Subscribe: Diabetes Journal publish ahead of print articles
Preview: Diabetes Journal publish ahead of print articles

Diabetes Journal publish ahead of print articles

Diabetes Journal publish ahead of print articles


Nrf2-mediated Antioxidant Defense and Peroxiredoxin 6 are Linked to Biosynthesis of Palmitic Acid Ester of 9-Hydroxystearic Acid


Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipid mediators with promising anti-diabetic and anti-inflammatory properties that are formed in white adipose tissue (WAT) via de novo lipogenesis, but their biosynthetic enzymes are unknown. Using a combination of lipidomics in WAT, QTL mapping and correlation analyses in rat BXH/HXB recombinant inbred strains, and response to oxidative stress in murine models, we elucidated the potential pathway of biosynthesis of several FAHFAs.

Comprehensive analysis of WAT samples identified ~160 regioisomers documenting the complexity of this lipid class. The linkage analysis highlighted several members of Nuclear factor, erythroid 2-like 2 (Nrf2)-mediated antioxidant defense system (Prdx6, Mgst1, Mgst3), lipid-handling proteins (Cd36, Scd6, Acnat1, Acnat2, Baat) and family of Flavin Containing Monooxygenase (Fmo) as the positional candidate genes. Transgenic expression of Nrf2 and deletion of Prdx6 genes resulted in reduction of palmitic acid ester of 9-hydroxystearic acid (9-PAHSA) and 11-PAHSA levels, while oxidative stress induced by an inhibitor of glutathione synthesis increased PAHSA levels nonspecifically.

Our results indicate that the synthesis of FAHFAs via carbohydrate-responsive element-binding protein (ChREBP)-driven de novo lipogenesis depends on the adaptive antioxidant system and suggest that FAHFAs may link activity of this system with insulin sensitivity in peripheral tissues.

Native Oxyntomodulin has Significant Glucoregulatory Effects Independent of Weight loss in Obese Humans with and Without Type 2 Diabetes


Oxyntomodulin (OXM), an enteroendocrine hormone, causes appetite suppression, increased energy expenditure, and weight loss in obese humans via activation of GLP-1 and glucagon receptors. However, the effects of OXM on glucose homeostasis remain ill-defined. To address this gap, we evaluated the effects of an intravenous (IV) infusion of native OXM on insulin secretory rates (ISR) and glycemic excursion in a graded glucose infusion (GGI) procedure in two separate randomized, placebo-controlled, single dose crossover trials in 12 overweight and obese subjects without diabetes, and in 12 obese subjects with Type 2 diabetes (T2DM) respectively, using the GLP-1 analog, liraglutide as a comparator in the T2DM. In both groups, in the GGI, 3.0 pmol/kg/min of OXM significantly increased ISR and blunted glycemic excursion relative to placebo. In the T2DM, the effects of OXM were comparable to those of liraglutide, including restoration of beta cell glucose responsiveness to that of non-obese subjects without diabetes. Our findings indicate that native OXM significantly augments glucose-dependent insulin secretion acutely in obese subjects with and without diabetes, with effects comparable to pharmacologic GLP-1R activation and independent of weight loss. Native OXM has potential to improve hyperglycemia via complementary and independent induction of insulin secretion and weight loss.

Superior Glycemic Control with a Glucose-Responsive Insulin Analog: Hepatic and Nonhepatic Impacts


We evaluated the hepatic and nonhepatic responses to glucose-responsive insulin (GRI). Eight dogs received GRI or regular human insulin (HI) in random order. A primed, continuous intravenous infusion of [3-3H]glucose began at -120 min. Basal sampling (-30 to 0 min) was followed by 2 study periods (150 min each), P1 and P2. At 0 min, somatostatin and GRI (36±3 pmol/kg/min) or HI (1.8 pmol/kg/min) were infused IV; basal glucagon was replaced intraportally. Glucose was infused intravenously to clamp plasma glucose at 80 mg/dL (P1) and 240 mg/dL (P2). Whole body insulin clearance (WBIC) and insulin concentrations were not different in P1 vs P2 with HI, but WBIC was 23% higher and arterial insulin 16% lower in P1 vs P2 with GRI. Net hepatic glucose output was similar between treatments in P1. In P2, both treatments induced net hepatic glucose uptake (2.1±0.5 [HI] vs 3.3±0.4 [GRI] mg/kg/min). Nonhepatic glucose uptake (nonHGU, mg/kg/min) in P1 and P2, respectively, differed between treatments (2.6±0.3 and 7.4±0.6 with HI; 2.0±0.2 and 8.1±0.8 with GRI). Thus, glycemia impacted GRI but not HI clearance, with resultant differential effects on HGU and nonHGU. GRI holds promise for decreasing hypoglycemia risk while enhancing glucose uptake under hyperglycemic conditions.

Reduced Nonexercise Activity Attenuates Negative Energy Balance in Mice Engaged in Voluntary Exercise


Exercise alone is often ineffective for treating obesity despite the associated increase in metabolic requirements. Decreased nonexercise physical activity has been implicated in this resistance to weight loss, but the mechanisms responsible are unclear. We quantified the metabolic cost of nonexercise activity, or "off-wheel" activity (OWA), and voluntary wheel running (VWR) and examined whether changes in OWA during VWR altered energy balance in chow-fed C57BL/6J mice (n = 12). Energy expenditure (EE), energy intake, and behavior (VWR and OWA) were continuously monitored for 4 days with locked running wheels followed by 9 days with unlocked running wheels. Unlocking the running wheels increased EE as a function of VWR distance. The metabolic cost of exercise (MCE; kcal/m traveled) decreased with increasing VWR speed. Unlocking the wheel led to a negative energy balance, but also decreased OWA, which was predicted to mitigate the expected change in energy balance by ~45%. A novel behavioral circuit involved repeated bouts of VWR, and roaming was discovered and represented novel predictors of VWR behavior. The integrated analysis described here reveals that the weight loss effects of voluntary exercise can be countered by a reduction in nonexercise activity.

Defining the Transcriptional Targets of Leptin Reveals a Role For Atf3 in Leptin Action


Leptin acts via its receptor (LepRb) to modulate gene expression in hypothalamic LepRb-expressing neurons, thereby controlling energy balance and glucose homeostasis. Despite the importance of the control of gene expression in hypothalamic LepRb neurons for leptin action, the transcriptional targets of LepRb signaling have remained undefined because LepRb cells contribute a small fraction to the aggregate transcriptome of the brain regions in which they reside. We thus employed Translating Ribosome Affinity Purification (TRAP) followed by RNA-seq to isolate and analyze mRNA from the hypothalamic LepRb neurons of wild-type or leptin-deficient (Lepob/ob) mice treated with vehicle or exogenous leptin. While the expression of most of the genes encoding the neuropeptides commonly considered to represent the main targets of leptin action were altered only following chronic leptin deprivation, our analysis revealed other transcripts that were coordinately regulated by leptin under multiple treatment conditions. Among these, acute leptin treatment increased expression of the transcription factor, Atf3, in LepRb neurons. Furthermore, ablation of Atf3 from LepRb neurons (Atf3LepRbKO mice) decreased leptin efficacy and promoted positive energy balance in mice. Thus, this analysis revealed the gene targets of leptin action, including Atf3, which represents a cellular mediator of leptin action.

Altered Brain Dynamics in Patients with Type 1 Diabetes During Working Memory Processing


It is now generally accepted that diabetes increases the risk for cognitive impairment, but the precise mechanisms are poorly understood. A critical problem in linking diabetes to cognitive impairment is that patients often have multiple comorbidities (e.g., obesity, hypertension) that have been independently linked to cognitive deficits. Herein, we focus on young adults with and without type 1 diabetes who were virtually free of such comorbidities. The two groups were matched on major health and demographic factors, and all participants completed a verbal working memory task during magnetoencephalographic (MEG) brain imaging. We hypothesized that patients would have altered neural dynamics in verbal working memory processing and that these differences would directly relate to clinical disease measures. Accordingly, we found that patients had significantly stronger neural responses in the superior parietal cortices during memory encoding, and significantly weaker activity in parietal-occipital regions during maintenance compared to controls. Moreover, disease duration and glycemic control were both significantly correlated with neural responses in various brain regions. In conclusion, young healthy adults with type 1 diabetes already have aberrant neural processing relative to their non-diabetic peers, employing compensatory responses to perform the task, and glucose management and duration may play a central role.

