 0.1% saponin in PBS with 0.5% BSA and 0.05% sodium azide. After the cells were postfixed with 1% paraformaldehyde, flow cytometric analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) with CytExpert software (version 2.0; Beckman Coulter).

      RNA Extraction, RT-PCR, and Quantitative PCR

      Total RNA from CD36 INS-1 cells was extracted, and cDNA was generated as described (23). Quantitative PCR was performed in 384-well plates on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) using TaqMan Gene Expression Assays with Universal PCR Master Mix (Thermo Fisher Scientific). Rat Hprt1 (assay identification number: Rn01527840_m1; Applied Biosystems) and Ppia (assay identification number: Rn00690933_m1) were used for normalization. Relative expression was calculated using the ΔΔ threshold cycle method.

      Transmission Electron Microscopy

      CD36 INS-1 and EndoC-βH1 cells were prepared for electron microscopy, examined, and analyzed as previously described (24). Granules (large dense-core vesicles) were defined as docked when the center of the granule was located within 100 nm and 120 nm from the plasma membrane for CD36 INS-1 and EndoC-βH1, respectively. The distance between the center of the granule and the plasma membrane was calculated using an in-house software programmed in MATLAB 7 (The MathWorks, Inc., Natick, MA).

      Statistical Analysis

      Data are presented as mean ± SE. The differences between two groups were analyzed by Student t test with two-tailed analysis. All statistical analyses were performed using Prism (version 7.0; GraphPad Software, San Diego, CA).

      Data and Resource Availability

      The data sets generated and/or analyzed during the current study are available from the corresponding authors on reasonable request. The CD36-overexpressing INS-1 cell line may be available from Prof. Anneli Björklund (Karolinska Institute, Stockholm, Sweden) upon reasonable request and with permission of the founder, C.B.W.

      Results

      Reduced Islet Insulin Secretion and β-Cell Exocytosis in Donors With T2D and Obesity

      We investigated insulin secretion capacity of pancreatic islets in donors with T2D and ND with normal body weight (BMI <25 kg/m2) or obesity (BMI ≥30 kg/m2). Islets of donors with T2D in both BMI categories showed reduced glucose-stimulated insulin secretion as compared with those of ND (Fig. 1A and B). Basal insulin secretion was decreased only in islets of donors with T2D with normal body weight (Fig. 1A). Accordingly, decreased insulin-stimulation index in islets of T2D was confirmed only among donors with obesity (Supplementary Fig. 1). Static incubation with K+ also revealed significantly decreased insulin secretion in islets of T2D as compared with ND among donors with obesity, albeit a similar basal insulin secretion (Fig. 1C and D), suggesting defects downstream of the ATP-dependent K+ channel. Indeed, capacitance recordings on single β-cells evoked by a train of 10 500-ms depolarizations from −70 to 0 mV demonstrated reduced Ca2+-dependent exocytosis in β-cells of obese donors with T2D as compared with those of obese donors with ND (Fig. 1E).

      Figure 1
      Figure 1

      Characteristics of pancreatic islets from donors with ND and T2D with normal body weight and with obesity. A and B: Insulin-release curves in response to dynamic glucose perfusion. Islets from donors with ND (blue) and T2D (red) with normal body weight (A) (ND, n = 38 donors; T2D, n = 14 donors) and with obesity (B) (ND, n = 11 donors; T2D, n = 12 donors) were subjected to dynamic perifusion at low (1.67 mmol/L) and high glucose (20 mmol/L) solutions. High glucose was added from 48 to 90 min, whereas other fractions were exposed to low glucose (1.67 mmol/L). Insulin secretion in static incubation at 1 mmol/L glucose (C) and 1 mmol/L glucose with 70 mmol/L K+ for 1 h (D) in islets from donors with ND and T2D with normal body weight (ND, n = 4 donors; T2D, n = 5 donors) and obesity (ND, n = 4 donors; T2D, n = 5 donors). E: Total cell capacitance changes (ΣΔCm), reflecting exocytosis, evoked by a train of 10 500-ms depolarizations from −70 to 0 mV in β-cells from donors with ND (n = 9 cells from 5 donors) and T2D (n = 7 cells from 3 donors) with high BMI (BMI ≥29 kg/m2). All data were normalized to the cell size by the measured whole-cell capacitance (Cs). The P values were determined by Student two-tailed t test (unpaired). *P < 0.05; **P < 0.01. fF, femtofarad; hr, hour; pF, picofarad.

