We investigated the link between enhancement of SI (by hyperinsulinemic-euglycemic clamp) and muscle metabolites after 12 weeks of aerobic (high-intensity interval training [HIIT]), resistance training (RT), or combined training (CT) exercise in 52 lean healthy individuals. Muscle RNA sequencing revealed a significant association between SI after both HIIT and RT and the branched-chain amino acid (BCAA) metabolic pathway. Concurrently with increased expression and activity of branched-chain ketoacid dehydrogenase enzyme, many muscle amino metabolites, including BCAAs, glutamate, phenylalanine, aspartate, asparagine, methionine, and γ-aminobutyric acid, increased with HIIT, supporting the substantial impact of HIIT on amino acid metabolism. Short-chain C3 and C5 acylcarnitines were reduced in muscle with all three training modes, but unlike RT, both HIIT and CT increased tricarboxylic acid metabolites and cardiolipins, supporting greater mitochondrial activity with aerobic training. Conversely, RT and CT increased more plasma membrane phospholipids than HIIT, suggesting a resistance exercise effect on cellular membrane protection against environmental damage. Sex and age contributed modestly to the exercise-induced changes in metabolites and their association with cardiometabolic parameters. Integrated transcriptomic and metabolomic analyses suggest various clusters of genes and metabolites are involved in distinct effects of HIIT, RT, and CT. These distinct metabolic signatures of different exercise modes independently link each type of exercise training to improved SI and cardiometabolic risk.

In contrast to the well-defined biological feedback loops controlling glucose, the mechanisms by which the body responds to changes in fatty acid availability are less clearly defined. Growth differentiating factor 15 (GDF15) suppresses the consumption of diets high in fat but is paradoxically increased in obese mice fed a high-fat diet. Given this interrelationship, we investigated whether diets high in fat could directly increase GDF15 independently of obesity. We found that fatty acids increase GDF15 levels dose dependently, with the greatest response observed with linolenic acid. GDF15 mRNA expression was modestly increased in the gastrointestinal tract; however, kidney GDF15 mRNA was ∼1,000-fold higher and was increased by more than threefold, with subsequent RNAscope analysis showing elevated expression within the cortex and outer medulla. Treatment of wild-type mice with linolenic acid reduced food intake and body mass; however, this effect disappeared in mice lacking the GDF15 receptor GFRAL. An equal caloric load of glucose did not suppress food intake or reduce body mass in either wild-type or GFRAL-knockout mice. These data indicate that fatty acids such as linolenic acid increase GDF15 and suppress food intake through a mechanism requiring GFRAL. These data suggest that a primary physiological function of GDF15 may be as a fatty acid sensor designed to protect cells from fatty acid overload.

Diabetes is characterized by elevation of plasma glucose due to an insufficiency of the hormone insulin and is associated with both inadequate insulin secretion and impaired insulin action. The Banting Medal for Scientific Achievement Commemorates the work of Sir Frederick Banting, a member of the team that first used insulin to treat a patient with diabetes almost exactly one hundred years ago on 11 January 1922. This article is based on my Banting lecture of 2022 and concerns the mechanism of glucose-stimulated insulin secretion from pancreatic β-cells, with an emphasis on the metabolic regulation of the KATP channel. This channel plays a central role in insulin release. Its closure in response to metabolically generated changes in the intracellular concentrations of ATP and MgADP stimulates β-cell electrical activity and insulin granule exocytosis. Activating mutations in KATP channel genes that impair the ability of the channel to respond to ATP give rise to neonatal diabetes. Impaired KATP channel regulation may also play a role in type 2 diabetes. I conjecture that KATP channel closure in response to glucose is reduced because of impaired glucose metabolism, which fails to generate a sufficient increase in ATP. Consequently, glucose-stimulated β-cell electrical activity is less. As ATP is also required for insulin granule exocytosis, both reduced exocytosis and less β-cell electrical activity may contribute to the reduction in insulin secretion. I emphasize that what follows is not a definitive review of the topic but a personal account of the contribution of my team to the field that is based on my Banting lecture.