Circulating Fetuin-A and Risk of Type 2 Diabetes: A Mendelian Randomization Analysis


Fetuin-A, a hepatic-origin protein, is strongly positively associated with risk of type 2 diabetes in human observational studies, but it is unknown whether this association is causal. We aimed to study the potential causal relation of circulating fetuin-A to risk of type 2 diabetes in a Mendelian Randomization study with SNPs located in the fetuin-A-encoding AHSG gene. We used data from eight European countries of the prospective EPIC-InterAct case-cohort study including 10,020 incident cases. Plasma fetuin-A concentration was measured in a subset of 965 subcohort participants and 654 cases. A genetic score of the AHSG SNPs was strongly associated with fetuin-A (28% explained variation). Using the genetic score as instrumental variable of fetuin-A, we observed no significant association of a 50 µg/ml higher fetuin-A concentration with diabetes risk (HR 1.02 [95%-CI 0.97, 1.07]). Combining our results with those from the Diabetes Genetics Replication And Meta-analysis (DIAGRAM) consortium (12,171 cases) also did not suggest a clear significant relation of fetuin-A with diabetes risk. In conclusion, although there is mechanistical evidence for an effect of fetuin-A on insulin sensitivity and secretion, this study doesn’t support a strong, relevant relationship between circulating fetuin-A and diabetes risk in the general population.

Adipocyte Long Noncoding RNA Transcriptome Analysis of Obese Mice Identified Lnc-leptin Which Regulates Leptin


Obesity induces profound transcriptome changes in adipocytes; recent evidence suggests that lncRNAs play key roles in this process. Here, we performed a comprehensive transcriptome study by RNA-Seq in adipocytes isolated from interscapular brown, inguinal and epididymal white adipose tissues in diet-induced obese mice. Our analysis reveals a set of obesity-dysregulated lncRNAs, many of which exhibit dynamic changes in fed vs. fasted state, potentially serving as novel molecular markers reflecting adipose energy status. Among the most prominent ones is Lnc-leptin, an lncRNA transcribed from an enhancer region upstream of Leptin. Expression of Lnc-leptin is sensitive to insulin and closely correlates to Leptin expression across diverse pathophysiological conditions. Functionally, induction of Lnc-leptin is essential for adipogenesis, and its presence is required for the maintenance of Leptin expression in vitro and in vivo. Direct interaction was detected between DNA loci of Lnc-leptin and Leptin in mature adipocytes, which diminished upon Lnc-leptin knockdown. Our study establishes Lnc-leptin as a new regulator of Leptin.

Detection and Characterization of CD8+ Autoreactive Memory Stem T Cells in Patients with Type 1 Diabetes


Stem memory T cells (Tscm) constitute the earliest developmental stage of memory T cells, displaying stem cell-like properties such as self-renewal capacity. Their superior immune reconstitution potential has sparked interest in cancer immune-therapy, vaccine development and immune reconstitution, whereas their role in autoimmunity is largely unexplored. Here we show that autoreactive CD8+ Tscm specific for β cell antigens GAD65, insulin and IGRP are present in patients with type 1 diabetes (T1D). In vitro, generation of autoreactive Tscm from naïve precursors required the presence of the homeostatic cytokine interleukin-7 (IL-7). IL-7 promotes glucose uptake via overexpression of the glucose transporter GLUT-1 and up-regulation of the glycolytic enzyme hexokinase II. Even though metabolism depends on glucose uptake, the subsequent oxidation of pyruvate in the mitochondria was necessary for Tscm generation from naïve precursors. In patients with T1D, high expression of GLUT1 was a hallmark of circulating Tscm and targeting glucose uptake via GLUT-1 using the selective inhibitor WZB117 resulted in inhibition of Tscm generation and expansion. Our results suggest that autoreactive Tscm are present in patients with T1D and can be selectively targeted by inhibition of glucose metabolism.

Postnatal Ontogenesis of the Islet Circadian Clock Plays a Contributory Role in {beta}-Cell Maturation Process


Development of cell replacement therapies in diabetes requires understanding of the molecular underpinnings of β-cell maturation. Circadian clock regulates diverse cellular functions important for regulation of β-cell function and turnover. However postnatal ontogenesis of the islet circadian clock and its potential role in β-cell maturation remain unknown. To address this, we studied wild type Sprague Dawley as well as Period1 luciferase transgenic rats (Per1:LUC) to determine circadian clock function, clock protein expression and diurnal insulin secretion during islet development and maturation process. We additionally studied β-cell-specific Bmal1 deficient mice to elucidate potential role of this key circadian transcription factor in β-cell functional and transcriptional maturation. We report that emergence of the islet circadian clock 1) occurs during early postnatal period, 2) depends on the establishment of global behavioral circadian rhythms and 3) leads to the induction of diurnal insulin secretion and gene expression. Islet cell maturation was also characterized by induction in the expression of circadian transcription factor BMAL1, deletion of which altered postnatal development of glucose-stimulated insulin secretion and associated transcriptional network. Postnatal development of the islet circadian clock contributes to early life β-cell maturation and should be considered for optimal design of future β-cell replacement strategies in diabetes.

Altered Odor-Induced Brain Activity as an Early Manifestation of Cognitive Decline in Patients with Type 2 Diabetes


Type 2 diabetes is reported to be associated with olfactory dysfunction and cognitive decline. However, whether and how olfactory neural circuit abnormalities involve cognitive impairment in diabetes remains uncovered. This study thus aimed to investigate olfactory network alterations and the associations of odor-induced brain activity with cognitive and metabolic parameters in type 2 diabetes. Participants with normal cognition, including 51 type 2 diabetes patients and 41 non-diabetic control subjects, underwent detailed cognitive assessment, olfactory behavior tests and odor-induced functional MRI measurements. Olfactory brain regions showing significantly different activation between two groups were extracted for functional connectivity analysis. Compared with the control, diabetic patients demonstrated significantly lower olfactory threshold score, decreased brain activation, and functional connectivity in the olfactory network. Positive associations of the disrupted functional connectivity with decreased neuropsychology test scores and reduced pancreatic function were observed in diabetic patients. Notably, the association between pancreatic function with executive function was mediated by olfactory behavior and olfactory functional connectivity. Our results suggested the alteration of olfactory network is present before clinical measurable cognitive decrements in type 2 diabetes, bridging the gap between central olfactory system and cognitive decline in diabetes.

Loss of OcaB Prevents Age-Induced Fat Accretion and Insulin Resistance by Altering B Lymphocyte Transition and Promoting Energy Expenditure


The current demographic shift toward an aging population has led to a robust increase in the prevalence of age-associated metabolic disorders. Interestingly, recent studies have demonstrated that the etiology of obesity-related insulin resistance developed upon aging differs from that induced by high-calorie diets. Whereas the role of adaptive immunity on the changes in energy metabolism driven by nutritional challenges has gained attention in the past years, its impact upon aging remains mostly unknown. Here, we found that follicular B2 lymphocytes and expression levels of the B cell-specific transcriptional coactivator OcaB increase with age in spleen and in intra-abdominal epididymal white adipose tissue (eWAT), concomitantly with higher circulating levels of immunoglobulin G (IgG) and impaired glucose homeostasis. Reduction of B cell maturation and immunoglobulin production - especially that of IgG2c - by ablation of OcaB prevented age-induced glucose intolerance and insulin resistance, and promoted energy expenditure by stimulating fatty acid utilization in eWAT and brown adipose tissue. Transfer of wild-type bone marrow in OcaB-/- mice replenished eWAT B2 cell population and IgG levels, which diminished glucose tolerance, insulin sensitivity, and energy expenditure, while increasing body weight gain in aged mice. Thus, these findings demonstrate that upon aging, modifications in B cell-driven adaptive immunity contribute to glucose intolerance and fat accretion.