      Differential Islet CD36 Expression Between Donors With T2D and ND With Obesity

      We next compared gene expression levels of FA transporters (CD36 and SLC27 family) in RNA-seq data of human pancreatic islets deposited in the Gene Expression Omnibus (GSE50398 and GSE108072) to investigate the role of facilitated FA uptake for defective insulin secretion observed in the islets of donors with T2D and obesity (Fig. 2A and Supplementary Fig. 2). Differences were observed in CD36 and SLC27A3 expression between ND (HbA1c <6% [42 mmol/mol]) and impaired glucose tolerance (IGT)/T2D (HbA1c ≥6.0% [42 mmol/mol] or those previously diagnosed with T2D) among donors with obesity. Within the group of FA transporters, CD36 was the most abundantly expressed in human islets. Interestingly, CD36 expression was lower in islets from the obese donors with ND as compared with the other three groups. We further validated the RNA-seq findings by Western blot analysis of human islets. CD36 protein expression in islets from obese donors with T2D was 70% higher than that in islets from obese donors with ND (Fig. 2B and C). We also confirmed CD36 protein expression in human β-cells using confocal microscopy (Fig. 2D). A semiquantitative analysis of the confocal images suggests that CD36 is expressed equally in the cytosol and on the plasma membrane (Supplementary Fig. 3). A flow cytometry analysis further revealed that CD36 is localized on the outer surface of the plasma membrane (Fig. 2E and F).

      Figure 2
      Figure 2

      Expression of CD36 in human pancreatic islets. A: CD36 mRNA levels in islets from donors with ND (HbA1c <6.0%) and IGT/T2D (HbA1c ≥6.0%) with normal body weight (BMI <25 kg/m2; ND, n = 39 donors; IGT/T2D, n = 29 donors) and with obesity (BMI ≥30 kg/m2; ND, n = 12 donors; IGT/T2D, n = 19 donors). The box extends from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. The whiskers go down to the smallest value and up to the largest. CD36 protein levels (B) and the Western blotting bands (C) in islets from donors with ND and T2D with normal body weight (ND, n = 4 donors; T2D, n = 5 donors) and with obesity (ND, n = 4 donors; T2D, n = 5 donors). The total protein images obtained using the Stain-Free technology are shown as a loading control (Loading). D: Immunocytochemical images of CD36 (green) expression in insulin-positive (red) islet cells from two different human donors. Scale bars, 5 µm. E and F: Flow cytometric analysis of human islet cells. Pancreatic islets were dissociated into a single cell suspension and extracellularly stained for CD36 (APC) followed by intracellular staining for insulin (Alexa 488) and glucagon (phycoerythrin [PE]). Insulin-positive glucagon-negative cells within a green square were defined as β-cells (E), and the β-cell surface CD36 (APC) expression was analyzed with anti-CD36 antibody (purple) and isotype control (gray) (F). The P values were determined by Student two-tailed t test (unpaired). *P < 0.05. CPM, counts per million.

      CD36 Impairs Exocytosis by Inhibiting Granule Docking in β-Cells

      To evaluate the mechanisms by which CD36 effects insulin secretion, we investigated insulin secretion in INS-1 cells carrying a Tet-on system for CD36 overexpression (25). Doxycycline induced CD36 expression dose dependently (Supplementary Fig. 4A), and the induced CD36 protein was located on the plasma membrane (Fig. 3A). We observed reduced glucose-stimulated insulin secretion (∼40%) in INS-1 cells at 72 h after the induction of CD36 compared with control (Fig. 3B). Likewise, membrane depolarization by K+ showed reduced insulin secretion by ∼30% in CD36-overexpressing cells (Fig. 3C). CD36 overexpression did not change cellular insulin content (Supplementary Fig. 4B). Capacitance measurements on single cells using the patch-clamp technique (Fig. 3D) revealed that exocytosis, evoked by a train of 10 depolarizations (Σall), was reduced by ∼25% in CD36-overexpressing cells, which did not reach significance due to endocytosis evoked by the latter depolarizations (Fig. 3E). The reduction in membrane capacitance increase was more prominent (and reached significance) during the first two depolarizations (Σ1–2), reflecting rapid exocytosis of docked and primed granules (Fig. 3E). The reduced exocytosis was not a cause of changes in the inward Ca2+ current (Fig. 3F).

