The aim of this study was to establish the contribution of insulin resistance to the morning (a.m.) versus afternoon (p.m.) lower glucose tolerance of people with type 2 diabetes (T2D). Eleven subjects with T2D (mean [SD] diabetes duration 0.79 [0.23] years, BMI 28.3 [1.8] kg/m2, A1C 6.6% [0.26%] [48.9 (2.9) mmol/mol]), treatment lifestyle modification only) and 11 matched control subjects without diabetes were monitored between 5:00 and 8:00 a.m. and p.m. (in random order) on one occasion (study 1), and on a subsequent occasion, they underwent an isoglycemic clamp (a.m. and p.m., both between 5:00 and 8:00, insulin infusion rate 10 mU/m2/min) (study 2). In study 1, plasma glucose, insulin, C-peptide, and glucagon were higher and insulin clearance lower in subjects with T2D a.m. versus p.m. and versus control subjects (P < 0.05), whereas free fatty acid, glycerol, and β-hydroxybutyrate were lower a.m. versus p.m. However, in study 2 at identical hyperinsulinemia a.m. and p.m. (∼150 pmol/L), glucose Ra and glycerol Ra were both less suppressed a.m. versus p.m. (P < 0.05) in subjects with T2D. In contrast, in control subjects, glucose Ra was more suppressed a.m. versus p.m. Leucine turnover was no different a.m. versus p.m. In conclusion, in subjects with T2D, insulin sensitivity for glucose (liver) and lipid metabolism has diurnal cycles (nadir a.m.) opposite that of control subjects without diabetes already at an early stage, suggesting a marker of T2D.

Hepatocyte nuclear factor 1α (HNF1α) plays essential roles in controlling development and metabolism; its mutations are clearly linked to the occurrence of maturity-onset diabetes of the young (MODY3) in humans. Lysine 117 (K117) to glutamic acid (E117) mutation in the HNF1α gene has been clinically associated with MODY3, but no functional data on this variant are available. Here, we addressed the role of lysine 117 in HNF1α function using a knock-in animal model and site-directed mutagenesis. HNF1α K117E homozygous mice exhibited dwarfism, hepatic dysfunction, renal Fanconi syndrome, and progressive wasting syndrome. These phenotypes were very similar to those of mice with complete HNF1α deficiency, suggesting that K117 is critical to HNF1α functions. K117E homozygotes developed diabetes in the early postnatal period. The relative deficiency of serum insulin levels and the normal response to insulin treatment in homozygous mice were markedly similar to those in the MODY3 disorder in humans. Moreover, K117E heterozygous mutant causes age-dependent glucose intolerance, which is similar to the pathogenesis of MODY3 as well. K117 mutants significantly reduced the overall transactivation and DNA binding capacity of HNF1α by disrupting dimerization. Collectively, our findings reveal a previously unappreciated role of POU domain of HNF1α in homodimerization and provide important clues for identifying the molecular basis of HNF1α-related diseases such as MODY3.

Following a diagnosis of type 1 diabetes (T1D), persisting C-peptide secretion leads to improved glycemic control and outcomes. Residual β-cell function is often assessed with serial mixed-meal tolerance tests, but these tests do not correlate well with clinical outcomes. Herein, we instead use β-cell glucose sensitivity (βGS) to assess changes in β-cell function, incorporating insulin secretion for a given serum glucose into the assessment of β-cell function. We evaluated changes in βGS in individuals enrolled in the placebo arm of 10 T1D trials performed at diabetes onset. We found that βGS showed a more rapid decline in children, as compared with adolescents and adults. Individuals in the top quartile of βGS baseline distribution had a slower rate in loss of glycemic control time over time. Notably, half of this group were children and adolescents. Finally, to identify predictors of glycemic control throughout follow-up, we ran multivariate Cox models and found that incorporating βGS significantly improved the overall model. Taken together, these data suggest that βGS may be of great utility in predicting those more likely to have a more robust clinical remission and may be of use in design of new-onset diabetes clinical trials and in evaluating response to therapies.

