Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET)-producing enzyme, promotes insulin sensitivity, and Cyp2c44-/- mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SCD)-fed male Cyp2c44-/- mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared with wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol (DAG) levels. Cyp2c44-/- mice had impaired glucose tolerance and increased hepatic plasma membrane-associated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SCD and high-fat diet (HFD)-fed Cyp2c44-/- mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SCD-fed Cyp2c44-/- mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44-/- hepatocytes. Finally, Cyp2c44-/- HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, DAG accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.

Observational studies have shown correlations between intramyocellular lipid (IMCL) content and muscle strength and contractile function in people with metabolically abnormal obesity. However, a clear physiologic mechanism for this association is lacking, and causation is debated. We combined immunofluorescent confocal imaging with force measurements on permeabilized muscle fibers from metabolically normal and metabolically abnormal mice and people with metabolically normal (defined as normal fasting plasma glucose and glucose tolerance) and metabolically abnormal (defined as prediabetes and type 2 diabetes) overweight/obesity to evaluate relationships among myocellular lipid droplet characteristics (droplet size and density) and biophysical (active contractile and passive viscoelastic) properties. The fiber type specificity of lipid droplet parameters varied by metabolic status and by species. It was different between mice and people across the board and different between people of different metabolic status. However, despite considerable quantities of IMCL in the metabolically abnormal groups, there were no significant differences in peak active tension or passive viscoelasticity between the metabolically abnormal and control groups in mice or people. Additionally, there were no significant relationships among IMCL parameters and biophysical variables. Thus, we conclude that IMCL accumulation per se does not impact muscle fiber biophysical properties or physically impede contraction.

Accumulating data suggest a role for the lysosomal protease cathepsin S (CTSS) in type 1 diabetes. Circulating CTSS is increased in type 1 diabetes; however, whether CTSS has protective or deleterious effects is unclear. The study's objectives were to examine the biomarker potential of CTSS in new-onset type 1 diabetes, and to investigate the expression and secretion of CTSS in human islets and β-cells. The CTSS level was analyzed in serum from children with new-onset type 1 diabetes and autoantibody-positive and -negative siblings by ELISA. The expression and secretion of CTSS were evaluated in isolated human islets and EndoC-βH5 cells by real-time qPCR, immunoblotting, and ELISA. The CTSS serum level was elevated in children with new-onset type 1 diabetes and positively associated with autoantibody status in healthy siblings. Human islets and EndoC-βH5 cells demonstrated induction and secretion of CTSS after exposure to proinflammatory cytokines, a model system of islet inflammation. Analysis of publicly available single-cell RNA sequencing data on human islets showed that elevated CTSS expression was exclusive for the β-cells in donors with type 1 diabetes as compared with nondiabetic donors. These findings suggest a potential of CTSS as a diagnostic biomarker in type 1 diabetes.

Bile acids (BAs) are cholesterol-derived compounds that regulate glucose, lipid, and energy metabolism. Despite their significance in glucose homeostasis, the association between specific BA molecular species and their synthetic pathways with diabetes is unclear. Here, we used a recently validated, stable-isotope dilution, high-performance liquid chromatography with tandem mass spectrometry method to quantify a panel of BAs in fasting plasma from 2,145 study participants and explored structural and genetic determinants of BAs linked to diabetes, insulin resistance, and obesity. Multiple 12α-hydroxylated BAs were associated with diabetes (adjusted odds ratio [aOR] range, 1.3-1.9; P < 0.05 for all) and insulin resistance (aOR range, 1.3-2.2; P < 0.05 for all). Conversely, multiple 6α-hydroxylated BAs and isolithocholic acid (iso-LCA) were inversely associated with diabetes and obesity (aOR range, 0.3-0.9; P < 0.05 for all). Genome-wide association studies revealed multiple genome-wide significant loci linked with 9 of the 14 diabetes-associated BAs, including a locus for iso-LCA (rs11866815). Mendelian randomization analyses showed genetically elevated deoxycholic acid levels were causally associated with higher BMI, and iso-LCA levels were causally associated with reduced BMI and diabetes risk. In conclusion, comprehensive, large-scale, quantitative mass spectrometry and genetics analyses show circulating levels of multiple structurally specific BAs, especially DCA and iso-LCA, are clinically associated with and genetically linked to obesity and diabetes.