A Novel Strategy to Prevent Advanced Atherosclerosis and Lower Blood Glucose in a Mouse Model of Metabolic Syndrome


Cardiovascular disease caused by atherosclerosis is the leading cause of mortality associated with type 2 diabetes and metabolic syndrome. Insulin therapy is often needed to improve glycemic control, but it does not clearly prevent atherosclerosis. Upon binding to the insulin receptor (IR), insulin activates distinct arms of downstream signaling. The IR-Akt arm is associated with blood glucose lowering and beneficial effects, while the IR-Erk arm might exert less desirable effects. We investigated whether selective activation of the IR-Akt arm, leaving the IR-Erk arm largely inactive, would result in protection from atherosclerosis in a mouse model of metabolic syndrome. The insulin mimetic peptide S597 lowered blood glucose and activated Akt in insulin target tissues, mimicking insulin’s effects, but only weakly activated Erk and even prevented insulin-induced Erk activation. Strikingly, S597 retarded atherosclerotic lesion progression through a process associated with protection from leukocytosis, thereby reducing lesional accumulation of inflammatory Ly6Chi monocytes. S597-mediated protection from leukocytosis was accompanied by reduced numbers of the earliest bone marrow hematopoietic stem cells and reduced IR-Erk activity in hematopoietic stem cells. This study provides a conceptually novel treatment strategy for advanced atherosclerosis associated with metabolic syndrome and type 2 diabetes.

Urinary IgG4 and Smad1 are Specific Biomarkers for Renal Structural and Functional Changes in Early Stage of Diabetic Nephropathy


Diabetic nephropathy (DN) is the major cause of end-stage kidney disease but early biomarkers of DN risk are limited. Herein we examine urinary immunoglobulin G4 (IgG4) and Smad1 as additional early DN biomarkers. 815 patients with type 2 diabetes mellitus were recruited; 554 patients fulfilled the criteria of more than 60 ml/min of estimated glomerular filtration rate (eGFR) and no macroalbuminuria at baseline with followed-up for 5 years. Patients without macroalbuminuria were also recruited for renal biopsies. Urinary IgG4 and Smad1 were determined by enzyme-linked immunoassays using specific antibodies. The assays’ specificities, sensitivity and reproducibility were confirmed. Increased urinary IgG4 was significantly associated with lower eGFR. The level of urinary IgG4 was also significantly correlated with surface density of peripheral glomerular basement membrane [Sv(PGBM/Glom)], while Smad1 was associated with the degree of the mesangial expansion, both classical pathological findings in DN. The baseline eGFR were not different between any groups, however increases in both urinary IgG4 and Smad1 levels at baseline significantly predicted the later development of eGFR decline in patients without macroalbuminuria. These data suggest that urinary IgG4 and Smad1 at relatively early stages of DN reflect underlying DN lesions and is relevant to later clinical outcomes.

Fibroblast Growth Factor 21 Protects Photoreceptor Function in Type 1 Diabetic Mice


Retinal neuronal abnormalities occur before vascular changes in diabetic retinopathy. Accumulating experimental evidence suggests that neurons control vascular pathology in diabetic and other neovascular retinal disease. Therefore, normalizing neuronal activity in diabetes may prevent vascular pathology. We investigated whether fibroblast growth factor 21 (FGF21) prevented retinal neuronal dysfunction in insulin-deficient diabetic mice. We found that in diabetic neural retina, photoreceptor rather than inner retinal function was most affected and administration of the long-acting FGF21 analog, PF-05231023 restored the retinal neuronal functional deficits detected by electroretinography. PF-05231023 administration protected against diabetes-induced disorganization of photoreceptor segments seen in retinal cross section with immunohistochemistry and attenuated the reduction in the thickness of photoreceptor segments measured by optical coherence tomography. PF-05231023, independent of its downstream metabolic modulator adiponectin, reduced inflammatory marker IL-1β mRNA levels. PF-05231023 activated the AKT-NRF2 pathway and reduced IL-1β expression in stressed photoreceptors. PF-05231023 administration did not change retinal expression of VEGFA, suggesting a novel therapeutic approach for the prevention of early diabetic retinopathy by protecting photoreceptor function in diabetes.

HLA-B*07, HLA-DRB1*07, HLA-DRB1*12 and HLA-C*03:02 Strongly Associate with Body Mass Index: Data from 1.3 Million Healthy Chinese Adults


Strong associations between HLA alleles and infectious and autoimmune diseases are well established. Although obesity is also associated with these diseases, the relationship between HLA and obesity has not been systematically investigated in a large cohort. In the current study, we analyzed the association of HLA alleles with body mass index (BMI) using data from 1.3 million healthy adult donors from the Chinese Marrow Donor Program (CMDP). A total of 23 HLA alleles including 12 low resolution and 11 high resolution alleles were significantly associated with BMI after correction for multiple testing. Alleles associated with high BMI were enriched in haplotypes which were common in both Chinese and European populations, whereas the alleles associated with low BMI were enriched in haplotypes common only in Asians. Alleles B*07, DRB1*07, DRB1*12 and C*03:02 provided the strongest associations with BMI (P = 6.89 x 10-10, 1.32 x 10-9, P =1.52x10-9 and 4.45 x 10-8 respectively), where B*07 and DRB1*07 also had evidence for sex specific effects (Pheterogeneity =0.0067 and 0.00058 respectively). These results, which identify associations between alleles of HLA-B, DRB1 and C with BMI in Chinese young adults, implicate a novel biological connection between HLA alleles and obesity.

Hepatic F-Box Protein FBXW7 Maintains Glucose Homeostasis Through Degradation of Fetuin A


Type 2 diabetes has become one of the most serious and long-term threats to human health. However, the molecular mechanism that links obesity to insulin resistance remains largely unknown. Here, we show that F-box and WD repeat domain-containing 7 (FBXW7), an E3 ubiquitin-protein ligase, is markedly downregulated in the liver of two obese mouse models and obese human subjects. We further identify a functional low-frequency human FBXW7 coding variant (p. Ala204Thr) in Chinese population, which is associated with elevated blood glucose and type 2 diabetes risk. Notably, mice with liver-specific knockout of FBXW7 develop hyperglycemia, glucose intolerance and insulin resistance even under normal chow diet. Conversely, overexpression of FBXW7 in the liver not only prevents the development of high-fat-diet-induced insulin resistance but also attenuates the disease signature of obese mice. Mechanistically, FBXW7 directly binds to hepatokine Fetuin A to induce its ubiquitination and subsequent proteasomal degradation, comprising an important mechanism maintaining glucose homeostasis. Thus, we provide evidence showing a beneficial role of FBXW7 in glucose homeostasis.

Sleeve Gastrectomy Improves Glycemia Independent of Weight Loss by Restoring Hepatic Insulin Sensitivity


Bariatric surgery dramatically improves glycemic control, yet the underlying molecular mechanisms remain controversial due to confounding weight loss. We performed sleeve gastrectomy (SG) on obese and diabetic leptin receptor-deficient mice (db/db). One week post-surgery, mice weighed 5% less and displayed improved glycemia compared to sham-operated controls and islets from SG mice displayed reduced expression of diabetes markers. One month post-surgery SG mice weighed more than pre-operatively, but remained near-euglycemic and displayed reduced hepatic lipid droplets. Pair-feeding of SG and sham db/db mice showed that surgery rather than weight loss was responsible for reduced glycemia following SG. While insulin secretion profiles from islets of sham and SG mice were indistinguishable, clamp studies revealed that SG causes a dramatic improvement in muscle and hepatic insulin sensitivity, accompanied by hepatic regulation of HNF4a and PPARα targets. We conclude that long-term weight loss following SG requires leptin signaling. Nevertheless, SG elicits a remarkable improvement in glycemia via insulin sensitization, independent of reduced feeding and weight loss.

