Obesity-induced lipid overload in cardiomyocytes contributes to profound oxidative stress and cardiomyopathy, culminating in heart failure. In this study, we investigate a novel mechanism whereby lipids accumulate in cardiomyocytes, and seek the relevant treatment strategies. P21-activated kinase 3 (PAK3) was elevated in obese human myocardium, and the murine hearts and cardiomyocytes upon diet- or fatty acid-induced stress, respectively. Mice with cardiac-specific overexpression of PAK3 were more susceptible to the development of cardiac dysfunction upon diet stress, at least partially, because of increased deposition of toxic lipids within the myocardium. Mechanistically, PAK3 promoted the nuclear expression of sterol regulatory element binding protein 1c (SREBP1c) through activation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase β-1 (S6K1) pathway in cardiomyocytes, resulting in abnormal lipid genes profile, accumulation of excessive lipids, and oxidative stress. More importantly, PAK3 knockdown attenuated fatty acid-induced lipotoxicity and cell death in rat and human cardiomyocytes. More importantly, the S6K1 or SREBP1c inhibitor alleviated PAK3-triggered intracellular lipid overload and cardiac dysfunction under obese stress. Collectively, we have demonstrated that PAK3 impairs myocardial lipid homeostasis, while inhibition of cardiac lipotoxicity mitigates cardiac dysfunction. Our study provides a promising therapeutic strategy for ameliorating obesity cardiomyopathy.

We postulated that type 2 diabetes (T2D) predisposes patients to exocrine pancreatic diseases through (epi)genetic mechanisms. We explored the methylome (using MethylationEPIC arrays) of the exocrine pancreas in 141 donors, assessing the impact of T2D. An epigenome-wide association study of T2D identified hypermethylation in an enhancer of the pancreatic lipase-related protein 1 (PNLIPRP1) gene, associated with decreased PNLIPRP1 expression. PNLIPRP1 null variants (found in 191,000 participants in the UK Biobank) were associated with elevated glycemia and LDL cholesterol. Mendelian randomization using 2.5M SNP Omni arrays in 111 donors revealed that T2D was causal of PNLIPRP1 hypermethylation, which in turn was causal of LDL cholesterol. Additional AR42J rat exocrine cell analyses demonstrated that Pnliprp1 knockdown induced acinar-to-ductal metaplasia, a known prepancreatic cancer state, and increased cholesterol levels, reversible with statin. This (epi)genetic study suggests a role for PNLIPRP1 in human metabolism and exocrine pancreatic function, with potential implications for pancreatic diseases.

The aim of this study was to investigate the associations among sex hormone-binding globulin (SHBG), visceral adipose tissue (VAT), liver fat content, and risk of type 2 diabetes (T2D). In the Netherlands Epidemiology of Obesity study, 5,690 women (53%) and men (47%) without preexisting diabetes were included and followed for incident T2D. SHBG concentrations were measured in all participants, VAT was measured using MRI, and liver fat content was measured using proton magnetic resonance spectroscopy in a random subset of 1,822 participants. We examined associations between SHBG and liver fat using linear regression and bidirectional Mendelian randomization analyses and between SHBG and T2D using Cox regression adjusted for confounding and additionally for VAT and liver fat to examine mediation. Mean age was 56 (SD 6) years, mean BMI was 30 (SD 4) kg/m2, median SHBG was 47 (interquartile range [IQR] 34-65) nmol/L in women and 34 (26-43) nmol/L in men, and median liver fat was 3.4% (IQR 1.6-8.2%) in women and 6.0% (2.9-13.5%) in men. Compared with the highest SHBG quartile, liver fat was 2.9-fold (95% CI 2.4, 3.4) increased in women and 1.6-fold (95% CI 1.3, 1.8) increased in men, and the hazard ratio of T2D was 4.9 (95% CI 2.4, 9.9) in women and 1.8 (1.1, 2.9) in men. Genetically predicted SHBG was associated with liver fat content (women: SD -0.45 [95% CI -0.55, -0.35]; men: natural logarithm, -0.25 [95% CI -0.34, -0.16]). VAT and liver fat together mediated 43% (women) and 60% (men) of the SHBG-T2D association. To conclude, in a middle-aged population with overweight, the association between low SHBG and increased risk of T2D was, for a large part, mediated by increased VAT and liver fat.

