GHRH agonist MR-409 protects ß-cells from streptozotocin-induced diabetes.

Patients with type 1 diabetes (T1D) suffer from insufficient functional ß-cell mass, which results from infiltration of inflammatory cells and cytokine-mediated ß-cell death. Previous studies demonstrated the beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R), such as MR-409 on preconditioning of islets in a transplantation model. However, the therapeutic potential and protective mechanisms of GHRH-R agonists on models of T1D diabetes have not been explored. Using in vitro and in vivo models of T1D, we assessed the protective propertie of the GHRH agonist, MR409 on ß-cells. The treatment of insulinoma cell lines and rodent and human islets with MR-409 induces Akt signaling by induction of insulin receptor substrate 2 (IRS2), a master regulator of survival and growth in ß-cells, in a PKA-dependent manner. The increase in cAMP/PKA/CREB/IRS2 axis by MR409 was associated with decrease in ß-cell death and improved insulin secretory function in mouse and human islets exposed to proinflammatory cytokines. The assessment of the effects of GHRH agonist MR-409 in a model of T1D induced by low-dose streptozotocin showed that mice treated with MR-409 exhibited better glucose homeostasis, higher insulin levels, and preservation of ß-cell mass. Increased IRS2 expression in ß-cells in the group treated with MR-409 corroborated the in vitro data and provided evidence for the underlying mechanism responsible for beneficial effects of MR-409 in vivo. Collectively, our data show that MR-409 is a novel therapeutic agent for the prevention and treatment of ß-cells death in T1D.

Hyperglucagonaemia in diabetes: altered amino acid metabolism triggers mTORC1 activation, which drives glucagon production.

AIM/HYPOTHESIS: Hyperglycaemia is associated with alpha cell dysfunction, leading to dysregulated glucagon secretion in type 1 and type 2 diabetes; however, the mechanisms involved are still elusive. The nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) plays a major role in the maintenance of alpha cell mass and function. We studied the regulation of alpha cell mTORC1 by nutrients and its role in the development of hyperglucagonaemia in diabetes. METHODS: Alpha cell mTORC1 activity was assessed by immunostaining for phosphorylation of its downstream target, the ribosomal protein S6, and glucagon, followed by confocal microscopy on pancreatic sections and flow cytometry on dispersed human and mouse islets and the alpha cell line, αTC1-6. Metabolomics and metabolic flux were studied by 13C glucose labelling in 2.8 or 16.7 mmol/l glucose followed by LC-MS analysis. To study the role of mTORC1 in mediating hyperglucagonaemia in diabetes, we generated an inducible alpha cell-specific Rptor knockout in the Akita mouse model of diabetes and tested the effects on glucose tolerance by IPGTT and on glucagon secretion. RESULTS: mTORC1 activity was increased in alpha cells from diabetic Akita mice in parallel to the development of hyperglycaemia and hyperglucagonaemia (two- to eightfold increase). Acute exposure of mouse and human islets to amino acids stimulated alpha cell mTORC1 (3.5-fold increase), whereas high glucose concentrations inhibited mTORC1 (1.4-fold decrease). The mTORC1 response to glucose was abolished in human and mouse diabetic alpha cells following prolonged islet exposure to high glucose levels, resulting in sustained activation of mTORC1, along with increased glucagon secretion. Metabolomics and metabolic flux analysis showed that exposure to high glucose levels enhanced glycolysis, glucose oxidation and the synthesis of glucose-derived amino acids. In addition, chronic exposure to high glucose levels increased the expression of Slc7a2 and Slc38a4, which encode amino acid transporters, as well as the levels of branched-chain amino acids and methionine cycle metabolites (~1.3-fold increase for both). Finally, conditional Rptor knockout in alpha cells from adult diabetic mice inhibited mTORC1, thereby inhibiting glucagon secretion (~sixfold decrease) and improving diabetes, despite persistent insulin deficiency. CONCLUSIONS/INTERPRETATION: Alpha cell exposure to hyperglycaemia enhances amino acid synthesis and transport, resulting in sustained activation of mTORC1, thereby increasing glucagon secretion. mTORC1 therefore plays a major role in mediating alpha cell dysfunction in diabetes. DATA AVAILABILITY: All sequencing data are available from the Gene Expression Omnibus (GEO) repository (accession no. GSE154126; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154126 ).

Trimethylguanosine synthase 1 is a novel regulator of pancreatic beta-cell mass and function.