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic β-cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic β-cell-specific Cpe knockout mice (βCpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in βCpeKO islets remained intact. High-fat diet-fed βCpeKO mice showed weight gain and glucose tolerance comparable with those of Wt littermates. Notably, β-cell area was increased in chow-fed βCpeKO mice and β-cell replication was elevated in βCpeKO islets. Transcriptomic analysis of βCpeKO β-cells revealed elevated glycolysis and Hif1α-target gene expression. On high glucose challenge, β-cells from βCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin injections, βCpeKO mice had accelerated development of hyperglycemia with reduced β-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining β-cell function during the development of hyperglycemia.

Congenital hyperinsulinism (HI) is a genetic disorder in which pancreatic β-cell insulin secretion is excessive and results in hypoglycemia that, without treatment, can cause brain damage or death. Most patients with loss-of-function mutations in ABCC8 and KCNJ11, the genes encoding the β-cell ATP-sensitive potassium channel (KATP), are unresponsive to diazoxide, the only U.S. Food and Drug Administration-approved medical therapy and require pancreatectomy. The glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin-(9-39) is an effective therapeutic agent that inhibits insulin secretion in both HI and acquired hyperinsulinism. Previously, we identified a highly potent antagonist antibody, TB-001-003, which was derived from our synthetic antibody libraries that were designed to target G protein-coupled receptors. Here, we designed a combinatorial variant antibody library to optimize the activity of TB-001-003 against GLP-1R and performed phage display on cells overexpressing GLP-1R. One antagonist, TB-222-023, is more potent than exendin-(9-39), also known as avexitide. TB-222-023 effectively decreased insulin secretion in primary isolated pancreatic islets from a mouse model of hyperinsulinism, Sur1-/- mice, and in islets from an infant with HI, and increased plasma glucose levels and decreased the insulin to glucose ratio in Sur1-/- mice. These findings demonstrate that targeting GLP-1R with an antibody antagonist is an effective and innovative strategy for treatment of hyperinsulinism.

With rising incidence and prevalence of type 2 diabetes, prevention including identification of prospective biomarkers becomes increasingly relevant. Although ketone bodies recently received a renewed interest as potential biomarkers, data linking these metabolites to diabetes risk are scarce. Therefore, the present prospective study investigated a potential association between fasting ketone bodies and incident type 2 diabetes in the general population. This study from the PREVEND cohort included 3,307 participants from the general population initially free of diabetes or impaired fasting glucose. Baseline fasting ketone body concentrations were measured by nuclear magnetic resonance spectroscopy. One hundred twenty-six participants (3.8%) developed type 2 diabetes during a median (interquartile range) follow-up of 7.3 (6.3-7.6) years. In Kaplan-Meier analysis, sex-stratified ketone body levels strongly positively associated with incident type 2 diabetes, which was confirmed in Cox regression analyses adjusted for several potential confounders. There was no significant interaction by sex. Both 3-β-hydroxybutyrate and acetoacetate+acetone individually associated with incident type 2 diabetes. In conclusion, fasting plasma ketone body levels are strongly positively associated with incident type 2 diabetes in the general population independent of several other recognized risk factors. These results may have important implications for diabetes prevention including dietary strategies.

To investigate whether glucoregulatory neurons in the hypothalamus can sense and respond to physiological variation in the blood glucose (BG) level, we combined continuous arterial glucose monitoring with continuous measures of the activity of a specific subset of neurons located in the hypothalamic ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) obtained using fiber photometry. Data were collected in conscious, free-living mice during a 1-h baseline monitoring period and a subsequent 2-h intervention period during which the BG level was raised either by consuming a chow or a high-sucrose meal or by intraperitoneal glucose injection. Cross-correlation analysis revealed that, following a 60- to 90-s delay, interventions that raise the BG level reliably associate with reduced VMNPACAP neuron activity (P < 0.01). In addition, a strong positive correlation between BG and spontaneous VMNPACAP neuron activity was observed under basal conditions but with a much longer (∼25 min) temporal offset, consistent with published evidence that VMNPACAP neuron activation raises the BG level. Together, these findings are suggestive of a closed-loop system whereby VMNPACAP neuron activation increases the BG level; detection of a rising BG level, in turn, feeds back to inhibit these neurons. To our knowledge, these findings constitute the first evidence of a role in glucose homeostasis for glucoregulatory neurocircuits that, like pancreatic β-cells, sense and respond to physiological variation in glycemia.