Type 1 diabetes (T1D) results from β-cell destruction due to autoimmunity. It has been proposed that β-cell loss is relatively quiescent in the early years after seroconversion to islet antibody positivity (stage 1), with accelerated β-cell loss only developing around 6-18 months prior to clinical diagnosis. This construct implies that immunointervention in this early stage will be of little benefit, since there is little disease activity to modulate. Here, we argue that the apparent lack of progression in early-stage disease may be an artifact of the modality of assessment used. When substantial β-cell function remains, the standard assessment, the oral glucose tolerance test, represents a submaximal stimulus and underestimates the residual function. In contrast, around the time of diagnosis, glucotoxicity exerts a deleterious effect on insulin secretion, giving the impression of disease acceleration. Once glucotoxicity is relieved by insulin therapy, β-cell function partially recovers (the honeymoon effect). However, evidence from recent trials suggests that glucose control has little effect on the underlying disease process. We therefore hypothesize that the autoimmune destruction of β-cells actually progresses at a more or less constant rate through all phases of T1D and that early-stage immunointervention will be both beneficial and desirable.

The generation of stem cell-derived β-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human β-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.

Persistent enterovirus B infection has been proposed as an important contributor to the etiology of type 1 diabetes. We leveraged extensive bulk RNA-sequencing (RNA-seq) data from α-, β-, and exocrine cells, as well as islet single-cell RNA-seq data from the Human Pancreas Analysis Program (HPAP), to evaluate the presence of enterovirus B sequences in the pancreas of patients with type 1 diabetes and prediabetes (no diabetes but positive for autoantibodies). We examined all available HPAP data for either assay type, including donors without diabetes and with type 1 and type 2 diabetes. To assess the presence of viral reads, we analyzed all reads not mapping to the human genome with the taxonomic classification system Kraken2 and its full viral database augmented to encompass representatives for all 28 enterovirus B serotypes for which a complete genome is available. As a secondary approach, we input the same sequence reads into the STAR aligner using these 28 enterovirus B genomes as the reference. No enterovirus B sequences were detected by either approach in any of the 243 bulk RNA libraries or in any of the 79 single-cell RNA libraries. While we cannot rule out the possibility of a very-low-grade persistent enterovirus B infection in the donors analyzed, our data do not support the notion of chronic viral infection by these viruses as a major driver of type 1 diabetes.

Maturation of postnatal β-cells is regulated in a cell-autonomous manner, and metabolically stressed β-cells regress to an immature state, ensuring defective β-cell function and the onset of type 2 diabetes. The molecular mechanisms connecting the nutritional transition to β-cell maturation remain largely unknown. Here, we report a mature form of miRNA (miR-203)/ZBTB20/MAFA regulatory axis that mediates the β-cell maturation process. We show that the level of the mature form of miRNA (miR-203) in β-cells changes during the nutritional transition and that miR-203 inhibits β-cell maturation at the neonatal stage and under high-fat diet conditions. Using single-cell RNA sequencing, we demonstrated that miR-203 elevation promoted the transition of immature β-cells into CgBHi endocrine cells while suppressing gene expressions associated with β-cell maturation in a ZBTB20/MAFA-dependent manner. ZBTB20 is an authentic target of miR-203 and transcriptionally upregulates MAFA expression. Manipulating the miR-203/ZBTB20/MAFA axis may therefore offer a novel strategy for boosting functional β-cell numbers to alleviate diabetes.

A hallmark of type 2 diabetes (T2D) is endocrine islet β-cell failure, which can occur via cell dysfunction, loss of identity, and/or death. How each is induced remains largely unknown. We used mouse β-cells deficient for myelin transcription factors (Myt TFs; including Myt1, -2, and -3) to address this question. We previously reported that inactivating all three Myt genes in pancreatic progenitor cells (MytPancΔ) caused β-cell failure and late-onset diabetes in mice. Their lower expression in human β-cells is correlated with β-cell dysfunction, and single nucleotide polymorphisms in MYT2 and MYT3 are associated with a higher risk of T2D. We now show that these Myt TF-deficient postnatal β-cells also dedifferentiate by reactivating several progenitor markers. Intriguingly, mosaic Myt TF inactivation in only a portion of islet β-cells did not result in overt diabetes, but this created a condition where Myt TF-deficient β-cells remained alive while activating several markers of Ppy-expressing islet cells. By transplanting MytPancΔ islets into the anterior eye chambers of immune-compromised mice, we directly show that glycemic and obesity-related conditions influence cell fate, with euglycemia inducing several Ppy+ cell markers and hyperglycemia and insulin resistance inducing additional cell death. These findings suggest that the observed β-cell defects in T2D depend not only on their inherent genetic/epigenetic defects but also on the metabolic load.