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in ß-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in ß-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human ß-cells from type 2 diabetes donors. Using mice with conditional (ßTGS1KO) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determined that TGS1 regulates ß-cell mass and function. Using unbiased approaches, we identified a link between TGS1 and endoplasmic reticulum stress and cell cycle arrest, as well as and how TGS1 regulates ß-cell apoptosis. We also found that deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, in addition to promoting several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating ß-cell mass and function. We propose that these observations can be used as a stepping-stone for the design of novel strategies focused on TGS1 as a therapeutic target for the treatment of diabetes.

Connecting From Afar: Implementation of Remote Data-Sharing for Patients With Type 1 Diabetes on Insulin Pump Therapy.

Quality Improvement Success Stories are published by the American Diabetes Association in collaboration with the American College of Physicians and the National Diabetes Education Program. This series is intended to highlight best practices and strategies from programs and clinics that have successfully improved the quality of care for people with diabetes or related conditions. Each article in the series is reviewed and follows a standard format developed by the editors of Clinical Diabetes. The following article describes an effort to improve the remote collection of insulin pump data in an academic center in South Florida.

Unbiased Phosphoproteome Mining Reveals New Functional Sites of Metabolite-Derived PTMs Involved in MASLD Development.

Post-translational modifications (PTMs) of proteins are paramount in health and disease. Phosphoproteome analysis by enrichment techniques is becoming increasingly attractive for biomedical research. Recent findings show co-enrichment of other phosphate-containing biologically relevant PTMs, but these results were obtained by closed searches focused on the modifications sought. Open searches are a breakthrough in high-throughput PTM analysis (OS-PTM), identifying practically all PTMs detectable by mass spectrometry, even unknown ones, with their modified sites, in a hypothesis-free and deep manner. Here we reanalyze liver phosphoproteome by OS-PTM, demonstrating its extremely complex nature. We found extensive Lys glycerophosphorylations (pgK), as well as modification with glycerylphosphorylethanolamine on Glu (gpetE) and flavin mononucleotide on His (fmnH). The functionality of these metabolite-derived PTMs is demonstrated during metabolic dysfunction-associated steatotic liver disease (MASLD) development in mice. MASLD elicits specific alterations in pgK, epgE and fmnH in the liver, mainly on glycolytic enzymes and mitochondrial proteins, suggesting an increase in glycolysis and mitochondrial ATP production from the early insulin-resistant stages. Thus, we show new possible mechanisms based on metabolite-derived PTMs leading to intrahepatic lipid accumulation during MASLD development and reinforce phosphoproteome enrichment as a valuable tool with which to study the functional implications of a variety of low-abundant phosphate-containing PTMs in cell physiology.

Novel roles of mTORC2 in regulation of insulin secretion by actin filament remodeling.

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in ß-cells has been extensively studied, less is known about mTORC2's function in ß-cells. Here, we show that mice with constitutive and inducible ß-cell-specific deletion of RICTOR (ßRicKO and ißRicKO mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in ßRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in ßRicKO islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.NEW & NOTEWORTHY The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling.

Glucose stimulates microRNA-199 expression in murine pancreatic ß-cells.

MicroRNA 199 (miR-199) negatively impacts pancreatic ß-cell function and its expression is highly increased in islets from diabetic mice as well as in plasma of diabetic patients. Here we investigated how miR-199 expression is regulated in ß-cells by assessing expression of miR-199 precursors (primiR-199a1, primiR-199a2, and primiR-199b) and mature miR-199 (miR-199-3p and miR-199-5p) and promoter transcriptional activity assays in mouse islets and mouse insulinoma cells (MIN6) under different stimuli. We found that mouse islets equally express miR-199-3p and miR-199-5p. However, the primiRNA expression levels differed; although primiR-199a1 expression was about 30% greater than that of primiR-199a2, primiR-199b is barely detected in islets. We observed a 2-fold increase in primiR-199a1 and primiR-199a2 mRNA levels in mouse islets cultured in 10 mm glucose compared with 5.5 mm glucose. Similar responses to glucose were observed in MIN6 cells. Exposure to 30 mm KCl to induce membrane depolarization and calcium influx increased expression of primiR-199a2 but not of primiR-199a1 in MIN6 cells, indicating that calcium influx was involved. Transcriptional activity studies in MIN6 cells also revealed that primiR-199a2 promoter activity was enhanced by glucose and reduced by 2-deoxy-D-glucose-induced starvation. KCl and the potassium channel blocker tolbutamide also stimulated primiR-199a2 promoter activity. Calcium channel blockade by nifedipine reduced primiR-199a2 promoter activity in MIN6 cells, and diazoxide-mediated calcium influx inhibition blunted glucose up-regulation of miR-199-3p in islets. In conclusion, we uncover that glucose acutely up-regulates miR-199 family expression in ß-cells. Glucose metabolism and calcium influx are involved in primiR-199a2 expression but not primiR-199a1 expression.