      Figure 3
      Figure 3

      Insulin secretion and exocytosis in CD36-overexpressing INS-1 cells. A: Immunocytochemical images of CD36 (green) in INS-1 cells without (DOX−) or with doxycycline-induced CD36 overexpression (DOX+). Na+-K+ ATPase (orange) was stained for a plasma membrane marker. Scale bars, 5 µm. Insulin secretion at 2.8 mmol/L or 16.7 mmol/L glucose for 1 h (B) and at 2.8 mmol/L glucose with 50 mmol/L K+ for 15 min (C). N = 5 for each group. Representative cell capacitance changes evoked by a train of 10 500-ms depolarizations from −70 to 0 mV (D) and the sum of capacitance increase (ΔCm) in response to the depolarization pulses of the train (E). N = 13 to 14 cells for each group. F: Inward Ca2+ currents evoked by depolarizations. Current (I)–voltage (V) relationship for the peak Ca2+ currents. N = 15 to 16 cells for each group. The P values were determined by repeated-measures one-way ANOVA followed by Tukey honest significant difference (B) and Student two-tailed t test (paired for C and unpaired for E). *P < 0.05, **P < 0.01 vs. DOX− under the same condition; ##P < 0.01 vs. 2.8 mmol/L glucose for each group (DOX− or DOX+). fF, femtofarad; hr, hour; pA, picoampere.

      We next performed transmission electron microscopy (Fig. 4A) to estimate the granule volume density per cell (Nv) and the surface density per cell (Ns), which are proportional to the total number of granules and the number of docked granules, respectively (24). While the total number of granules within the cells was not affected by CD36 overexpression (Fig. 4B), the number of granules within 100 nm from the plasma membrane, regarded as the docked granule pool, was significantly reduced (Fig. 4C and D). We found expression of exocytotic genes Snap25 and Vamp2 to be downregulated by CD36 overexpression (Fig. 4E). Moreover, the downregulation of Stxbp1 was almost significant (P = 0.057). CD36 overexpression significantly reduced STXBP1 and SNAP25 protein levels (Fig. 4F).

      Figure 4
      Figure 4

      Insulin granule localization and exocytotic protein levels in CD36-overexpressing INS-1 cells. A: Electron micrographs of INS-1 cells without (DOX−) or with doxycycline-induced CD36 overexpression (DOX+). Original magnification ×6,000 (left) and ×43,000 (right). BD: Granule-distribution analyses on the electron micrographs. Nv (B) and Ns (C) were measured as volume density and surface density, respectively. The granules for which the center was located within 100 nm (a half-length of the mean granule diameter) from the plasma membrane were defined as docked. Relative distribution of granules at shown distance intervals from the center of granule to the plasma membrane is shown in D. The distance shown in the x-axis is the upper border of each fraction. N = 34–38 cells for each group. E: mRNA expression levels of exocytotic genes. The expression levels were normalized to Ppia and Hprt. N = 6 for each group. F: Western blot analysis of exocytotic proteins. The total protein images obtained using the Stain-Free technology are shown as a loading control (Loading). N = 4 for each group. The P values were determined by Student two-tailed t test (unpaired). *P < 0.05; **P < 0.01; ***P < 0.001.