Many people living with diabetes also have nonalcoholic fatty liver disease (NAFLD). Interleukin-6 (IL-6) is involved in both diseases, interacting with both membrane-bound (classical) and circulating (trans-signaling) soluble receptors. We investigated whether secretion of IL-6 trans-signaling coreceptors are altered in NAFLD by diabetes and whether this might associate with the severity of fatty liver disease. Secretion patterns were investigated with use of human hepatocyte, stellate, and monocyte cell lines. Associations with liver pathology were investigated in two patient cohorts: 1) biopsy-confirmed steatohepatitis and 2) class 3 obesity. We found that exposure of stellate cells to high glucose and palmitate increased IL-6 and soluble gp130 (sgp130) secretion. In line with this, plasma sgp130 in both patient cohorts positively correlated with HbA1c, and subjects with diabetes had higher circulating levels of IL-6 and trans-signaling coreceptors. Plasma sgp130 strongly correlated with liver stiffness and was significantly increased in subjects with F4 fibrosis stage. Monocyte activation was associated with reduced sIL-6R secretion. These data suggest that hyperglycemia and hyperlipidemia can directly impact IL-6 trans-signaling and that this may be linked to enhanced severity of NAFLD in patients with concomitant diabetes.

Maternal consumption of a Western-style diet (mWD) during pregnancy alters fatty acid metabolism and reduces insulin sensitivity in fetal skeletal muscle. The long-term impact of these fetal adaptations and the pathways underlying disordered lipid metabolism are incompletely understood. Therefore, we tested whether a mWD chronically fed to lean, insulin-sensitive adult Japanese macaques throughout pregnancy and lactation would impact skeletal muscle oxidative capacity and lipid metabolism in adolescent offspring fed a postweaning (pw) Western-style diet (WD) or control diet (CD). Although body weight was not different, retroperitoneal fat mass and subscapular skinfold thickness were significantly higher in pwWD offspring consistent with elevated fasting insulin and glucose. Maximal complex I (CI)-dependent respiration in muscle was lower in mWD offspring in the presence of fatty acids, suggesting that mWD impacts muscle integration of lipid with nonlipid oxidation. Abundance of all five oxidative phosphorylation complexes and VDAC, but not ETF/ETFDH, were reduced with mWD, partially explaining the lower respiratory capacity with lipids. Muscle triglycerides increased with pwWD; however, the fold increase in lipid saturation, 1,2-diacylglycerides, and C18 ceramide compared between pwCD and pwWD was greatest in mWD offspring. Reductions in CI abundance and VDAC correlated with reduced markers of oxidative stress, suggesting that these reductions may be an early-life adaptation to mWD to mitigate excess reactive oxygen species. Altogether, mWD, independent of maternal obesity or insulin resistance, results in sustained metabolic reprogramming in offspring muscle despite a healthy diet intervention.

Insulin activates insulin receptor (IR) signaling and subsequently triggers IR endocytosis to attenuate signaling. Cell division regulators MAD2, BUBR1, and p31comet promote IR endocytosis on insulin stimulation. Here, we show that genetic ablation of the IR-MAD2 interaction in mice delays IR endocytosis, increases IR levels, and prolongs insulin action at the cell surface. This in turn causes a defect in insulin clearance and increases circulating insulin levels, unexpectedly increasing glucagon levels, which alters glucose metabolism modestly. Disruption of the IR-MAD2 interaction increases serum fatty acid concentrations and hepatic fat accumulation in fasted male mice. Furthermore, disruption of the IR-MAD2 interaction distinctly changes metabolic and transcriptomic profiles in the liver and adipose tissues. Our findings establish the function of cell division regulators in insulin signaling and provide insights into the metabolic functions of IR endocytosis.

Both long- and short-term glycemic variability have been associated with incident diabetes complications. We evaluated their relative and potential additive effects on incident renal complications in the Action to Control Cardiovascular Risk in Diabetes trial. A marker of short-term glycemic variability, 1,5-anhydroglucitol (1,5-AG), was measured in 4,000 random 12-month postrandomization plasma samples (when hemoglobin A1c [HbA1c] was stable). Visit-to-visit fasting plasma glucose coefficient of variation (CV-FPG) was determined from 4 months postrandomization until the end point of microalbuminuria or macroalbuminuria. Using Cox proportional hazards models, high CV-FPG and low 1,5-AG were independently associated with microalbuminuria after adjusting for clinical risk factors. However, only the CV-FPG association remained after additional adjustment for average HbA1c. Only CV-FPG was a significant risk factor for macroalbuminuria. This post hoc analysis indicates that long-term rather than short-term glycemic variability better predicts the risk of renal disease in type 2 diabetes.