The regulatory G protein signaling complex, Gß5-R7, promotes glucose- and extracellular signal-stimulated insulin secretion.

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gß5-R7, a protein complex consisting of the atypical G protein ß subunit Gß5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit ß 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gß5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gß5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Islet pericytes convert into profibrotic myofibroblasts in a mouse model of islet vascular fibrosis.

AIMS/HYPOTHESIS: Islet vascular fibrosis may play an important role in the progression of type 2 diabetes, but there are no mouse models allowing detailed mechanistic studies to understand how a dysfunctional islet microvasculature contributes to diabetes pathogenesis. Here we report that the transgenic AktTg mouse, unlike other mouse strains, shows an increased deposition of extracellular matrix (ECM) proteins in perivascular regions, allowing us to study the cellular mechanisms that lead to islet vascular fibrosis. METHODS: Using immunohistochemistry, we labelled the islet microvasculature and ECM in pancreas sections of AktTg mice and human donors and performed lineage tracing to follow the fate of islet pericytes. We compared islet microvascular responses in living pancreas slices from wild-type and AktTg mice. RESULTS: We found that vascular pericytes proliferate extensively, convert into profibrotic myofibroblasts and substantially contribute to vascular fibrosis in the AktTg mouse model. The increased deposition of collagen I, fibronectin and periostin within the islet is associated with diminished islet perfusion as well as impaired capillary responses to noradrenaline (norepinephrine) and to high glucose in living pancreas slices. CONCLUSIONS/INTERPRETATION: Our study thus illustrates how the AktTg mouse serves to elucidate a cellular mechanism in the development of islet vascular fibrosis, namely a change in pericyte phenotype that leads to vascular dysfunction. Because beta cells in the AktTg mouse are more numerous and larger, and secrete more insulin, in future studies we will test the role beta cell secretory products play in determining the phenotype of pericytes and other cells residing in the islet microenvironment under physiological and pathophysiological conditions. Graphical abstract.

Lactational metformin exposure programs offspring white adipose tissue glucose homeostasis and resilience to metabolic stress in a sex-dependent manner.

We previously demonstrated that exposing mouse dams to metformin during gestation results in increased beta-cell mass at birth and increased beta-cell insulin secretion in adult male offspring. Given these favorable changes after a gestational maternal metformin exposure, we wanted to understand the long-term metabolic impact on offspring after exposing dams to metformin during the postnatal window. The newborn period provides a feasible clinical window for intervention and is important for beta-cell proliferation and metabolic tissue development. Using a C57BL/6 model, we administered metformin to dams from the day of birth to postnatal day 21. We monitored maternal health and offspring growth during the lactation window, as well as adult glucose homeostasis through in vivo testing. At necropsy we assessed pancreas and adipocyte morphology using histological and immunofluorescent staining techniques. We found that metformin exposure programmed male and female offspring to be leaner with a higher proportion of small adipocytes in the gonadal white adipose tissue (GWAT). Male, but not female, offspring had an improvement in glucose tolerance as young adults concordant with a mild increase in insulin secretion in response to glucose in vivo. These data demonstrate long-term metabolic programming of offspring associated with maternal exposure to metformin during lactation.

Maternal low-protein diet on the last week of pregnancy contributes to insulin resistance and ß-cell dysfunction in the mouse offspring.

Maternal low-protein diet (LP) throughout gestation affects pancreatic ß-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for ß-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal ß-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with ß-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient ß-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.

Sexual dimorphism in hypothalamic inflammation in the offspring of dams exposed to a diet rich in high fat and branched-chain amino acids.