Improved Murine-MHC-Deficient HLA-Transgenic NOD-Mouse Models for Type 1 Diabetes Therapy Development


Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.β2m-/- mice transgenically expressing human disease associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8+ T-cell responses mediated by these human class I variants. NOD.β2m-/--A2.1 mice were previously used to identify β-cell autoantigens presented by this human class I variant to pathogenic CD8+ T-cells, and for testing therapies to attenuate such effectors. However, NOD.β2m-/- mice also lack non-classical MHC I family members, including FcRn required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we utilized CRISPR/Cas9 to directly ablate the NOD H2-Kd and H2-Db classical class I variants either individually or in tandem (cMHCI-/-). Ablation of the H2-Ag7 class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II-/-). NOD-cMHCI-/- mice retained non-classical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or B39 restored pathogenic CD8+ T-cell development and T1D susceptibility to NOD-cMHCI-/- mice. These next generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.

Amylin Selectively Signals onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus


Amylin phosphorylates ERK (p-ERK) in the area postrema (AP) to reduce eating and synergizes with leptin to phosphorylate STAT3 in the arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei, to reduce food intake and body weight. The current studies assessed potential amylin and amylin-leptin ARC/VMN interactions on ERK signaling and their roles in postnatal hypothalamic pathway development. In amylin knockout (KO) mice, the density of agouti-related protein (AgRP) immunoreactive (IR) fibers in the hypothalamic paraventricular nucleus (PVN) was increased, while the density of α-melanocyte stimulating hormone (αMSH) fibers was decreased. In mice deficient of the amylin receptor components, RAMP1/3, both AgRP and αMSH-IR fiber densities were decreased, while only αMSH-IR fiber density was decreased in rats injected neonatally in the ARC/VMN with an AAV shRNA against the amylin core receptor. Amylin induced pERK in ARC neurons, 60% of which was present in POMC-expressing neurons with none in NPY neurons. An amylin-leptin interaction was shown by an additive effect on ARC ERK signaling in neonatal rats and a 44% decrease in amylin-induced pERK in the ARC of leptin receptor deficient and of ob/ob mice. Together these results suggest that amylin directly acts, through a p-ERK-mediated process, on POMC neurons to enhance ARC-PVN αMSH pathway development.

Fetal Genotype and Maternal Glucose have Independent and Additive Effects on Birth Weight


Maternal glycemia is a key determinant of birth weight but recent large-scale genome wide-association studies demonstrated an important contribution of fetal genetics. It is not known whether fetal genotype modifies the impact of maternal glycemia, or whether it acts through insulin-mediated growth. We tested the effects of maternal fasting plasma glucose (FPG) and a fetal genetic score for birth weight on birth weight and fetal insulin in 2,051 European mother-child pairs from the Exeter Family Study of Childhood Health (EFSOCH) and Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. The fetal genetic score influenced birth weight independently of maternal FPG and impacted on growth at all levels of maternal glycemia. For mothers with FPG in the top tertile, the frequency of large for gestational age (LGA, birth weight ≥90th centile) was 31.1% for offspring with the highest tertile genetic score and only 14.0% with the lowest tertile genetic score. Unlike maternal glucose, the fetal genetic score was not associated with cord insulin or C-peptide. Similar results were seen for HAPO participants of non-European ancestry (n=2,842 pairs). This work demonstrates that for any level of maternal FPG, fetal genetics have a major impact on fetal growth and act predominantly through independent mechanisms.

Circulating miRNA Profiles Associated With Hyperglycemia in Patients With Type 1 Diabetes Mellitus.


We investigated plasma microRNA (miRNA) profiles associated with variation of hyperglycemia, measured as hemoglobin A1c (HbA1c), in two panels of patients with type 1 diabetes (T1D). Using the HTG Molecular Diagnostics EdgeSeq platform, 2,083 miRNAs were measured in plasma from 71 patients included in a screening panel. Quantitative real-time polymerase chain reaction was used to measure the candidate miRNAs in plasma from 95 patients included in an independent replication panel. We found 10 miRNAs replicated in both panels and four with high statistical significance. The strongest positive correlations with HbA1c were found with miR-125b-5p (rs= 0.40, p=6.0x10-5) and miR-365a-3p (rs=0.35, p=5.9x10-4). The strongest negative correlations were found with miR-5190 (rs=-0.30, p=0.003) and miR-770-5p (rs=-0.27, p=0.008). Pathway analysis revealed that 50 KEGG pathways were significantly enriched by genes targeted by these 4 miRNAs. The axon guidance signaling pathway was enriched (p<1x10-7) by genes targeted by all 4 miRNAs. And three other pathways (the Rap1 signaling, focal adhesion and neurotrophin signaling) were also significantly enriched but with genes targeted by only by 3 of the identified miRNAs. In conclusion, our study identified 4 circulating miRNAs that were influenced by variation in hyperglycemia. Dis-regulation of these miRNAs, which are associated with hyperglycemia in patients with T1D, may contribute to the development of diabetic complications. However, there are multitudes of possible mechanisms/pathways through which dis-regulation of these miRNAs may impact risk of diabetic complications.

RAGE Deletion Confers Renoprotection by Reducing Responsiveness to Transforming Growth Factor-{beta} and Increasing Resistance to Apoptosis


Signalling via the receptor of advanced glycation end-products (RAGE) although complex and not fully elucidated in the setting of diabetes, is considered a key injurious pathway in the development of diabetic nephropathy (DN). We report here that RAGE deletion resulted in increased expression of fibrotic (collagen I and IV, fibronectin) and the inflammatory marker, MCP-1 in primary mouse mesangial cells (MC) and in kidney cortex. RNA-seq analysis in MCs from RAGE-/- and wild type mice confirmed these observations. Nevertheless, despite these gene expression changes a decreased responsiveness to transforming growth factor-β was identified in RAGE-/- mice. Furthermore, RAGE deletion conferred a more proliferative phenotype in MCs and reduced susceptibility to staurosporine-induced apoptosis. RAGE restoration experiments in RAGE-/- MCs largely reversed these gene expression changes resulting in reduced expression of fibrotic and inflammatory markers. This study highlights that protection against DN in RAGE KO mice is likely in part to be due the result of decreased responsiveness to growth factor stimulation and an anti-apoptotic phenotype in mesangial cells. Furthermore, it extends our understanding of the role of RAGE in the progression of DN since RAGE appears to play a key role in modulating the sensitivity of the kidney to injurious stimuli such as prosclerotic cytokines.

Role of Protein Phosphatase 1 and Inhibitor of Protein Phosphatase-1 in Nitric Oxide-Dependent Inhibition of the DNA Damage Response in Pancreatic {beta}-Cells.


Nitric oxide is produced at micromolar levels by pancreatic β-cells during exposure to proinflammatory cytokines. While classically viewed as damaging, nitric oxide also activates pathways that promote β-cell survival. We have shown that nitric oxide, in a cell type selective manner, inhibits the DNA damage response (DDR) and, in doing so, protects β-cells from DNA damage-induced apoptosis. This study explores potential mechanisms by which nitric oxide inhibits DDR signaling. We show that inhibition of DDR signaling (measured by H2AX formation and the phosphorylation of KAP1) is selective for nitric oxide, as other forms of reactive oxygen/nitrogen species do not impair DDR signaling. The kinetics and broad range of DDR substrates that are inhibited suggest that protein phosphatase activation may be one mechanism by which nitric oxide attenuates DDR signaling in β-cells. While protein phosphatase 1 (PP1) is a primary regulator of DDR signaling and an inhibitor of protein phosphatase-1 (IPP-1) is selectively expressed only in β-cells, disruption of either IPP-1 or PP1 does not modify the inhibitory actions of nitric oxide on DDR signaling in β-cells. These findings support a PP1-independent mechanism by which nitric oxide selectively impairs DDR signaling and protects β-cells from DNA damage-induced apoptosis.

Metabolic Syndrome is Associated with Impaired Diastolic Function Independently of Mri-Derived Myocardial Extracellular Volume: the Mesa Study


The relationship of MetS (metabolic syndrome) and insulin resistance (one of its key pathophysiological mediators) with diastolic dysfunction and myocardial fibrosis is not well understood. This study aimed to evaluate the association of MetS with diastolic function and myocardial extracellular matrix (ECM) using cardiac magnetic resonance imaging (CMR) in a large community-based population.

