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.

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.

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.

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.

Exercise is a first-line treatment for type 2 diabetes and preserves β-cell function by hitherto unknown mechanisms. We postulated that proteins from contracting skeletal muscle may act as cellular signals to regulate pancreatic β-cell function. We used electric pulse stimulation (EPS) to induce contraction in C2C12 myotubes and found that treatment of β-cells with EPS-conditioned medium enhanced glucose-stimulated insulin secretion (GSIS). Transcriptomics and subsequent targeted validation revealed growth differentiation factor 15 (GDF15) as a central component of the skeletal muscle secretome. Exposure to recombinant GDF15 enhanced GSIS in cells, islets, and mice. GDF15 enhanced GSIS by upregulating the insulin secretion pathway in β-cells, which was abrogated in the presence of a GDF15 neutralizing antibody. The effect of GDF15 on GSIS was also observed in islets from GFRAL-deficient mice. Circulating GDF15 was incrementally elevated in patients with pre- and type 2 diabetes and positively associated with C-peptide in humans with overweight or obesity. Six weeks of high-intensity exercise training increased circulating GDF15 concentrations, which positively correlated with improvements in β-cell function in patients with type 2 diabetes. Taken together, GDF15 can function as a contraction-induced protein that enhances GSIS through activating the canonical signaling pathway in a GFRAL-independent manner.

Obesity is a global health threat, and the induction of white adipose tissue (WAT) browning presents a promising therapeutic method for it. Recent publications revealed the essential role of protein arginine methyltransferase 4 (PRMT4) in lipid metabolism and adipogenesis, but its involvement in WAT browning has not been investigated. Our initial studies found that the expression of PRMT4 in adipocytes was upregulated in cold-induced WAT browning but downregulated in obesity. Besides, PRMT4 overexpression in inguinal adipose tissue accelerated WAT browning and thermogenesis to protect against high-fat diet-induced obesity and metabolic disruptions. Mechanistically, our work demonstrated that PRMT4 methylated peroxisome proliferator-activated receptor-γ (PPARγ) on Arg240 to enhance its interaction with the coactivator PR domain-containing protein 16 (PRDM16), leading to the increased expression of thermogenic genes. Taken together, our results uncover the essential role of the PRMT4/PPARγ/PRDM16 axis in the pathogenesis of WAT browning.

Lactate is an important metabolic substrate for sustaining brain energy requirements when glucose supplies are limited. Recurring exposure to hypoglycemia (RH) raises lactate levels in the ventromedial hypothalamus (VMH), which contributes to counterregulatory failure. However, the source of this lactate remains unclear. The current study investigates whether astrocytic glycogen serves as the major source of lactate in the VMH of RH rats. By decreasing the expression of a key lactate transporter in VMH astrocytes of RH rats, we reduced extracellular lactate concentrations, suggesting excess lactate was locally produced from astrocytes. To determine whether astrocytic glycogen serves as the major source of lactate, we chronically delivered either artificial extracellular fluid or 1,4-dideoxy-1,4-imino-d-arabinitol to inhibit glycogen turnover in the VMH of RH animals. Inhibiting glycogen turnover in RH animals prevented the rise in VMH lactate and the development of counterregulatory failure. Lastly, we noted that RH led to an increase in glycogen shunt activity in response to hypoglycemia and elevated glycogen phosphorylase activity in the hours following a bout of hypoglycemia. Our data suggest that dysregulation of astrocytic glycogen metabolism following RH may be responsible, at least in part, for the rise in VMH lactate levels.

Type 1 diabetes (T1D) is caused by the immune-mediated loss of pancreatic β-cells that produce insulin. The latest advances in stem cell (SC) β-cell differentiation methods have made a cell replacement therapy for T1D feasible. However, recurring autoimmunity would rapidly destroy transplanted SC β-cells. A promising strategy to overcome immune rejection is to genetically engineer SC β-cells. We previously identified Renalase (Rnls) as a novel target for β-cell protection. Here we show that Rnls deletion endows β-cells with the capacity to modulate the metabolism and function of immune cells within the local graft microenvironment. We used flow cytometry and single-cell RNA sequencing to characterize β-cell graft-infiltrating immune cells in a mouse model for T1D. Loss of Rnls within transplanted β-cells affected both the composition and the transcriptional profile of infiltrating immune cells in favor of an anti-inflammatory profile with decreased antigen-presenting capacity. We propose that changes in β-cell metabolism mediate local immune regulation and that this feature could be exploited for therapeutic goals.

Sphingolipids are thought to promote skeletal muscle insulin resistance. Deoxysphingolipids (dSLs) are atypical sphingolipids that are increased in the plasma of individuals with type 2 diabetes and cause β-cell dysfunction in vitro. However, their role in human skeletal muscle is unknown. We found that dSL species are significantly elevated in muscle of individuals with obesity and type 2 diabetes compared with athletes and lean individuals and are inversely related to insulin sensitivity. Furthermore, we observed a significant reduction in muscle dSL content in individuals with obesity who completed a combined weight loss and exercise intervention. Increased dSL content in primary human myotubes caused a decrease in insulin sensitivity associated with increased inflammation, decreased AMPK phosphorylation, and altered insulin signaling. Our findings reveal a central role for dSL in human muscle insulin resistance and suggest dSLs as therapeutic targets for the treatment and prevention of type 2 diabetes.