Diabetic endothelial dysfunction associated with diminished endothelial nitric oxide (NO) synthase (eNOS) activity accelerates the development of atherosclerosis and cardiomyopathy. However, the approaches to restore eNOS activity and endothelial function in diabetes remain limited. The current study shows that enhanced expression of Krüppel-like factor 2 (KLF2), a shear stress-inducible transcription factor, effectively improves endothelial function through increasing NO bioavailability. KLF2 expression is suppressed in diabetic mouse aortic endothelium. Running exercise and simvastatin treatment induce endothelial KLF2 expression in db/db mice. Adenovirus-mediated endothelium-specific KLF2 overexpression enhances both endothelium-dependent relaxation and flow-mediated dilatation, while it attenuates oxidative stress in diabetic mouse arteries. KLF2 overexpression increases the phosphorylation of eNOS at serine 1177 and eNOS dimerization. RNA-sequencing analysis reveals that KLF2 transcriptionally upregulates genes that are enriched in the cyclic guanosine monophosphate-protein kinase G-signaling pathway, cAMP-signaling pathway, and insulin-signaling pathway, all of which are the upstream regulators of eNOS activity. Activation of the phosphoinositide 3-kinase-Akt pathway and Hsp90 contributes to KLF2-induced increase of eNOS activity. The present results suggest that approaches inducing KLF2 activation, such as physical exercise, are effective to restore eNOS activity against diabetic endothelial dysfunction.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes.

Diabetic retinopathy (DR), one of the most common microangiopathic complications in diabetes, causes severe visual damage among working-age populations. Retinal vascular endothelial cells, the key cell type in DR pathogenesis, are responsible for abnormal retinal angiogenesis in advanced stages of DR. The roles of exosomes in DR have been largely unknown. In this study, we report the first evidence that exosomes derived from the vitreous humor of patients with proliferative DR (PDR-exo) promote proliferation, migration, and tube formation of human retinal vascular endothelial cells (HRVECs). We identified long noncoding RNA (lncRNA) LOC100132249 enrichment in PDR-exo via high-throughput sequencing. This lncRNA, also mainly derived from HRVECs, promoted angiogenesis both in vitro and in vivo. Mechanistically, LOC100132249 acted as a competing endogenous sponge of miRNA-199a-5p (miR-199a-5p), thus regulating the endothelial-mesenchymal transition promoter SNAI1 via activation of the Wnt/β-catenin pathway and ultimately resulting in endothelial dysfunction. In conclusion, our findings underscored the pathogenic role of endothelial-derived exosomes via the LOC100132249/miR-199a-5p/SNAI1 axis in DR angiogenesis and may shed light on new therapeutic strategies for future treatment of DR.