The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 individuals with congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and located near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth and 24 years, and the severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose half-saturation concentration values and an increased enzyme activity index compared with wild-type GK. We performed functional evaluation of islets from the pancreata of three children with GCK-HI who required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal, and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused glucokinase hyperinsulinism (GCK-HI) islets was decreased, and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage.

Prediabetes is a metabolic condition associated with gut microbiome composition, although mechanisms remain elusive. We searched for fecal metabolites, a readout of gut microbiome function, associated with impaired fasting glucose (IFG) in 142 individuals with IFG and 1,105 healthy individuals from the UK Adult Twin Registry (TwinsUK). We used the Cooperative Health Research in the Region of Augsburg (KORA) cohort (318 IFG individuals, 689 healthy individuals) to replicate our findings. We linearly combined eight IFG-positively associated metabolites (1-methylxantine, nicotinate, glucuronate, uridine, cholesterol, serine, caffeine, and protoporphyrin IX) into an IFG-metabolite score, which was significantly associated with higher odds ratios (ORs) for IFG (TwinsUK: OR 3.9 [95% CI 3.02-5.02], P < 0.0001, KORA: OR 1.3 [95% CI 1.16-1.52], P < 0.0001) and incident type 2 diabetes (T2D; TwinsUK: hazard ratio 4 [95% CI 1.97-8], P = 0.0002). Although these are host-produced metabolites, we found that the gut microbiome is strongly associated with their fecal levels (area under the curve >70%). Abundances of Faecalibacillus intestinalis, Dorea formicigenerans, Ruminococcus torques, and Dorea sp. AF24-7LB were positively associated with IFG, and such associations were partially mediated by 1-methylxanthine and nicotinate (variance accounted for mean 14.4% [SD 5.1], P < 0.05). Our results suggest that the gut microbiome is linked to prediabetes not only via the production of microbial metabolites but also by affecting intestinal absorption/excretion of host-produced metabolites and xenobiotics, which are correlated with the risk of IFG. Fecal metabolites enable modeling of another mechanism of gut microbiome effect on prediabetes and T2D onset.

Nerve conduction velocity (NCV) abnormalities are the forerunners of diabetic peripheral neuropathy (DPN). Therefore, this study aimed to analyze the effect of glucose profile quality on NCV in children and young adults with type 1 diabetes. Fifty-three children age 5 to 23 years with type 1 diabetes were recruited to participate in the study, which was conducted prospectively at the Children's Hospital of Eastern Switzerland from 2016 to 2022. Glycemic targets were recorded, and a cross-sectional nerve conduction study analyzing the peroneal, tibial, median motor, and median sensory nerves was performed. Data were compared with those of a control group of 50 healthy children. In the age- and height-matched diabetes subgroup aged 10-16 years, all four nerves showed significantly slower NCV, most pronounced for the peroneal nerve. Because height has a retarding effect on peroneal NCV, NCV was adjusted for height (dNCV). Peroneal dNCV correlated negatively with long-term glycated hemoglobin and highly significantly with glucose variability. Because high glucose variability clearly increases the risk of neuropathy, together with but also independently of the mean glucose level, this aspect of glycemic control should be given more attention in the care of individuals with diabetes.

The ability of metabolically active tissues to increase glucose uptake in response to insulin is critical to whole-body glucose homeostasis. This report describes the Dual Tracer Test, a robust method involving sequential retro-orbital injection of [14C]2-deoxyglucose ([14C]2DG) alone, followed 40 min later by injection of [3H]2DG with a maximal dose of insulin to quantify both basal and insulin-stimulated 2DG uptake in the same mouse. The collection of both basal and insulin-stimulated measures from a single animal is imperative for generating high-quality data since differences in insulin action may be misinterpreted mechanistically if basal glucose uptake is not accounted for. The approach was validated in a classic diet-induced model of insulin resistance and a novel transgenic mouse with reduced GLUT4 expression that, despite ubiquitous peripheral insulin resistance, did not exhibit fasting hyperinsulinemia. This suggests that reduced insulin-stimulated glucose disposal is not a primary contributor to chronic hyperinsulinemia. The Dual Tracer Test offers a technically simple assay that enables the study of insulin action in many tissues simultaneously. By administering two tracers and accounting for both basal and insulin-stimulated glucose transport, this assay halves the required sample size for studies in inbred mice and demonstrates increased statistical power to detect insulin resistance, relative to other established approaches, using a single tracer. The Dual Tracer Test is a valuable addition to the metabolic phenotyping toolbox.