Branched-chain amino acid (BCAAs: leucine, isoleucine, and valine) contribute to the development of obesity-associated insulin resistance in the context of consumption of a high-fat diet (HFD) in humans and rodents. Maternal diet is a major determinant of offspring health, and there is strong evidence that maternal HFD alters hypothalamic developmental programming and disrupts offspring energy homeostasis in rodents. In this study, we exposed pregnant and lactating C57BL/6JB female mice to either HFD, HFD with supplemented BCAA (HFD+BCAA), or standard diet (SC), and we studied offspring metabolic phenotypes. Both maternal HFD and HFD supplemented with BCAA had similar effect rendering the offspring metabolic imbalance and impairing their ability to cope with HFD when challenged during aging. The metabolic effects of HFD challenge were more profound in females, worsening female offspring ability to cope with an HFD challenge by activating hypothalamic inflammation in aging. Moreover, the sex differences in hypothalamic estrogen receptor α (ER-α) expression levels were lost in female offspring upon HFD challenge, supporting a link between ER-α levels and hypothalamic inflammation in offspring and highlighting the programming potential of hypothalamic inflammatory responses and maternal nutrition.

Cyclin C stimulates ß-cell proliferation in rat and human pancreatic ß-cells.

Activation of pancreatic ß-cell proliferation has been proposed as an approach to replace reduced functional ß-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic ß-cell proliferation. We hypothesized that cyclin C overexpression would induce ß-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional ß-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [(3)H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected ß-cell death by TUNEL, ß-cell differentiation by RT-PCR, and ß-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human ß-cell proliferation. This augmented proliferation did not induce ß-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing ß-cell regeneration.

Exposure of mouse embryonic pancreas to metformin enhances the number of pancreatic progenitors.

AIMS/HYPOTHESIS: Developing beta cells are vulnerable to nutrient environmental signals. Early developmental processes that alter the number of pancreatic progenitors can determine the number of beta cells present at birth. Metformin, the most widely used oral agent for treating diabetes, alters intracellular energy status in part by increasing AMP-activated protein kinase (AMPK) signalling. This study examined the effect of metformin on developing pancreas and beta cells. METHODS: Pancreatic rudiments from CD-1 mice at embryonic day 13.0 (E13.0) were cultured with metformin, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR, an AMPK activator) or vehicle control in vitro. In another set of studies, pregnant C57BL/6 mice were treated with metformin throughout gestation. Embryonic (E14.0) and neonatal pancreases were then analysed for their morphometry. RESULTS: In vitro metformin treatment led to an increase in the proliferation and number of pancreatic duodenal homeobox 1-positive (PDX1(+)) progenitors. These results were reproduced by in vitro culture of embryonic pancreas rudiments with AICAR, suggesting that AMPK activation was involved. Similarly, metformin administration to pregnant dams induced an increase in both PDX1(+) and neurogenin 3-positive progenitors in the embryonic pancreas at E14.0 and these changes resulted in an increased beta cell fraction in neonates. CONCLUSIONS/INTERPRETATION: These results indicate that exposure to metformin during gestation modulates the early steps of beta cell development (prior to E14.0) towards an increase in the number of pancreatic and endocrine progenitors. These changes ultimately result in a higher beta cell fraction at birth. These findings are of clinical importance given that metformin is currently used for the treatment of gestational diabetes.

Long-lived crowded-litter mice exhibit lasting effects on insulin sensitivity and energy homeostasis.

The action of nutrients on early postnatal growth can influence mammalian aging and longevity. Recent work has demonstrated that limiting nutrient availability in the first 3 wk of life [by increasing the number of pups in the crowded-litter (CL) model] leads to extension of mean and maximal lifespan in genetically normal mice. In this study, we aimed to characterize the impact of early-life nutrient intervention on glucose metabolism and energy homeostasis in CL mice. In our study, we used mice from litters supplemented to 12 or 15 pups and compared those to control litters limited to eight pups. At weaning and then throughout adult life, CL mice are significantly leaner and consume more oxygen relative to control mice. At 6 mo of age, CL mice had low fasting leptin concentrations, and low-dose leptin injections reduced body weight and food intake more in CL female mice than in controls. At 22 mo, CL female mice also have smaller adipocytes compared with controls. Glucose and insulin tolerance tests show an increase in insulin sensitivity in 6 mo old CL male mice, and females become more insulin sensitive later in life. Furthermore, ß-cell mass was significantly reduced in the CL male mice and was associated with reduction in ß-cell proliferation rate in these mice. Together, these data show that early-life nutrient intervention has a significant lifelong effect on metabolic characteristics that may contribute to the increased lifespan of CL mice.

O-GlcNAcylation modulates mTORC1 and autophagy in ß-cells, driving diabetes progression.