It is not completely clear which organs are responsible for glucagon elimination in humans, and disturbances in the elimination of glucagon could contribute to the hyperglucagonemia observed in chronic liver disease and chronic kidney disease (CKD). Here, we evaluated kinetics and metabolic effects of exogenous glucagon in individuals with stage 4 CKD (n = 16), individuals with Child-Pugh A-C cirrhosis (n = 16), and matched control individuals (n = 16), before, during, and after a 60-min glucagon infusion (4 ng/kg/min). Individuals with CKD exhibited a significantly lower mean metabolic clearance rate of glucagon (14.0 [95% CI 12.2;15.7] mL/kg/min) compared with both individuals with cirrhosis (19.7 [18.1;21.3] mL/kg/min, P < 0.001) and control individuals (20.4 [18.1;22.7] mL/kg/min, P < 0.001). Glucagon half-life was significantly prolonged in the CKD group (7.5 [6.9;8.2] min) compared with individuals with cirrhosis (5.7 [5.2;6.3] min, P = 0.002) and control individuals (5.7 [5.2;6.3] min, P < 0.001). No difference in the effects of exogenous glucagon on plasma glucose, amino acids, or triglycerides was observed between groups. In conclusion, CKD, but not liver cirrhosis, leads to a significant reduction in glucagon clearance, supporting the kidneys as a primary site for human glucagon elimination.

A ketogenic diet (KD) can induce weight loss and improve glycemic regulation, potentially reducing the risk of type 2 diabetes development. To elucidate the underlying mechanisms behind these beneficial effects of a KD, we investigated the impact of a KD on organ-specific insulin sensitivity (IS) in skeletal muscle, liver, and adipose tissue. We hypothesized that a KD would increase IS in skeletal muscle. The study included 11 individuals with obesity who underwent a randomized, crossover trial with two 3-week interventions: 1) a KD and 2) a standard diet. Skeletal muscle IS was quantified as the increase in glucose disposal during a hyperinsulinemic-euglycemic clamp (HEC). Hepatic IS and adipose tissue IS were quantified as the relative suppression of endogenous glucose production (EGP) and the relative suppression of palmitate flux during the HEC. The KD led to a 2.2-kg weight loss and increased insulin-stimulated glucose disposal, whereas the relative suppression of EGP during the HEC was similar. In addition, the KD decreased insulin-mediated suppression of lipolysis. In conclusion, a KD increased skeletal muscle IS in individuals with obesity.

Growth arrest-specific 6 (GAS6) is a secreted protein that acts as a ligand for TAM receptors (TYRO3, AXL, and MERTK). In humans, GAS6 circulating levels and genetic variations in GAS6 are associated with hyperglycemia and increased risk of type 2 diabetes. However, the mechanisms by which GAS6 influences glucose metabolism are not understood. Here, we show that Gas6 deficiency in mice increases insulin sensitivity and protects from diet-induced insulin resistance. Conversely, increasing GAS6 circulating levels is sufficient to reduce insulin sensitivity in vivo. GAS6 inhibits the activation of the insulin receptor (IR) and reduces insulin response in muscle cells in vitro and in vivo. Mechanistically, AXL and IR form a complex, while GAS6 reprograms signaling pathways downstream of IR. This results in increased IR endocytosis following insulin treatment. This study contributes to a better understanding of the cellular and molecular mechanisms by which GAS6 and AXL influence insulin sensitivity.

Overnutrition has gradually become the primary causative factor in nonalcoholic fatty liver disease (NAFLD). However, how nutritional signals are integrated to orchestrate the transcriptional programs important for NAFLD progression remains poorly understood. We identified hepatic BAF60b as a lipid-sensitive subunit of the switch/sucrose nonfermentable chromatin-remodeling complex that is negatively associated with liver steatosis in mice and humans. Hepatic BAF60b deficiency promotes high-fat diet (HFD)-induced liver steatosis in mice, whereas transgenic expression of BAF60b in the liver attenuates HFD-induced obesity and NAFLD, both accompanied by a marked regulation of peroxisome proliferator-activated receptor γ (PPARγ) expression. Mechanistically, through motif analysis of liver assay for transposase-accessible chromatin sequencing and multiple validation experiments, we identified C/EBPβ as the transcription factor that interacts with BAF60b to suppress Pparγ gene expression, thereby controlling hepatic lipid accumulation and NAFLD progression. This work identifies hepatic BAF60b as a negative regulator of liver steatosis through C/EBPβ-dependent chromatin remodeling.