Besides the secretion of fatty acids, lipolytic stimulation of adipocytes results in the secretion of triglyceride-rich extracellular vesicles and some free proteins (e.g., fatty acid binding protein 4) that, in sum, affect adipose homeostasis as well as the development of metabolic disease. At the mechanistic level, lipolytic signals activate p53 in an adipose triglyceride lipase-dependent manner, and pharmacologic inhibition of p53 attenuates adipocyte-derived extracellular vesicle (AdEV) protein and FABP4 secretion. Mass spectrometry analyses of the lipolytic secretome identified proteins involved in glucose and fatty acid metabolism, translation, chaperone activities, and redox control. Consistent with a role for p53 in adipocyte protein secretion, activation of p53 by the MDM2 antagonist nutlin potentiated AdEV particles and non-AdEV protein secretion from cultured 3T3-L1 or OP9 adipocytes while the levels of FABP4 and AdEV proteins were significantly reduced in serum from p53-/- mice compared with wild-type controls. The genotoxin doxorubicin increased AdEV protein and FABP4 secretion in a p53-dependent manner and DNA repair-depleted ERCC1-/Δ-haploinsufficient mice expressed elevated p53 in adipose depots, along with significantly increased serum FABP4. In sum, these data suggest that lipolytic signals, and cellular stressors such as DNA damage, facilitate AdEV protein and FABP4 secretion by adipocytes in a p53-dependent manner.

Obesity and insulin resistance are risk factors for the pathogenesis of type 2 diabetes (T2D). Here, we report that hepatic TGF-β1 expression positively correlates with obesity and insulin resistance in mice and humans. Hepatic TGF-β1 deficiency decreased blood glucose levels in lean mice and improved glucose and energy dysregulations in diet-induced obese (DIO) mice and diabetic mice. Conversely, overexpression of TGF-β1 in the liver exacerbated metabolic dysfunctions in DIO mice. Mechanistically, hepatic TGF-β1 and Foxo1 are reciprocally regulated: fasting or insulin resistance caused Foxo1 activation, increasing TGF-β1 expression, which, in turn, activated protein kinase A, stimulating Foxo1-S273 phosphorylation to promote Foxo1-mediated gluconeogenesis. Disruption of TGF-β1→Foxo1→TGF-β1 looping by deleting TGF-β1 receptor II in the liver or by blocking Foxo1-S273 phosphorylation ameliorated hyperglycemia and improved energy metabolism in adipose tissues. Taken together, our studies reveal that hepatic TGF-β1→Foxo1→TGF-β1 looping could be a potential therapeutic target for prevention and treatment of obesity and T2D.

Understanding the mechanisms linking steatosis to fibrosis is needed to establish a promising therapy against nonalcoholic fatty liver disease (NAFLD). The aim of this study was to clarify clinical features and hepatic gene expression signatures that predict and contribute to liver fibrosis development during the long-term real-world histological course of NAFLD in subjects with and without diabetes. A pathologist scored 342 serial liver biopsy samples from 118 subjects clinically diagnosed with NAFLD during a 3.8-year (SD 3.45 years, maximum 15 years) course of clinical treatment. At the initial biopsy, 26 subjects had simple fatty liver, and 92 had nonalcoholic steatohepatitis (NASH). In the trend analysis, the fibrosis-4 index (P < 0.001) and its components at baseline predicted the future fibrosis progression. In the generalized linear mixed model, an increase in HbA1c, but not BMI, was significantly associated with fibrosis progression (standardized coefficient 0.17 [95% CI 0.009-0.326]; P = 0.038) for subjects with NAFLD and diabetes. In gene set enrichment analyses, the pathways involved in zone 3 hepatocytes, central liver sinusoidal endothelial cells (LSECs), stellate cells, and plasma cells were coordinately altered in association with fibrosis progression and HbA1c elevation. Therefore, in subjects with NAFLD and diabetes, HbA1c elevation was significantly associated with liver fibrosis progression, independent of weight gain, which may be a valuable therapeutic target to prevent the pathological progression of NASH. Gene expression profiles suggest that diabetes-induced hypoxia and oxidative stress injure LSECs in zone 3 hepatocytes, which may mediate inflammation and stellate cell activation, leading to liver fibrosis.