Type 2 diabetes (T2D) arises when pancreatic ß-cells fail to produce sufficient insulin to control blood glucose appropriately. Aberrant nutrient sensing by O-GlcNAcylation and mTORC1 is linked to T2D and the failure of insulin-producing ß-cells. However, the nature of their crosstalk in ß-cells remains unexplored. Recently, O-GlcNAcylation, a post-translation modification controlled by enzymes OGT/OGA, emerged as a pivotal regulator for ß-cell health; deficiency in either enzyme causes ß-cell failure. The present study investigates the previously unidentified connection between nutrient sensor OGT and mTORC1 crosstalk to regulate ß-cell mass and function in vivo. We show reduced OGT and mTORC1 activity in islets of preclinical ß-cell dysfunction model and obese human islets. Using loss or gain of function of OGT, we identified that O-GlcNAcylation positively regulates mTORC1 signaling in ß-cells. O-GlcNAcylation negatively modulates autophagy, as the removal of OGT increases autophagy, while the deletion of OGA decreases it. Increasing mTORC1 signaling, via deletion of TSC2, alleviates the diabetic phenotypes by increasing ß-cell mass but not ß-cell function in OGT deficient mice. Downstream phospho-protein signaling analysis reveal diverging impact on MKK4 and calmodulin signaling between islets with OGT, TSC2, or combined deletion. These data provide new evidence of OGT's significance as an upstream regulator of mTORC1 and autophagy, crucial for the regulation of ß-cell function and glucose homeostasis.

Predictors of lack of glycemic control in persons with type 2 diabetes.

BACKGROUND: Professional guidelines recommend an HbA1c < 7% for most people with diabetes and < 8.5% for those with relaxed glycemic goals. However, many people with type 2 diabetes mellitus (T2DM) are unable to achieve the desired HbA1c goal. This study evaluated factors associated with lack of improvement in HbA1c over 3 years. METHODS: All patients with T2DM treated within a major academic healthcare system during 2015-2020, who had at least one HbA1c value > 8.5% within 3 years from their last HbA1c were included in analysis. Patients were grouped as improved glycemic control (last HbA1c ≤ 8.5%) or lack of improvement (last HbA1c > 8.5%). Multivariate logistic regression analysis was performed to assess independent predictors of lack of improvement in glycemic control. RESULTS: Out of 2,232 patients who met the inclusion criteria, 1,383 had an improvement in HbA1c while 849 did not. In the fully adjusted model, independent predictors of lack of improvement included: younger age (odds ratio, 0.89 per 1-SD [12 years]; 95% CI, 0.79-1.00), female gender (1.30, 1.08-1.56), presence of hypertension (1.29, 1.08-1.55), belonging to Black race (1.32, 1.04-1.68, White as reference), living in low income area (1.86,1.28-2.68, high income area as reference), and insurance coverage other than Medicare (1.32, 1.05-1.66). Presence of current smoking was associated with a paradoxical improvement in HbA1c (0.69, 0.47-0.99). In a subgroup analysis, comparing those with all subsequent HbA1c values > 8.5% (N = 444) to those with all subsequent HbA1c values < 8.5% (N = 341), similar factors were associated with lack of improvement, but smoking was no longer significant. CONCLUSION: We conclude that socioeconomic factors like race, type of insurance coverage and living in low-income areas are associated with lack of improvement in HbA1c over a period of 3-years in people with T2DM. Intervention strategies focused on low-income neighborhoods need to be designed to improve diabetes management.

Defining the In Vivo Role of mTORC1 in Thyrocytes by Studying the TSC2 Conditional Knockout Mouse Model.