Diabetic retinopathy is the most common microvascular complication caused by chronic hyperglycemia and is a leading cause of blindness; however, the underlying molecular mechanism has not been clearly elucidated. Therefore, we investigated whether regulation of AMPK and GAPDH by transglutaminase 2 (TGase2) is important for hyperglycemia-induced microvascular leakage in the diabetic retina. In human retinal endothelial cells (HRECs) and diabetic mouse retinas, we found that TGase2, activated by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels, played an essential role in hyperglycemia-induced vascular leakage. ROS generation and TGsae2 activation were involved in hyperglycemia-induced AMPK dephosphorylation, which resulted in vascular endothelial-cadherin (VE-cadherin) disassembly and increased fluorescein isothiocyanate-dextran extravasation. Furthermore, high glucose-induced TGase2 activation suppressed GAPDH activity, determined by an on-chip activity assay, through inhibition of AMPK, which induced VE-cadherin disassembly and endothelial permeability in HRECs. Overall, our findings suggest that inhibition of AMPK and GAPDH by TGase2 plays a pivotal role in hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

Inducible pluripotent stem cell-derived human β-like cells (BLCs) hold promise for both therapy and disease modeling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single-cell electrophysiological tools to evaluate function of BLCs from pioneer protocols that can be easily adapted to more differentiated BLCs. The multi-electrode arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs, like primary β-cells, are electrically coupled and produce slow potential (SP) signals that are closely linked to insulin secretion. We also used high-resolution single-cell patch clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents, and found that they were comparable with those in primary β- and EndoC-βH1 cells. The KATP channel conductance is greater than in human primary β-cells, which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes-protective SLC30A8 allele (p.Lys34Serfs50*) and found that BLCs with this allele have stronger electrical coupling activity. Our data suggest that BLCs can be used to evaluate the functional impact of genetic variants on β-cell function and coupling.

The islets of Langerhans reside within the endocrine pancreas as highly vascularized microorgans that are responsible for the secretion of key hormones, such as insulin and glucagon. Islet function relies on a range of dynamic molecular processes that include Ca2+ waves, hormone pulses, and complex interactions between islet cell types. Dysfunction of these processes results in poor maintenance of blood glucose homeostasis and is a hallmark of diabetes. Recently, the development of optogenetic methods that rely on light-sensitive molecular actuators has allowed perturbation of islet function with near physiological spatiotemporal acuity. These actuators harness natural photoreceptor proteins and their engineered variants to manipulate mouse and human cells that are not normally light-responsive. Until recently, optogenetics in islet biology has primarily focused on controlling hormone production and secretion; however, studies on further aspects of islet function, including paracrine regulation between islet cell types and dynamics within intracellular signaling pathways, are emerging. Here, we discuss the applicability of optogenetics to islets cells and comprehensively review seminal as well as recent work on optogenetic actuators and their effects in islet function and diabetes mellitus.

We investigated whether characterization of full-length GAD (f-GADA) antibody (GADA) responses could identify early insulin requirement in adult-onset diabetes. In 179 f-GADA-positive participants diagnosed with type 2 diabetes, we assessed associations of truncated GADA (t-GADA) positivity, f-GADA IgG subclasses, and f-GADA affinity with early insulin requirement (<5 years), type 1 diabetes genetic risk score (T1D GRS), and C-peptide. t-GADA positivity was lower in f-GADA-positive without early insulin in comparison with f-GADA-positive type 2 diabetes requiring insulin within 5 years, and T1D (75% vs. 91% and 95% respectively, P < 0.0001). t-GADA positivity (in those f-GADA positive) identified a group with a higher T1D genetic susceptibility (mean T1D GRS 0.248 vs. 0.225, P = 0.003), lower C-peptide (1,156 pmol/L vs. 4,289 pmol/L, P = 1 × 10-7), and increased IA-2 antigen positivity (23% vs. 6%, P = 0.03). In survival analysis, t-GADA positivity was associated with early insulin requirement compared with those only positive for f-GADA, independently from age of diagnosis, f-GADA titer, and duration of diabetes (adjusted hazard ratio 5.7 [95% CI 1.4, 23.5], P = 0.017). The testing of t-GADA in f-GADA-positive individuals with type 2 diabetes identifies those who have genetic and clinical characteristics comparable to T1D and stratifies those at higher risk of early insulin requirement.