Mitochondrial metabolism and oxidative respiration are crucial for pancreatic β-cell function and stimulus secretion coupling. Oxidative phosphorylation (OxPhos) produces ATP and other metabolites that potentiate insulin secretion. However, the contribution of individual OxPhos complexes to β-cell function is unknown. We generated β-cell-specific, inducible OxPhos complex knock-out (KO) mouse models to investigate the effects of disrupting complex I, complex III, or complex IV on β-cell function. Although all KO models had similar mitochondrial respiratory defects, complex III caused early hyperglycemia, glucose intolerance, and loss of glucose-stimulated insulin secretion in vivo. However, ex vivo insulin secretion did not change. Complex I and IV KO models showed diabetic phenotypes much later. Mitochondrial Ca2+ responses to glucose stimulation 3 weeks after gene deletion ranged from not affected to severely disrupted, depending on the complex targeted, supporting the unique roles of each complex in β-cell signaling. Mitochondrial antioxidant enzyme immunostaining increased in islets from complex III KO, but not from complex I or IV KO mice, indicating that severe diabetic phenotype in the complex III-deficient mice is causing alterations in cellular redox status. The present study highlights that defects in individual OxPhos complexes lead to different pathogenic outcomes.

The functional state of adipocytes plays a central role in regulating numerous important metabolic functions, including energy and glucose homeostasis. While white adipocytes store excess calories as fat (triglycerides) and release free fatty acids as a fuel source in times of need, brown and beige adipocytes (so-called thermogenic adipocytes) convert chemical energy stored in substrates (e.g., fatty acids or glucose) into heat, thus promoting energy expenditure. Like all other cell types, adipocytes express many G protein-coupled receptors (GPCRs) that are linked to four major functional classes of heterotrimeric G proteins (Gs, Gi/o, Gq/11, and G12/13). During the past few years, novel experimental approaches, including the use of chemogenetic strategies, have led to a series of important new findings regarding the metabolic consequences of activating or inhibiting distinct GPCR/G protein signaling pathways in white, brown, and beige adipocytes. This novel information should guide the development of novel drugs capable of modulating the activity of specific adipocyte GPCR signaling pathways for the treatment of obesity, type 2 diabetes, and related metabolic disorders.

The mechanisms accounting for the functional changes of α- and β-cells over the course of type 1 diabetes (T1D) development are largely unknown. Permitted by our established technology of high spatiotemporal resolution imaging of cytosolic Ca2+ ([Ca2+]c) dynamics on fresh pancreas tissue slices, we tracked the [Ca2+]c dynamic changes, as the assessment of function, in islet α- and β-cells of female nonobese diabetic (NOD) mice during the development of spontaneous diabetes. We showed that, during the phases of islet inflammation, 8 mmol/L glucose-induced synchronized short [Ca2+]c events in β-cells were diminished, whereas long [Ca2+]c events were gradually more triggerable at substimulatory 4 and 6 mmol/L glucose. In the islet destruction phase, the synchronized short [Ca2+]c events in a subset of β-cells resumed at high glucose condition, while the long [Ca2+]c events were significantly elevated already at substimulatory glucose concentrations. In the α-cells, the glucose sensitivity of the [Ca2+]c events persisted throughout the course of T1D development. At the late islet destruction phase, the α-cell [Ca2+]c events exhibited patterns of synchronicity. Our work has uncovered windows of functional recovery in β-cells and potential α-cells functional synchronization in NOD mice over the course of T1D development.

In obesity, CD11c+ innate immune cells are recruited to adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. We found that ablation of Gnas, the gene that encodes Gαs, in CD11c expressing cells protects mice from obesity, glucose intolerance, and insulin resistance. Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and did not require adaptive immunity. Loss of cAMP signaling was associated with increased adipose tissue norepinephrine and cAMP signaling, and prevention of catecholamine resistance. The adipose tissue had reduced expression of catecholamine transport and degradation enzymes, suggesting that the elevated norepinephrine resulted from decreased catabolism. Collectively, our results identified an important role for cAMP signaling in CD11c+ innate immune cells in whole-body metabolism by controlling norepinephrine levels in white adipose tissue, modulating catecholamine-induced lipolysis and increasing thermogenesis, which, together, created a lean phenotype.