Background: The thyroid gland is susceptible to abnormal epithelial cell growth, often resulting in thyroid dysfunction. The serine-threonine protein kinase mechanistic target of rapamycin (mTOR) regulates cellular metabolism, proliferation, and growth through two different protein complexes, mTORC1 and mTORC2. The PI3K-Akt-mTORC1 pathway's overactivity is well associated with heightened aggressiveness in thyroid cancer, but recent studies indicate the involvement of mTORC2 as well. Methods: To elucidate mTORC1's role in thyrocytes, we developed a novel mouse model with mTORC1 gain of function in thyrocytes by deleting tuberous sclerosis complex 2 (TSC2), an intracellular inhibitor of mTORC1. Results: The resulting TPO-TSC2KO mice exhibited a 70-80% reduction in TSC2 levels, leading to a sixfold increase in mTORC1 activity. Thyroid glands of both male and female TPO-TSC2KO mice displayed rapid enlargement and continued growth throughout life, with larger follicles and increased colloid and epithelium areas. We observed elevated thyrocyte proliferation as indicated by Ki67 staining and elevated cyclin D3 expression in the TPO-TSC2KO mice. mTORC1 activation resulted in a progressive downregulation of key genes involved in thyroid hormone biosynthesis, including thyroglobulin (Tg), thyroid peroxidase (Tpo), and sodium-iodide symporter (Nis), while Tff1, Pax8, and Mct8 mRNA levels remained unaffected. NIS protein expression was also diminished in TPO-TSC2KO mice. Treatment with the mTORC1 inhibitor rapamycin prevented thyroid mass expansion and restored the gene expression alterations in TPO-TSC2KO mice. Although total thyroxine (T4), total triiodothyronine (T3), and TSH plasma levels were normal at 2 months of age, a slight decrease in T4 and an increase in TSH levels were observed at 6 and 12 months of age while T3 remained similar in TPO-TSC2KO compared with littermate control mice. Conclusions: Our thyrocyte-specific mouse model reveals that mTORC1 activation inhibits thyroid hormone (TH) biosynthesis, suppresses thyrocyte gene expression, and promotes growth and proliferation.

Endothelial c-Myc knockout disrupts metabolic homeostasis and triggers the development of obesity.

Introduction: Obesity is a major risk factor associated with multiple pathological conditions including diabetes and cardiovascular disease. Endothelial dysfunction is an early predictor of obesity. However, little is known regarding how early endothelial changes trigger obesity. In the present work we report a novel endothelial-mediated mechanism essential for regulation of metabolic homeostasis, driven by c-Myc. Methods: We used conditional knockout (EC-Myc KO) and overexpression (EC-Myc OE) mouse models to investigate the endothelial-specific role of c-Myc in metabolic homeostasis during aging and high-fat diet exposure. Body weight and metabolic parameters were collected over time and tissue samples collected at endpoint for biochemical, pathology and RNA-sequencing analysis. Animals exposed to high-fat diet were also evaluated for cardiac dysfunction. Results: In the present study we demonstrate that EC-Myc KO triggers endothelial dysfunction, which precedes progressive increase in body weight during aging, under normal dietary conditions. At endpoint, EC-Myc KO animals showed significant increase in white adipose tissue mass relative to control littermates, which was associated with sex-specific changes in whole body metabolism and increase in systemic leptin. Overexpression of endothelial c-Myc attenuated diet-induced obesity and visceral fat accumulation and prevented the development of glucose intolerance and cardiac dysfunction. Transcriptome analysis of skeletal muscle suggests that the protective effects promoted by endothelial c-Myc overexpression are associated with the expression of genes known to increase weight loss, energy expenditure and glucose tolerance. Conclusion: Our results show a novel important role for endothelial c-Myc in regulating metabolic homeostasis and suggests its potential targeting in preventing obesity and associated complications such as diabetes type-2 and cardiovascular dysfunction.

Time-dependent effects of endogenous hyperglucagonemia on glucose homeostasis and hepatic glucagon action.

Elevation of glucagon levels and increase in α cell proliferation is associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications for understanding abnormal responses to hypoglycemia in patients with diabetes and provide novel avenues for diabetes management. Using mice with inducible induction of Rheb1 in α cells (αRhebTg mice), we showed that short-term activation of mTORC1 signaling is sufficient to induce hyperglucagonemia through increased glucagon secretion. Hyperglucagonemia in αRhebTg mice was also associated with an increase in α cell size and mass expansion. This model allowed us to identify the effects of chronic and short-term hyperglucagonemia on glucose homeostasis by regulating glucagon signaling in the liver. Short-term hyperglucagonemia impaired glucose tolerance, which was reversible over time. Liver glucagon resistance in αRhebTg mice was associated with reduced expression of the glucagon receptor and genes involved in gluconeogenesis, amino acid metabolism, and urea production. However, only genes regulating gluconeogenesis returned to baseline upon improvement of glycemia. Overall, these studies demonstrate that hyperglucagonemia exerts a biphasic response on glucose metabolism: Short-term hyperglucagonemia lead to glucose intolerance, whereas chronic exposure to glucagon reduced hepatic glucagon action and improved glucose tolerance.