Treatment with glucagon-like peptide 1 receptor agonists reduces liver steatosis and cardiometabolic risk (CMR). Few data are available on lipid metabolism, and no information is available on the postprandial lipidomic profile. Thus, we investigated how exenatide treatment changes lipid metabolism and composition during fasting and after a mixed-meal tolerance test (MMTT) in adults with severe obesity without diabetes. Thirty individuals (26 females and 4 males, 30-60 years old, BMI >40 kg/m2, HbA1c 5.76%) were assigned (1:1) to diet with exenatide 10 μg twice daily treatment (n = 15) or without treatment as control (n = 15) for 3 months. Fasting and postprandial lipidomic profile (by liquid chromatography quadrupole time-of-flight mass spectrometry) and fatty acid metabolism (following a 6-h MMTT/tracer study) and composition (by gas chromatography-mass spectrometry) were evaluated before and after treatment. Both groups had slight weight loss (-5.5% vs. -1.9%, exenatide vs. control; P = 0.052). During fasting, exenatide, compared with control, reduced some ceramides (CERs) and lysophosphatidylcholines (LPCs) previously associated with CMR, while relatively increasing unsaturated phospholipid species (phosphatidylcholine [PC], LPC) with protective effects on CMR, although concentrations of total lipid species were unchanged. During MMTT, both groups showed suppressed lipolysis equal to baseline, but exenatide significantly lowered free fatty acid clearance and postprandial triacyclglycerol (TAG) concentrations, particularly saturated TAGs with 44-54 carbons. Exenatide also reduced some postprandial CERs, PCs, and LPCs previously linked to CMR. These changes in lipidomic profile remained statistically significant after adjusting for weight loss. Exenatide improved fasting and postprandial lipidomic profiles associated with CMR mainly by reducing saturated postprandial TAGs and CERs independently of weight loss and diabetes.

The stress response protein regulated in development and DNA damage response 1 (REDD1) has emerged as a key player in the pathogenesis of diabetes. Diabetes upregulates REDD1 in a variety of insulin-sensitive tissues, where the protein acts to inhibit signal transduction downstream of the insulin receptor. REDD1 functions as a cytosolic redox sensor that suppresses Akt/mTORC1 signaling to reduce energy expenditure in response to cellular stress. Whereas a transient increase in REDD1 contributes to an adaptive cellular response, chronically elevated REDD1 levels are implicated in disease progression. Recent studies highlight the remarkable benefits of both whole-body and tissue-specific REDD1 deletion in preclinical models of type 1 and type 2 diabetes. In particular, REDD1 is necessary for the development of glucose intolerance and the consequent rise in oxidative stress and inflammation. Here, we review studies that support a role for chronically elevated REDD1 levels in the development of diabetes complications, reflect on limitations of prior therapeutic approaches targeting REDD1 in patients, and discuss potential opportunities for future interventions to improve the lives of people living with diabetes. This article is part of a series of Perspectives that report on research funded by the American Diabetes Association Pathway to Stop Diabetes program.

Increased plasma levels of glucagon (hyperglucagonemia) promote diabetes development but are also observed in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). This may reflect hepatic glucagon resistance toward amino acid catabolism. A clinical test for measuring glucagon resistance has not been validated. We evaluated our glucagon sensitivity (GLUSENTIC) test, which consists of 2 study days: a glucagon injection and measurements of plasma amino acids and an infusion of mixed amino acids and subsequent calculation of the GLUSENTIC index (primary outcome measure) from measurements of glucagon and amino acids. To distinguish glucagon-dependent from insulin-dependent actions on amino acid metabolism, we also studied patients with type 1 diabetes (T1D). The δ-decline in total amino acids was 49% lower in MASLD following exogenous glucagon (P = 0.01), and the calculated GLUSENTIC index was 34% lower in MASLD (P < 0.0001) but not T1D (P > 0.99). In contrast, glucagon-induced glucose increments were similar in control participants and participants with MASLD (P = 0.41). The GLUSENTIC test and index may be used to measure glucagon resistance in individuals with obesity and MASLD.

Insulin is a key regulator of amino acid metabolism. Many plasma amino acids, including lysine and its metabolite, α-aminoadipic acid (α-AA), a predictor for developing diabetes, are elevated in insulin resistance (IR). In 18 overweight women with IR and polycystic ovary syndrome compared with 12 lean control women, high physiological insulin during a euglycemic clamp failed to normalize many elevated amino acid metabolites, including branched-chain and aromatic amino acids, α-aminobutyric acid, and lysine, but normalized α-AA. To understand the underpinnings of differential responses of lysine and its metabolic product α-AA to high physiological insulin in IR compared with control participants, we developed a kinetic model using [α-15N1]-lysine and [13C1]-α-AA as tracers and measured the two tracers simultaneously in α-AA by innovative mass spectrometry. High insulin increased lysine conversion to α-AA in the IR and control groups but failed to normalize plasma lysine concentrations in IR due to a decrease in lysine metabolic clearance rate (MCR). In contrast, despite higher conversion rates of lysine to α-AA by high insulin, α-AA concentration decreased in IR because of the sustained greater MCR of α-AA. The abnormal amino acids and metabolites, even while on high physiological insulin, could potentially explain many functional derangements in IR.