This cross-sectional analysis included 1,582 participants from the Multi-Ethnic Study of Atherosclerosis with left ventricular ejection fraction ≥50% and no past history of cardiac events. Diastolic function was assessed using tagged-CMR parameters including end-diastolic strain rate (EDSR) and strain relaxation index (SRI). ECM was evaluated using extracellular volume (ECV) quantification.

Participants’ mean age was 67.4±8.6 years and 48.1% were males. MetS was present in 533 individuals (33.7%) and type 2 diabetes in 250 (15.8%). In the multivariable analyses, MetS (irrespective of the presence of type 2 diabetes) and higher insulin resistance were associated with impaired diastolic function (higher SRI and lower EDSR), independently of ECV.

In conclusion, MetS, irrespective of the presence of type 2 diabetes, was independently associated with impaired diastole. These functional myocardial changes seem to result from intrinsic cardiomyocytes’ alterations, irrespective of the myocardial interstitium (including fibrosis).

Perilipin 3 Deficiency Stimulates Thermogenic Beige Adipocytes Through PPAR{alpha} Activation


Beige adipocytes can dissipate energy as heat. Elaborate communication between metabolism and gene expression is important in the regulation of beige adipocytes. While lipid droplet (LD)-binding proteins play important roles in adipose tissue biology, it remains unknown whether perilipin 3 (Plin3) is involved in the regulation of beige adipocyte formation and thermogenic activities. Here, we demonstrate that Plin3 ablation stimulates beige adipocytes and thermogenic gene expression in inguinal white adipose tissue (iWAT). Compared to wild-type mice, Plin3 knockout mice were cold-tolerant and displayed enhanced basal and stimulated lipolysis in iWAT, inducing PPARα activation. In adipocytes, Plin3 deficiency promoted PPARα target gene and UCP1 expression and multilocular LD formation upon cold stimulus. Moreover, FGF21 expression and secretion were upregulated, which was attributable to activated PPARα in Plin3-deficient adipocytes. These data suggest that Plin3 acts as an intrinsic protective factor preventing futile beige adipocyte formation by limiting lipid metabolism and thermogenic gene expression.

Regulation of KATP Channel Trafficking in Pancreatic {beta} Cells by Protein Histidine Phosphorylation


Protein histidine phosphatase 1 (PHPT-1) is an evolutionarily conserved 14 kDa protein that dephosphorylates phosphohistidine. PHPT-1-/- mice were generated to gain insight into the role of PHPT-1 and histidine phosphorylation/dephosphorylation in mammalian biology. PHPT-1-/- mice exhibited neonatal hyperinsulinemic hypoglycemia due to impaired trafficking of KATP channels to the plasma membrane in pancreatic β cells in response to low glucose and leptin and resembled patients with congenital hyperinsulinism (CHI). The defect in KATP channel trafficking in PHPT-1-/- β cells was due to the failure of PHPT-1 to directly activate transient receptor potential channel 4 (TRPC4) resulting in decreased Ca2+ influx and impaired downstream activation of AMPK. Thus, these studies demonstrate a critical role for PHPT-1 in normal pancreatic β cell function and raise the possibility that mutations in PHPT-1 and/or TRPC4 may account for yet to be defined cases of CHI.

Elevated Medium Chain-Acylcarnitines are Associated with Gestational Diabetes, and Early Progression to Type-2 Diabetes, and Induce Pancreatic {beta}-Cell Dysfunction


Specific circulating metabolites have emerged as important risk factors for the development of diabetes. The acylcarnitines (acylCs) are a family of metabolites known to be elevated in type-2 diabetes (T2D) and linked to peripheral insulin resistance. However, the impact of AcylCs on pancreatic β-cell function is not well understood. Here, we profiled circulating acylCs in two diabetes cohorts: 1) women with gestational diabetes (GDM) and 2) women with recent GDM who later developed impaired glucose tolerance (IGT), new-onset T2D or returned to normoglycemia within a two-year follow-up period. We observed a specific elevation in serum medium chain (M)-acylCs, particularly hexanoyl- and octanoylcarnitine among women with GDM and individuals with T2D without alteration in long (L)-acylCs. Mice treated with M-acylCs exhibited glucose intolerance, attributed to impaired insulin secretion. Murine and human islets exposed to elevated levels of M-acylCs developed defects in glucose-stimulated insulin secretion and this was directly linked to reduced mitochondrial respiratory capacity and subsequent ability to couple glucose metabolism to insulin secretion. In conclusion, our study reveals that an elevation in circulating M-acylCs is associated with GDM and early stages of T2D onset, and that this elevation directly impairs β-cell function.

CDKN2A/B T2D GWAS Risk-SNPs Impact Locus Gene Expression and Proliferation in Human Islets


Genome-wide association studies link the CDKN2A/B locus with T2D risk, but mechanisms increasing risk remain unknown. The CDKN2A/B locus encodes cell cycle inhibitors p14, p15, and p16, MTAP, and ANRIL, a lncRNA. The goal of this study was to determine whether CDKN2A/B T2D risk-SNPs impact locus gene expression, insulin secretion, or beta cell proliferation, in human islets. Islets from non-diabetic donors (n=95) were tested for SNP genotype (rs10811661, rs2383208, rs564398, rs10757283), gene expression (p14, p15, p16, MTAP, ANRIL, PCNA, KI67, CCND2), insulin secretion (n=61) and beta cell proliferation (n=47). Intriguingly, locus genes were co-regulated in islets in two physically overlapping cassettes: p14-p16-ANRIL, which increased with age, and MTAP-p15, which did not. Risk-alleles at rs10811661 and rs2383208 were differentially associated with expression of ANRIL, but not p14, p15, p16 or MTAP, in age-dependent fashion, such that younger homozygous-risk donors had higher ANRIL expression, equivalent to older donor levels. We identified several risk-SNP haplotype combinations that may impact locus gene expression, suggesting possible mechanisms by which SNPs impact locus biology. Risk-allele carriers at ANRIL coding SNP rs564398 had reduced beta cell proliferation index. In conclusion, CDKN2A/B locus SNPs may impact T2D risk by modulating islet gene expression and beta cell proliferation.

Catestatin Inhibits Obesity-Induced Macrophage Infiltration and Inflammation in the Liver and Suppresses Hepatic Glucose Production Leading to Improved Insulin Sensitivity


The activation of Kupffer cells (KCs) and monocyte (Mc)-derived recruited macrophages (McMs) in the liver contributes to obesity-induced insulin resistance and type 2 diabetes. Diet-induced obese (DIO) mice treated with Chromogranin A (CgA) peptide catestatin (CST) showed several positive results. These included decreased hepatic/plasma lipids and plasma insulin, diminished expression of gluconeogenic genes, attenuated expression of pro-inflammatory genes, increased expression of anti-inflammatory genes in McMs, and inhibition of the infiltration of McMs resulting in improvement of insulin sensitivity. Systemic CST knockout (CST-KO) mice on normal chow diet (NCD) ate more food, gained weight, and displayed elevated blood glucose and insulin levels. Supplementation of CST normalized glucose and insulin levels. To verify that the CST deficiency caused macrophages to be very pro-inflammatory in CST-KO-NCD mice and produced glucose intolerance, we tested the effects of FACS-sorted F4/80+Ly6C- cells (representing KCs) and F4/80-Ly6C+ cells (representing McMs) on hepatic glucose production (HGP). Both basal and glucagon-induced HGP was markedly increased in hepatocytes co-cultured with KCs and McMs from NCD-fed CST-KO mice, and the effect was abrogated upon pre-treatment of CST-KO-Ms with CST. Thus, we provide a novel mechanism of HGP suppression through CST-mediated inhibition of macrophage infiltration and function.

PDGF-BB Carried by Endothelial Cell-derived Extracellular Vesicles Reduces Vascular Smooth Muscle Cell Apoptosis in Diabetes


Endothelial cell-derived extracellular vesicles (CD31EVs) are a new entity for therapeutic/prognostic purposes. The roles of CD31EVs as mediators of smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2D) is investigated herein.