      CD36 Suppresses the Insulin Receptor Substrate/Phosphatidylinositol 3-Kinase/AKT Signaling Pathway

      Several reports suggest that exocytotic gene and protein expression in β-cells is tightly regulated by the insulin-signaling phosphatidylinositol 3-kinase (PI3K)/AKT pathway and its downstream target transcription factors (e.g., FoxO1) (26,27). We therefore examined the insulin-signaling PI3K/AKT pathway in CD36-overexpressing cells. While there were no changes in insulin receptor β protein levels (Fig. 5A), downstream insulin receptor substrate (IRS) 1 and IRS2 protein levels were reduced in CD36-overexpressing cells (Fig. 5B). Next, we found reduced phosphorylation at Ser473 of AKT (Supplementary Fig. 4C), a key regulatory molecule of the insulin-signaling pathway, indicating AKT inactivation. The AKT phosphorylation stimulated by exogenous insulin was also blunted by CD36 overexpression (Fig. 5C). This will result in retention of the transcription factor FoxO1 in the nucleus (28,29). FoxO1 is known as a potent transcriptional repressor of several exocytotic genes, including Snap25 (26). Indeed, CD36 overexpression led to a significant increase in nuclear localization of FoxO1 (Fig. 5D and E).

      Figure 5
      Figure 5

      Analysis of insulin-signaling cascade in CD36-overexpressing INS-1 cells. Protein levels of insulin receptor β subunit (IRβ) (A) and IRS1 and IRS2 (B) in INS-1 cells without (DOX−) or with doxycycline-induced CD36 overexpression (DOX+). Normalization factor was used to adjust the target band intensity. N = 4 for each group. C: Phosphorylation levels of AKT Ser473 (pSer473) under a short-term (10-min) stimulation of insulin with various doses (1 and 10 nmol/L). Phosphorylated protein signals were normalized to the total protein levels. N = 4 for each group. D: Immunocytochemical analysis of FoxO1 (red) localization. Nuclei were stained with DAPI (blue). The bar graph shows nuclear FoxO1 fluorescence intensity normalized to the whole-cell intensity. Scale bars, 5 µm. N = 21 cells for each group. E: Western blot analysis of cytoplasmic (Cyt) and nuclear (Nuc) protein levels of FoxO1. N = 4 for each group. The total protein images obtained using the Stain-Free technology are shown as a loading control (Loading). The P values were determined by Student two-tailed t test (unpaired). *P < 0.05, ***P < 0.001 vs. DOX− under the same condition; #P < 0.05, ##P < 0.01 vs. cells without the stimulation of insulin (0 nmol/L) for each group (DOX− or DOX+).

      CD36 Downregulates IRS1 Through Activation of Novel Protein Kinase C

      To address the mechanism underlying the IRS downregulation in CD36-overexpressing cells, we evaluated Irs1 and Irs2 gene expression. In accordance with a previous report that FoxO1 in the nucleus can directly suppress Ins2 gene expression by binding the Irs2 promoter (30), Irs2 gene expression was downregulated in CD36-overexpressing cells (Fig. 6A). However, Irs1 transcript was not altered by CD36 overexpression in contrast to the reduced protein level (Figs. 5B and 6A), suggesting that CD36 overexpression downregulates IRS1 protein in a different way from IRS2. Because intracellular lipid accumulation has been suggested to promote IRS1 protein degradation through the activation of IκB kinase or protein kinase C (PKC) (31,32), we investigated phosphorylation of IRS1 at Ser307 and Ser612, phosphorylation targets of IκB kinase and PKC, respectively (33,34). In CD36-overexpressing cells, phosphorylation at Ser612 was increased, while no difference was observed at Ser307 as compared with the control cells (Fig. 6B), suggesting that CD36-induced IRS1 degradation is likely dependent on PKC activation. Among various PKC isoforms, two novel subtype PKCs (nPKCs; i.e., PKCδ and PKCε) are postulated to have negative impacts on β-cell function (3537). We therefore evaluated membrane translocation (and activation) of PKCδ and PKCε from the cytosol to the plasma membrane. CD36 overexpression caused a twofold increase in membrane-associated PKCε protein level (Fig. 6C and D).