Understanding differences in adipose gene expression between individuals with different levels of clinical traits may reveal the genes and mechanisms leading to cardiometabolic diseases. However, adipose is a heterogeneous tissue. To account for cell-type heterogeneity, we estimated cell-type proportions in 859 subcutaneous adipose tissue samples with bulk RNA sequencing (RNA-seq) using a reference single-nuclear RNA-seq data set. Cell-type proportions were associated with cardiometabolic traits; for example, higher macrophage and adipocyte proportions were associated with higher and lower BMI, respectively. We evaluated cell-type proportions and BMI as covariates in tests of association between >25,000 gene expression levels and 22 cardiometabolic traits. For >95% of genes, the optimal, or best-fit, models included BMI as a covariate, and for 79% of associations, the optimal models also included cell type. After adjusting for the optimal covariates, we identified 2,664 significant associations (P ≤ 2e-6) for 1,252 genes and 14 traits. Among genes proposed to affect cardiometabolic traits based on colocalized genome-wide association study and adipose expression quantitative trait locus signals, 25 showed a corresponding association between trait and gene expression levels. Overall, these results suggest the importance of modeling cell-type proportion when identifying gene expression associations with cardiometabolic traits.

Costimulation serves as a critical checkpoint for T-cell activation, and several genetic variants affecting costimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism (rs763361) in the CD226 gene encoding a costimulatory receptor increases susceptibility to multiple autoimmune diseases, including type 1 diabetes. We previously found that Cd226 knockout protected NOD mice from disease, but the impact of CD226 on individual immune subsets remained unclear. Our prior reports implicate regulatory T cells (Tregs), as human CD226+ Tregs exhibit reduced suppressive function. Hence, we hypothesized that genomic Cd226 gene deletion would increase Treg stability and that Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Indeed, crossing NOD.Cd226-/- and a NOD Treg-lineage tracing strain resulted in decreased pancreatic Foxp3-deficient "ex-Tregs." We generated a novel Treg-conditional knockout (TregΔCd226) strain that displayed decreased insulitis and diabetes incidence. CD226-deficient pancreatic Tregs had increased expression of the coinhibitory counter-receptor T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT). Moreover, NOD splenocytes treated with TIGIT-Fc fusion protein exhibited reduced T-cell proliferation and interferon-γ production following anti-CD3/CD28 stimulation. This study demonstrates that a CD226/TIGIT imbalance contributes to Treg instability in NOD mice and highlights the potential for therapeutic targeting this costimulatory pathway to halt autoimmunity.

Extracellular (e)ATP, a potent proinflammatory molecule, is released by dying/damaged cells at the site of inflammation and is degraded by the membrane ectonucleotidases CD39 and CD73. In this study, we sought to unveil the role of eATP degradation in autoimmune diabetes. We then assessed the effect of soluble CD39 (sCD39) administration in prevention and reversal studies in NOD mice as well as in mechanistic studies. Our data showed that eATP levels were increased in hyperglycemic NOD mice compared with prediabetic NOD mice. CD39 and CD73 were found expressed by both α- and β-cells and by different subsets of T cells. Importantly, prediabetic NOD mice displayed increased frequencies of CD3+CD73+CD39+ cells within their pancreata, pancreatic lymph nodes, and spleens. The administration of sCD39 into prediabetic NOD mice reduced their eATP levels, abrogated the proliferation of CD4+- and CD8+-autoreactive T cells, and increased the frequency of regulatory T cells, while delaying the onset of T1D. Notably, concomitant administration of sCD39 and anti-CD3 showed a strong synergism in restoring normoglycemia in newly hyperglycemic NOD mice compared with monotherapy with anti-CD3 or with sCD39. The eATP/CD39 pathway plays an important role in the onset of T1D, and its targeting might represent a potential therapeutic strategy in T1D.