We demonstrated that, unlike non-diabetic, diabetic serum-derived-EVs (D-CD31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycaemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between anti-apoptotic/pro-apoptotic signals: increase of bcl-2 and decrease of bak/bax. D-CD31EV cargo analysis demonstrated that D-CD31EVs are enriched in membrane-bound-platelet-derived-growth-factor-BB (mbPDGF-BB). Thus, we postulated that mbPDGF-BB transfer by D-CD31EVs could account for VSMC resistance to apoptosis. By depleting CD31EVs of PDGF-BB or blocking the PDGF-BB-receptorβ on VSMCs, we demonstrated that mbPDGF-BB contributes to D-CD31EV-mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of PDGF-BB-mediated miR-296-5p expression. In fact, while PDGF-BB-treatment recapitulated D-CD31EV-mediated anti-apoptotic program and VSMC resistance to apoptosis, PDGF-BB-depleted CD31EVs failed. D-CD31EVs also increased VSMC migration and recruitment to neovessels, by means of PDGF-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of T2D individuals, express low bak/bax and high bcl-2 and miR-296-5p levels.

This study identifies the mbPDGF-BB in D-CD31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis.

Long Non-coding RNA LncSHGL Recruits hnRNPA1 to Suppress Hepatic Gluconeogenesis and Lipogenesis


Mammalian genomes encode a huge number of LncRNAs with unknown functions. This study determined the role and mechanism of a new LncRNA, LncRNA Suppressor of Hepatic Gluconeogenesis and Lipogenesis (LncSHGL), in regulating hepatic glucose/lipid metabolism. In the livers of obese mice and NAFLD patient, the expression levels of mouse LncSHGL and its human homologous LncRNA B4GALT1-AS1 were reduced. Hepatic LncSHGL restoration improved hyperglycemia, insulin resistance and steatosis in obese diabetic mice, whereas hepatic LncSHGL inhibition promoted fasting hyperglycemia and lipid deposition in normal mice. LncSHGL overexpression increased Akt phosphorylation, and repressed gluconeogenic and lipogenic gene expression in obese mouse livers, whereas LncSHGL inhibition exerted the opposite effects in normal mouse livers. Mechanistically, LncSHGL recruited hnRNPA1 to enhance the translation efficiency of CALM mRNAs to increase CaM protein level without affecting their transcription, leading to the activation of PI3K/Akt pathway and repression of mTOR/SREBP-1C pathway independent of insulin and calcium in hepatocytes. Hepatic hnRNPA1 overexpression also activated CaM/Akt pathway and repressed mTOR/SREBP-1C pathway to ameliorate hyperglycemia and steatosis in obese mice. In conclusion, LncSHGL is a novel insulin-independent suppressor of hepatic gluconeogenesis and lipogenesis. Activating LncSHGL/hnRNPA1 axis represents a potential strategy for the treatment of type 2 diabetes and steatosis.

Perilipin 5 Deletion Unmasks an Endoplasmic Reticulum Stress - Fibroblast Growth Factor 21 Axis in Skeletal Muscle


Lipid droplets are critical for the regulation of lipid metabolism, and dysregulated lipid metabolism contributes to the pathogenesis of several diseases including type 2 diabetes. We generated mice with muscle-specific deletion of the lipid droplet-associated protein, perilipin 5 (PLIN5, Plin5MKO), and investigated PLIN5’s role in regulating skeletal muscle lipid metabolism, intracellular signalling and whole-body metabolic homeostasis. High-fat feeding induced changes in muscle lipid metabolism of Plin5MKO mice, which included increased fatty acid oxidation and oxidative stress, but surprisingly, a reduction in inflammation and endoplasmic reticulum (ER) stress. These muscle-specific effects were accompanied by whole-body glucose intolerance, adipose tissue insulin resistance, and reduced circulating insulin and C-peptide levels in Plin5MKO mice. This coincided with reduced secretion of fibroblast growth factor (FGF) 21 from skeletal muscle and liver, resulting in reduced circulating FGF21. Intriguingly, muscle-secreted factors from Plin5MKO, but not wild-type mice, reduced hepatocyte FGF21 secretion. Exogenous correction of FGF21 levels restored glycemic control and insulin secretion in Plin5MKO mice. These results show that changes in lipid metabolism resulting from PLIN5 deletion reduce ER stress in muscle, decrease FGF21 production by muscle and liver, and impair glycemic control. Further, these studies highlight the importance for muscle-liver cross talk in metabolic regulation.

ELOVL4-Mediated Production of Very Long Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability


Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. While TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long chain (VLC ≥26) ceramides in TJs using diabetes induced loss of blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase ELOVL4. ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited VEGF and IL-1b induced permeability and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins; ZO-1 and claudin-5. Intravitreal delivery of hELOVL4-AAV2 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that omega-linked acyl-VLC ceramides co-localize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in DR through increase in VLC ceramides and stabilization of TJs.

Loss of B Cell Anergy in Type 1 Diabetes is Associated with High Risk HLA and non-HLA Disease Susceptibility Alleles


Although B cells reactive with islet autoantigens are silenced by tolerance mechanisms in healthy individuals, they can become activated and contribute to development of type 1 diabetes. We previously demonstrated that high-affinity insulin-binding B cells (IBCs) occur exclusively in the anergic (BND) compartment in peripheral blood of healthy subjects. Consistent with their activation early in disease development, high-affinity IBCs are absent from the BND compartment of some first-degree relatives (FDRs) as well as all autoantibody positive pre-diabetic and new-onset type 1 diabetes patients, a time when they are found in pancreatic islets. Loss of BND IBCs is associated with loss of the entire BND B cell compartment, consistent with provocation by an environmental trigger or predisposing genetic factors. To investigate potential mechanisms operative in subversion of B cell tolerance, we explored associations between HLA and non-HLA type 1 diabetes-associated risk allele genotypes and loss of BNDs in FDRs. We found that high-risk HLA alleles and a subset of non-HLA risk alleles, i.e. PTPN2 (rs1893217), INS (rs689) and IKZF3 (rs2872507), relevant to B and T cell development and function, are associated with loss of anergy. Hence, our results suggest a role for risk-conferring alleles in perturbation of B cell anergy during development of type 1 diabetes.

Molecular Pathways for Immune Recognition of Preproinsulin Signal Peptide in Type 1 Diabetes


The signal peptide region of preproinsulin (PPI) contains epitopes targeted by human leucocyte antigen-A (HLA-A)-restricted (HLA-A0201, A2402) cytotoxic T-cells as part of the pathogenesis of β-cell destruction in type 1 diabetes. We extended PPI epitope discovery to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles revealing that 4/6 alleles present epitopes derived from the signal peptide region. During co-translational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical, proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter-associated-with–antigen-processing (TAP)-dependent, and ER-luminal (TAP-independent) epitopes, each presented by different HLA class I molecules, and N-terminal trimmed by ER aminopeptidase 1 (ERAP1) for optimal presentation. In vivo, TAP expression is significantly up-regulated and correlated with HLA class I hyper-expression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.

Highly Proliferative Alpha-Cell Related Islet Endocrine Cells in Human Pancreata


The proliferative response of non-β islet endocrine cells in response to type 1 diabetes (T1D) remains undefined. We quantified islet endocrine cell proliferation in a large collection of non-diabetic control and T1D human pancreata across a wide range of ages. Surprisingly, islet endocrine cells with abundant proliferation were present in many adolescent and young adult T1D pancreata. But, the proliferative islet endocrine cells were also present in similar abundance within control samples. We queried the proliferating islet cells with antisera against various islet hormones. Although PP, somatostatin, and ghrelin cells did not exhibit frequent proliferation, glucagon-expressing α-cells were highly proliferative in many adolescent and young adult samples. Notably, α-cells only comprised a fraction (~1/3) of the proliferative islet cells within those samples; most proliferative cells did not express islet hormones. The proliferative hormone negative cells uniformly contained immunoreactivity for ARX (indicating α-cell fate) and cytoplasmic Sox9 (Sox9Cyt). These hormone negative cells represented the majority of islet endocrine Ki67+ nuclei and were conserved from infancy through young adulthood. Our studies reveal a novel population of highly proliferative ARX+ Sox9Cyt+ hormone negative cells and suggest the possibility of previously unrecognized islet development and/or lineage plasticity within adolescent and adult human pancreata.