      Figure 6
      Figure 6

      Analysis of background mechanism underlying IRS downregulation in CD36-overexpressing INS-1 cells. A: mRNA expression levels of Irs1 and Irs2. The expression levels were normalized to Ppia and Hprt. N = 6 for each group. B: Phosphorylation levels of IRS1 Ser307 (pSer307) and Ser612 (pSer612). Phosphorylated protein levels were normalized to the total protein levels. N = 4 for each group. Western blot analysis of cytoplasmic (Cyt) and membrane (Mem) protein levels of PKCδ (C) and PKCε (D). N = 3 for each group. The total protein images obtained using the Stain-Free technology are shown as a loading control (Loading). DOX− and DOX+ represent INS-1 cells without and with doxycycline-induced CD36 overexpression, respectively. The P values were determined by Student two-tailed t test (unpaired). *P < 0.05; **P < 0.01. N.D., not determined.

      Blockade of CD36 Function Improves Granule Docking and First-Phase Insulin Secretion

      Finally, we investigated if blockade of CD36 function can ameliorate insulin secretion and exocytosis in β-cells. For this, we used EndoC-βH1 cells, a clonal human β-cell line (16), expressing CD36 protein (Supplementary Fig. 5). Instead of a chemical CD36 inhibitor sulfo-N-succinimidyl oleate, which blunts β-cell function after 72 h of coincubation (Supplementary Fig. 6), we treated the cells with a CD36-neutralizing IgG antibody (FA6.125) reported to inhibit the known functions of CD36, including FA uptake (38). Treatment with the antibody significantly increased first-phase (15-min) insulin secretion at 20 mmol/L glucose (Fig. 7A). Moreover, the antibody treatment significantly increased protein levels of STX1A, SNAP25, VAMP2, and STXBP1 (Fig. 7B) and resulted in an increased number of docked granules (Fig. 7CE). Detailed analysis revealed that the increase in Nv is mainly due to an enlargement of the granule pool closer to the plasma membrane (Fig. 7F), including the docked granules (Ns) and the pool regarded as the “almost” docked granules (39). In parallel, increased IRS1/2 protein levels and downstream AKT activation were observed under the blockade of CD36 function (Fig. 7G and H).

      Figure 7
      Figure 7

      Effects of anti-CD36–neutralizing antibody on EndoC-βH1 cell functions. A: Insulin secretion at 20 mmol/L glucose for 15 min (fold change, 20 mmol/L glucose vs. 1 mmol/L glucose). N = 4 for each group. B: Exocytotic protein levels. Normalization factor (the total reference lane protein/total target lane protein) is the correction factor used to calculate the adjusted volume of target band. N = 4 for each group. CF: Granule-distribution analyses on the electron micrographs of EndoC-βH1 cells. Original magnification ×43,000 (C). Nv (D) and Ns (E) were measured as volume density and surface density, respectively. The granules for which the center was located within 120 nm from the plasma membrane (a half-length of the mean granule diameter) were defined as docked. Relative distribution of granules at shown distance intervals from the center of granule to the plasma membrane is shown in F. The distance shown in the x-axis is the upper border of each fraction. N = 20 cells for each group. G: IRS1 and IRS2 protein levels. N = 4 for each group. H: Phosphorylation levels of AKT Ser473 (pSer473). Phosphorylated protein signals were normalized to the total protein levels. N = 4 for each group. The total protein images obtained using the Stain-Free technology are shown as a loading control (Loading). The P values were determined by Student two-tailed t test (unpaired). *P < 0.05; **P < 0.01; ***P < 0.001.

      Discussion

      We present in this study novel human islet data and delineate the signaling pathway in CD36-dependent effects on insulin secretion. Hence, we extend previous findings of CD36 on insulin secretion (14,25). Our data show not only differential glucose-stimulated insulin secretion in islets but also a difference in β-cell exocytosis between donors with T2D and ND with obesity, suggesting that the process of exocytosis is involved in the regulation of insulin secretion capacity to compensate for insulin resistance in obesity. Indeed, CD36, which is differentially expressed in islets between donors with T2D and ND with obesity, impairs insulin secretion through the reduction of exocytotic proteins crucial for granule docking and priming in β-cells. This occurs through the downregulation of IRS1/2 and suppression of the insulin-signaling PI3K/AKT pathway and following nuclear retention of FoxO1.

      One of the main functions of CD36 is a facilitated influx of long-chain FAs (LCFAs) across the plasma membrane (40). LCFA-induced β-cell dysfunction is well established (41,42), and long-term palmitate exposure of islets impairs exocytosis by dissociating Ca2+ channels from secretory granules (9). In contrast, CD36 overexpression inhibited exocytosis through the reduction of exocytotic protein levels and subsequent impairment of granule docking. Hence, defective exocytosis caused by CD36 is most likely a consequence of a different cellular mechanism than previously described as β-cell lipotoxicity. CD36 actually can bind not only LCFAs but also other ligands including phospholipids, ceramide, and lipoproteins (40). Considering the finding that CD36 overexpression could activate diacylglycerol (DAG)–dependent nPKCs (PKCε), FAs or other glycerolipids used for DAG synthesis are possible candidates to initiate the cellular cascade resulting in reduced exocytotic protein levels through CD36. Several studies have demonstrated the role of Fox family transcription factors on the regulation of exocytosis-related gene expression in β-cells (26,43). As FoxO1 is an established target of the insulin-signaling PI3K/AKT pathway, inhibition of the pathway actually reduces exocytotic gene and protein expression, as shown in previous studies (26,27,44). Not only FoxO1 but also other unknown transcription factors downstream of the PI3K/AKT pathway have been suggested to regulate exocytotic gene expression (e.g., constitutively active AKT [GagAkt] increased the expression of multiple exocytotic genes) (26). Furthermore, CD36-mediated LCFA influx may induce intracellular ceramide accumulation by its de novo synthesis, which has been reported to cause AKT inactivation in β-cells (45). It has also been suggested that extracellular ceramide-induced AKT inactivation is dependent on a CD36-initiated inflammatory signaling cascade in β-cells (46). These previous findings support the model in which CD36 participates in exocytotic protein reduction through the suppression of the insulin-signaling PI3K/AKT pathway and its downstream transcription factors (Fig. 8).

      Figure 8
      Figure 8

      Model describing the possible involvement of CD36 in β-cell granule docking and exocytosis. CD36-mediated facilitation of lipid influx (including FAs) increases intracellular DAG, which leads to the translocation of nPKCs to the plasma membrane (activation of nPKCs). The activated nPKCs elicit abnormal phosphorylation of IRS1 and consequently attenuate PI3K/AKT signaling. The following retention of FoxO1 in the nucleus results in the transcriptional inhibition of Irs2 and exocytotic genes, which in turn impairs granule docking and priming with impaired exocytosis of insulin granules and reduced insulin secretion in β-cells as a consequence. β-Ox, β-oxidation; InsR, insulin receptor; TAG, triacylglycerol.

      Are our findings relevant for human T2D? We and others have earlier shown that reduced expression of exocytotic genes in islets occurs in T2D (17,19,47). In addition, restoration of exocytotic proteins in human β-cells could improve granule docking and exocytosis (19). These data are in line with possible in vivo effects of CD36 on first-phase insulin secretion, because it has been suggested that the reduced first-phase insulin secretion in T2D is due to the reduced pool of docked and primed insulin granules (48). Considering the overall roles of CD36 for insulin secretion as shown in the current study, obese individuals who have higher CD36 expression in β-cells may have a lower insulin secretion capacity due to exocytotic defects and consequently might be more prone to develop T2D. Such speculation could be partly supported by the finding that CD36 expression in β-cells has been upregulated prior to the incidence of T2D, as there was no difference in CD36 gene expression between IGT and T2D islets of obese donors in the RNA-seq study (Supplementary Fig. 7). In contrast, lower CD36 expression in β-cells might be advantageous to avoid T2D. The finding that obese donors with lower CD36 expression in islets seem to be protected against T2D encouraged us to test the therapeutic potential of CD36 functional blockade to improve β-cell function, especially exocytosis. The EndoC-βH1 cell line closely resembles human β-cells, especially in stimulus-secretion coupling (49). The cell line is also regarded as an appropriate screening platform for novel drug candidates for diabetes (50). We could detect CD36 protein in EndoC-βH1 cells, and the blockade of CD36 function on the cells led to increased exocytotic protein levels, improved granule docking, and enhanced first-phase insulin secretion. We believe the blockage of CD36 function could be an attractive approach to improve insulin secretion, especially in obesity-related T2D. A further study to test the CD36-neutralizing antibody on human islets of obese donors with T2D is warranted as the next step.