Disruption of Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) Integrity Contributes to Muscle Insulin Resistance in Mice and Humans


Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently involved in the control of hepatic insulin action and glucose homeostasis, but with conflicting results. Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alterations in insulin resistant states, the relevance of MAM integrity in muscle insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin signaling could help to clarify this controversy. Here, we show in skeletal muscle of different mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria interactions, as an early event preceding mitochondrial dysfunction and insulin resistance. Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a reduction of structural and functional ER-mitochondria interactions. Importantly, experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-induced alterations of insulin signaling and action, whereas disruption of MAM integrity alters the action of the hormone. Lastly, we found an association between altered insulin signaling and ER-mitochondria interactions in human myotubes from obese subjects with or without T2D, compared to healthy lean subjects. Collectively, our data reveal a new role of MAM integrity in insulin action of skeletal muscle, and highlight MAM disruption as an essential subcellular alteration associated with muscle insulin resistance in mice and humans. Therefore, reduced ER-mitochondria coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of obesity and T2D.

Modest Decreases in Endogenous All-Trans-Retinoic Acid Produced by a Mouse Rdh10 Heterozygote Provoke Major Abnormalities in Adipogenesis and Lipid Metabolism


Pharmacological dosing of all-trans-retinoic acid (atRA) controls adiposity in rodents by inhibiting adipogenesis and inducing fatty acid oxidation. Retinol dehydrogenases (Rdh) catalyze the first reaction that activate retinol into atRA. This study examined post-natal contributions of Rdh10 to atRA biosynthesis and physiological functions of endogenous atRA. Embryonic fibroblasts from Rdh10 heterozygote hypomorphs or with a total Rdh10 knockout exhibit decreased atRA biosynthesis and escalated adipogenesis. atRA or a RAR pan-agonist reversed the phenotype. Eliminating one Rdh10 copy in vivo (Rdh10+/-) yielded a modest decrease (≤25%) in the atRA concentration of liver and adipose, but increased adiposity in male and female mice fed a high-fat diet, increased liver steatosis, glucose intolerance and insulin resistance in males fed a high-fat diet, and activated bone marrow adipocyte formation in females, regardless of dietary fat. Chronic dosing with low dose atRA corrected the metabolic defects. These data resolve physiological actions of endogenous atRA, reveal sex-specific effects of atRA in vivo, and establish importance of Rdh10 to metabolic control by atRA. The consequences of a modest decrease in tissue atRA suggest that impaired retinol activation may contribute to diabesity, and low-dose atRA therapy may ameliorate adiposity and its sequelae of glucose intolerance and insulin resistance.

SGK1/FOXO3 Signaling in Hypothalamic POMC Neurons Mediates Glucocorticoid-Increased Adiposity


Although central nervous system has been implicated in glucocorticoid-induced fat mass gain, the underlying mechanisms are poorly understood. The aim of our current study was to investigate the possible involvement of hypothalamic serum- and glucocorticoid-regulated kinase 1 (SGK1) in glucocorticoid-increased adiposity. It is well-known that SGK1 expression is induced by acute glucocorticoid treatment, interestingly, we found its expression was decreased in the arcuate nucleus of the hypothalamus, including POMC neurons, following chronic dexamethasone (Dex) treatment. To study a role of SGK1 in POMC neurons, mice with development or adult-onset SGK1 deletion in POMC neurons (PSKO) were then produced. As observed in Dex-treated mice, PSKO mice exhibited increased adiposity and decreased energy expenditure. Consistently, mice overexpressing constitutively active SGK1 in POMC neurons (PSOE) had the opposite phenotype and prevented from Dex-increased adiposity. Finally, Dex decreased hypothalamic α-melanocyte stimulating hormone (α-MSH) content and its precursor Pomc expression via SGK1/Forkhead box O3 (FOXO3) signaling and intracerebroventricular injection of α-MSH or adenovirus-mediated FOXO3 knockdown in ARC largely reversed the metabolic alterations in PSKO mice. These results demonstrate that POMC SGK1/FOXO3 signaling mediates glucocorticoid-increased adiposity, providing new insights into mechanistic link between glucocorticoid and fat accumulation and important hints for possible treatment targets for obesity.

The Diabetes Gene and Wnt Pathway Effector TCF7L2 Regulates Adipocyte Development and Function


The gene encoding for transcription factor 7-like 2 (TCF7L2) is the strongest type 2 diabetes (T2DM) candidate gene discovered to date. The TCF7L2 protein is a key transcriptional effector of the Wnt/β-catenin signaling pathway, which is an important developmental pathway that negatively regulates adipogenesis. However, the precise role that TCF7L2 plays in the development and function of adipocytes remains largely unknown. Using a combination of in vitro approaches, we first show that TCF7L2 protein is increased during adipogenesis in 3T3-L1 cells and primary adipocyte stem cells (ASC), and that TCF7L2 expression is required for the regulation of Wnt signaling during adipogenesis. Inactivating TCF7L2 protein by removing the HMG-box DNA binding domain in mature adipocytes in vivo leads to whole-body glucose intolerance and hepatic insulin resistance. This phenotype is associated with increased subcutaneous adipose tissue mass, adipocyte hypertrophy and inflammation. Finally, in humans with impaired glucose tolerance (IGT) and adipocyte insulin resistance we demonstrate that TCF7L2 mRNA expression is downregulated, highlighting the translational potential of these findings. In summary our data indicate that TCF7L2 has key roles in adipose tissue development and function that may reveal, at least in part, how TCF7L2 contributes to the pathophysiology of T2DM.

Skeletal Muscle-Specific Deletion of MKP-1 Reveals a p38 MAPK/JNK/Akt Signaling Node that Regulates Obesity-Induced Insulin Resistance


Stress responses promote obesity and insulin resistance, in part, by activating the stress-responsive mitogen-activated protein kinases (MAPKs), p38 MAPK and c-Jun NH2 kinase (JNK). Stress also induces expression of MAPK phosphatase-1 (MKP-1), which inactivates both JNK and p38 MAPK. However, the equilibrium between JNK/p38 MAPK and MKP-1 signaling in the development of obesity and insulin resistance is unclear. Skeletal muscle is a major tissue involved in energy expenditure and glucose metabolism. In skeletal muscle, MKP-1 is upregulated in high fat-diet fed mice and in skeletal muscle of obese humans. Mice lacking skeletal muscle expression of MKP-1 (MKP1-MKO) showed increased skeletal muscle p38 MAPK and JNK activities and were resistant to the development of diet-induced obesity. MKP1-MKO mice exhibited increased whole-body energy expenditure that was associated with elevated levels of myofiber-associated mitochondrial oxygen consumption. miR-21, a negative regulator of PTEN expression, was upregulated in skeletal muscle of MKP1-MKO mice resulting in increased Akt activity consistent with enhanced insulin sensitivity. Our results demonstrate that skeletal muscle MKP-1 represents a critical signaling node through which inactivation of the p38 MAPK/JNK module promotes obesity and insulin resistance.

Diabetes Reduces Severity of Aortic Aneurysms Depending on the Presence of Cell Division Autoantigen 1 (CDA1)


Diabetes is a negative risk factor for aortic aneurysm, but the underlying explanation for this phenomenon is unknown. We have previously demonstrated that Cell Division Autoantigen 1 (CDA1), which enhances TGF-β signaling, is upregulated in diabetes. We hypothesized that CDA1 plays a key role in conferring the protective effect of diabetes against aortic aneurysms. Male wildtype, CDA1 knockout, Apolipoprotein E (ApoE) knockout and CDA1/ApoE double knockout (dKO) mice were rendered diabetic. Whereas aneurysms were not observed in diabetic ApoE knockout and wildtype mice, 40% of diabetic dKO mice developed aortic aneurysms. These aneurysms were associated with attenuated aortic TGF-β signaling, reduced expression of various collagens as well as increased aortic macrophage infiltration and matrix metalloproteinase12 expression. In the well characterized model of angiotensin II (AngII) induced aneurysm formation, concomitant diabetes reduced fatal aortic rupture and attenuated suprarenal aortic expansion, changes not seen in dKO mice. Furthermore, aortic CDA1 expression was downregulated ~70% within biopsies from human abdominal aortic aneurysms. The identification that diabetes is associated with upregulation of vascular CDA1 and that CDA1 deletion in diabetic mice promotes aneurysm formation provides evidence that CDA1 plays a role in diabetes to reduce susceptibility to aneurysm formation.