      The importance of insulin signaling for β-cell function, especially in early phase insulin secretion, was previously demonstrated in β-cell–specific insulin receptor–deficient mice (51). However, a key molecule responsible for modulating β-cell insulin signaling in T2D has not been investigated previously. Intriguingly, the proposed mechanism to suppress β-cell insulin signaling by CD36 is considered similar to other insulin target organs (15,52), but the outcomes are unique to β-cells (i.e., defective exocytosis and reduced first-phase insulin secretion). CD36, therefore, may be a common denominator linking the two major etiologies of T2D (i.e., insulin resistance and defective insulin secretion). Taken together, our findings further highlight the pathogenic role of β-cell CD36 in the development of T2D, especially in relation to obesity.

      Article Information

      Acknowledgments. The authors thank Anna-Maria Veljanovska Ramsay and Britt-Marie Nilsson (both from Lund University Diabetes Centre, Malmö, Sweden) as well as Momoyo Kawahara and Miyuki Takatori (both from Nippon Medical School, Tokyo, Japan) for technical assistance. The authors also thank Anneli Björklund (Karolinska Institute, Stockholm, Sweden) for providing the CD36-overexpressing INS-1 cell line and EXODIAB and the Nordic Network for Clinical Islet Transplantation for providing human islets and human islet data.

      Funding. This study is financially supported by the Swedish Foundation for Strategic Research (IRC15-0067 to Lund University Diabetes Centre-Industrial Research Centre), Swedish Research Council (2009-1039 to EXODIAB; 349-2006-237 to Lund University Diabetes Centre; and project grants 2016-02124 and 2019-01406 to L.E.), Japan Society for the Promotion of Science (to M.N., J.L.S.E., and A.A.), European Foundation for the Study of Diabetes and Japan Diabetes Society (to M.N.), Insamlingsstiftelsen Diabetes Wellness Network Sverige (720-2964 JDWG to M.N.), Uehara Memorial Foundation (to M.N.), Scandinavia-Japan Sasakawa Foundation (to M.N.), Sumitomo Life Welfare Foundation (to M.N.), Nippon Medical School Alumni Association (to M.N.), Lotte Shigemitsu Prize (to A.A.), Albert Påhlsson Foundation (to J.L.S.E. and L.E.), Region Skåne-regional grant (ALF) (to L.E.), Novo Nordisk Foundation (to L.E.), and Swedish Diabetes Foundation (DIA2016-130 to L.E.).

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

      Author Contributions. L.E. supervised the project. M.N. and L.E. designed experiments, conducted data analysis, and wrote the manuscript. M.N., A.A., J.K.O., A.E., and A.W. performed experiments. J.L.S.E. analyzed RNA-seq data. H.S., C.B.W., and S.O. contributed with discussion and edited the manuscript. L.E. and M.N. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

      • Received September 19, 2019.
      • Accepted March 9, 2020.



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      Diabetic Foodie: What’s Next? – Diabetic Foodie

      By electricdiet / June 2, 2020


      Change is good. At least that’s what I keep telling myself.

      Last fall, I attended the DiabetesSisters PODS Leadership conference. Shawn Shepheard of The Leadership Advantage led a session about creating our vision for the future. We each wrote down our biggest opportunities and what challenges might keep them from becoming reality. It was the kind of quality time you rarely get the luxury of spending on yourself. The experience forced me to think long and hard about Diabetic Foodie’s future.

      After the conference, I reached out to Shawn and asked if he could help me work on a 2020 business plan for Diabetic Foodie. I felt it needed some new energy. He agreed and we had a call that was life-changing.

      The best time for new beginnings is now

      Discovering My Unique Abilities

      Shawn suggested I spend some time thinking about the things I’m passionate about that I love to do. He said people who are “successful” and “happy” spend more than 80% of their time using these unique abilities. Through a series of exercises, I determined I was at about 17%. Yikes.

      Next, I tried to figure out how I could mold Diabetic Foodie into something that allowed me to use my unique abilities most of the time. To make that work, I would need to hire a bunch of people to do the things I didn’t want to do, and I would have to manage them all. Management is something I’m fairly good at, but have no passion for. It’s not one of my unique abilities.

      I taught a couple of classes at my local Lifelong Learning Institute and really enjoyed it. Around the same time, I had a conversation with my friend from DiabetesSisters, Christel Oerum of Diabetes Strong, about my work with Shawn. Christel mentioned she was interested in starting a food blog.

      What’s Next for Shelby?

      By January I was making plans to offer my first in-person cooking class. I had located a commercial kitchen to use and scheduled an Instant Pot class for March 21. Nine people signed up. My lifelong best friend passed away on March 7 and her memorial service was planned for March 21, so I rescheduled my cooking class for May 16. Almost immediately stay-at-home orders were issued for my state (Virginia) through June 10. Both the memorial service and the class were postponed indefinitely.

      In business-speak, I pivoted. I offered my Instant Pot class online via Zoom. Cameras, lighting, and the configuration of my kitchen posed challenges, but it was kind of fun trying to figure it all out. And, thank goodness, my audience was very forgiving!

      As staying home became the “new normal,” I started hearing from people who lived alone. Their social outlet, eating in restaurants with friends and family, had been taken away. They were looking for connection. I realized that while we couldn’t share food together, we could share a food experience. We could cook together online and have fun conversation along the way. Cook & Chat with Shelby was born.

      Sometimes you just have to let go and see what happens

      My ultimate dream is to combine cooking classes with travel experiences, but the pandemic won’t allow that to happen for a while. The good news is that I have plenty of time to work out the details!

      If you want to keep up with what I’ve got cooking (so to speak), please reach out via my website (currently a work in progress) or on Instagram or Twitter. I’d also love it if you’d sign up for the Cook & Chat with Shelby mailing list.

      I want to Cook and Chat

       

      What’s Next for Diabetic Foodie?

      What does this mean for Diabetic Foodie? Remember that conversation I had with Christel? We both realized that Diabetes Strong taking over the reins of Diabetic Foodie made perfect sense for both of us. To that end, as of June 1, 2020, I’ll be leaving Diabetic Foodie in the oh-so-capable hands of the team at Diabetes Strong. You can expect great things in the future.

      Please welcome the new owners of Diabetic Foodie and show them all of the love you’ve thrown my way over the last 10 years.

      I’m so grateful for all of you and everything you have taught me. Until we meet again…
      Shelby





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      Hearts of Palm Tater Tots

      By electricdiet / May 31, 2020


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

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

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

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

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

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

      Print

      Hearts of Palm Tater Tots

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


      Scale

      Ingredients

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

      Instructions

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

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

      Notes

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

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

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





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      Shredded Chicken Salad (Instant Pot)

      By electricdiet / May 29, 2020


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

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

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

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

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

      How to make shredded chicken salad

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

      Seasoned chicken breast in the instant pot

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

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

      Close-up of instant pot settings

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

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

      Shredded chicken in the instant pot

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

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

      Finished salad served on a white plate

      Adjusting the carb count

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

      Why I use an Instant pot

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

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

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

      Tip for making the perfect salad

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

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

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

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

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

      More healthy salad recipes

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

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

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

      Recipe Card

      Shredded Chicken Salad (Instant Pot)

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

      Prep Time:15 minutes

      Cook Time:25 minutes

      Total Time:40 minutes

      Servings:4

      Shredded Chicken Salad

      Instructions

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

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

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

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

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

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

      Nutrition Info Per Serving

      Nutrition Facts

      Shredded Chicken Salad (Instant Pot)

      Amount Per Serving

      Calories 330 Calories from Fat 123

      % Daily Value*

      Fat 13.7g21%

      Saturated Fat 1.5g8%

      Trans Fat 0g

      Polyunsaturated Fat 3.5g

      Monounsaturated Fat 6.6g

      Cholesterol 86.7mg29%

      Sodium 192.9mg8%

      Potassium 832.7mg24%

      Carbohydrates 15.6g5%

      Fiber 5.4g22%

      Sugar 4.1g5%

      Protein 37.4g75%

      Net carbs 10.2g

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

      Course: Salad

      Cuisine: American

      Keyword: instant pot salad, low-carb salad



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