Sirt1- A Guardian of the Development of Diabetic Retinopathy


Diabetic retinopathy is a multifactorial disease, and the exact mechanism of its pathogenesis remains obscure. A multifunctional deacetylase Sirtuin 1 (Sirt1), is implicated in regulation of many cellular functions and transcription of genes, and retinal Sirt1 is inhibited in diabetes. Our aim is to determine the role of Sirt1 in the development of diabetic retinopathy, and elucidate the molecular mechanism of its downregulation. Using mice overexpressing Sirt1, diabetic for 8 month, structural, functional and metabolic abnormalities were investigated in vascular and neuronal retina. The role of epigenetics in Sirt1 transcriptional suppression was investigated in the retinal microvessels. Compared to wildtype diabetic mice, retinal vasculature from Sirt1 diabetic mice did not present any increase in the number of apoptotic cells, degenerative capillaries and decrease in vascular density. Sirt1 diabetic mice were also protected from mitochondrial damage, and they had normal ERG responses and ganglion cell layer thickness. Wildtype diabetic mice had Sirt1 promoter DNA hypermethylated, which was alleviated in Sirt1 diabetic mice, suggesting the role of epigenetics in its transcriptional suppression. Thus, strategies targeting amelioration of Sirt1 inhibition have potential to maintain retinal vascular and neuronal homeostasis, providing opportunities to retard the development of diabetic retinopathy in its early stages.

Potein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes


Bone loss and fractures are under-recognized complications of type 1 diabetes, and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cGMP/protein kinase G (PKG) signal transduction pathway, and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to O-Glc-N-acetylation of NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat—a NO-independent activator of oxidized guanylate cyclase—increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of anti-oxidant genes, and down-regulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

Dissection of Glomerular Transcriptional Profile in Patients with Diabetic Nephropathy: SRGAP2a Protects Podocyte Structure and Function


Podocytes play a pivotal role in maintaining glomerular filtration function through their interdigitated foot processes. However, the mechanisms that govern the podocyte cytoskeletal rearrangement still remain unclear. Through analyzing transcriptional profile of renal biopsy from diabetic nephropathy (DN) patients and control donors, we identify Slit-Robo GTP activating protein 2a (SRGAP2a) as one of the main ‘hub’ genes that are strongly associated with proteinuria and glomerular filtration in type 2 DN patients. Immunofluorescnce staining and western blot analysis reveal that human and mouse SRGAP2a is primarily localized at podocytes and largely co-localized with synaptopodin. Moreover, podocyte SRGAP2a is downregulated in DN patients and db/db mice at both mRNA and protein level. SRGAP2a reduction is also observed in cultured podocytes treated with TGF-β or high concentration of glucose. Functional and mechanistic studies show that SRGAP2a suppresses podocyte motility through inactivating RhoA/Cdc42 but not Rac1. The protective role of SRGAP2a in podocyte function is also confirmed in zebrafish, in which knockdown of SRGAP2a, a SRGAP2 ortholog in zebrafish, recapitulated podocyte foot process effacement. Finally, increasing podocyte SRGAP2a level in db/db mice via administration of adenovirus expressing SRGAP2a significantly mitigates podocyte injury and proteinuria. Our results demonstrate that SRGAP2a protects podocytes via suppressing podocyte migration.

Novel lncRNA Erbb4-IR Promotes Diabetic Kidney Injury in db/db Mice by Targeting miR-29b


TGF-β/Smad signaling plays an important role in diabetic nephropathy. The present study identified a novel Smad3-dependent long non-coding RNA (lncRNA) Erbb4-IR in the development of type-2 diabetic nephropathy (T2DN) in db/db mice. We found that Erbb4-IR was highly expressed in T2DN of db/db mice and was specifically induced by AGE via a Smad3-dependent mechanism. The functional role of Erbb4-IR in T2DN was revealed by kidney-specific silencing of Erbb4-IR to protect against the development of T2DN such as elevated microalbuminuria, serum creatinine and progressive renal fibrosis in db/db mice, and to block AGE-induced collagen I and IV expression in mouse mesangial cells (mMCs) and mouse tubular epithelial cells (mTECs). Mechanistically, we identified that the Erbb4-IR-miR-29b axis was a key mechanism of T2DN because Erbb4-IR was able to bind the 3’UTR of miR-29b genomic sequence to suppress miR-29b expression at transcriptional level. In contrast, silencing of renal Erbb4-IR increased miR-29b and therefore protected kidney from progressive renal injury in db/db mice and prevented mTECs and mMCs from AGE-induced loss of miR-29b and fibrotic response in vitro. Collectively, we identify that Erbb4-IR is a Smad3-dependent lncRNA that promotes renal fibrosis in T2DN by suppressing miR-29b. Targeting Erbb4-IR may represent a novel therapy for T2DN.

Low Neonatal Plasma N-6/N-3 Pufa Ratios Regulate Offspring Adipogenic Potential and Condition Adult Obesity Resistance


Adipose tissue expansion (ATE) progresses rapidly during postnatal life, influenced by both prenatal maternal factors and postnatal developmental cues. The ratio of n-6 relative to n-3 polyunsaturated fatty acids (PUFA) is thought to regulate perinatal adipogenesis, but the cellular mechanisms and long-term effects are not well understood. We lowered the fetal and postnatal n-6/n-3 PUFA ratio exposure in wildtype offspring, under standard maternal dietary fat amounts, to test effects of low n-6/n-3 ratios on offspring adipogenesis and adipogenic potential. Relative to wildtype pups receiving high perinatal n-6/n-3 ratios, subcutaneous adipose tissue in 14-day old wildtype pups receiving low n-6/n-3 ratios had more adipocytes that were smaller in size, decreased Ppar2, Fabp4, Plin-1 and several lipid metabolism mRNAs, and coincident hypermethylation of the PPAR2 proximal promoter and elevated circulating adiponectin. As adults, offspring that received low perinatal n-6/n-3 ratios were DIO-resistant, had lower positive energy balance and energy intake, greater lipid fuel preference and non-resting energy expenditure, half the body fat, and better glucose clearance. Together, our findings support a model in which low early life n-6/n-3 ratios remodel adipose morphology to increase circulating adiponectin, resulting in a persistent adult phenotype with improved metabolic flexibility that prevents diet induced obesity.

Overexpression of GPR40 in Pancreatic {beta}-Cells Augments Glucose Stimulated Insulin Secretion and Improves Glucose Tolerance in Normal and Diabetic Mice



GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We have generated transgenic mice overexpressing the human GPR40 gene (hGPR40-Tg) under control of the mouse insulin II promoter and have used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis.

Research Design and Methods:

Normal (C57BL/6J) and diabetic (KK) mice overexpressing the human GPR40 gene under control of the insulin II promoter were generated, and their glucose metabolism and islet function were analyzed.


In comparison with nontransgenic littermates, hGPR40-Tg mice exhibited improved oral glucose tolerance with an increase in insulin secretion. Although islet morphological analysis showed no obvious differences between hGPR40-Tg and nontransgenic (NonTg) mice, isolated islets from hGPR40-Tg mice enhanced insulin secretion in response to high glucose (16 mM) than those from NonTg mice with unchanged low glucose (3 mM)-stimulated insulin secretion. In addition, hGPR40-Tg islets significantly increased insulin secretion against a naturally occurring agonist palmitate in the presence of 11 mM glucose. hGPR40-Tg mice were also found to be resistant to high fat diet-induced glucose intolerance, and hGPR40-Tg harboring KK mice showed augmented insulin secretion and improved oral glucose tolerance compared to nontransgenic littermates.


Our results suggest that GPR40 may have a role in regulating glucose-stimulated insulin secretion and plasma glucose levels in vivo, and that pharmacological activation of GPR40 may provide a novel insulin secretagogue beneficial for the treatment of type 2 diabetes.