We aimed to clarify the relationship between intra- and periorgan fats, visceral fat, and subcutaneous fat. We used abdominal computed tomography to evaluate intra- and periorgan fat accumulations in the pancreas, liver, spleen, renal parenchyma, renal sinus, and skeletal muscle. The relationships between these fats, visceral fat, and subcutaneous fat were examined by using partial correlation and covariance analysis, adjusting for BMI. We found that visceral fat and all intra- and periorgan fat accumulations were positively correlated, whereas subcutaneous fat and accumulations of all intra- and periorgan fats and visceral fat were negatively correlated. Individuals with excessive visceral fat accumulation had significantly greater accumulations of fat in the pancreas, liver, renal sinus, and skeletal muscle than those without excessive visceral fat accumulation (P = 0.01, 0.006, 0.008, and 0.02, respectively). In conclusion, all intra- and periorgan fat accumulations show a positive correlation with visceral fat and a negative correlation with subcutaneous fat, independent of BMI.

Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17β-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.

Obesity is associated with chronic inflammation and metabolic complications, including insulin resistance (IR). Immune cells drive inflammation through the rewiring of intracellular metabolism. However, the impact of obesity-related IR on the metabolism and functionality of circulating immune cells, like monocytes, remains poorly understood. To increase insight into the interindividual variation of immunometabolic signatures among individuals and their role in the development of IR, we assessed systemic and tissue-specific IR and circulating immune markers, and we characterized metabolic signatures and cytokine secretion of circulating monocytes from 194 individuals with a BMI ≥25 kg/m2. Monocyte metabolic signatures were defined using extracellular acidification rates (ECARs) to estimate glycolysis and oxygen consumption rates (OCRs) for oxidative metabolism. Although monocyte metabolic signatures and function based on cytokine secretion varied greatly among study participants, they were strongly associated with each other. The ECAR-to-OCR ratio, representing the balance between glycolysis and oxidative metabolism, was negatively associated with fasting insulin levels, systemic IR, and liver-specific IR. These results indicate that monocytes from individuals with IR were relatively more dependent on oxidative metabolism, whereas monocytes from more insulin-sensitive individuals were more dependent on glycolysis. Additionally, circulating CXCL11 was negatively associated with the degree of systemic IR and positively with the ECAR-to-OCR ratio in monocytes, suggesting that individuals with high IR and a monocyte metabolic dependence on oxidative metabolism also have lower levels of circulating CXCL11. Our findings suggest that monocyte metabolism is related to obesity-associated IR progression and deepen insights into the interplay between innate immune cell metabolism and IR development in humans.

Forkhead box O1 (FOXO1) regulates muscle growth, but the metabolic role of FOXO1 in skeletal muscle and its mechanisms remain unclear. To explore the metabolic role of FOXO1 in skeletal muscle, we generated skeletal muscle-specific Foxo1 inducible knockout (mFOXO1 iKO) mice and fed them a high-fat diet to induce obesity. We measured insulin sensitivity, fatty acid oxidation, mitochondrial function, and exercise capacity in obese mFOXO1 iKO mice and assessed the correlation between FOXO1 and mitochondria-related protein in the skeletal muscle of patients with diabetes. Obese mFOXO1 iKO mice exhibited improved mitochondrial respiratory capacity, which was followed by attenuated insulin resistance, enhanced fatty acid oxidation, and improved skeletal muscle exercise capacity. Transcriptional inhibition of FOXO1 in peroxisome proliferator-activated receptor δ (PPARδ) expression was confirmed in skeletal muscle, and deletion of PPARδ abolished the beneficial effects of FOXO1 deficiency. FOXO1 protein levels were higher in the skeletal muscle of patients with diabetes and negatively correlated with PPARδ and electron transport chain protein levels. These findings highlight FOXO1 as a new repressor in PPARδ gene expression in skeletal muscle and suggest that FOXO1 links insulin resistance and mitochondrial dysfunction in skeletal muscle via PPARδ.

Body fat distribution is a predictor of metabolic health in obesity. In this Classics in Diabetes article, we revisit a 1985 Diabetes article by Swedish investigators Ohlson et al. This work was one of the first prospective population-based studies that established a relationship between abdominal adiposity and the risk for developing diabetes. Here, we discuss evolving concepts regarding the link between regional adiposity and diabetes and other chronic disorders. Moreover, we highlight fundamental questions that remain unresolved.

The early pathogenetic mechanism of diabetic retinopathy (DR) and its treatment remain unclear. Therefore, we used streptozotocin-induced diabetic mice to investigate the early pathogenic alterations in DR and the protective effect of sodium-glucose cotransporter 2 (SGLT2) inhibitors against these alterations. Retinal vascular leakage was assessed by dextran fluorescence angiography. Retinal thickness and vascular leakage were increased 2 and 4 weeks after onset of diabetes, respectively. Immunostaining showed that morphological change of microglia (amoeboid form) was observed at 2 weeks. Subsequently, increased angiopoietin-2 expression, simultaneous loss of pericytes and endothelial cells, decreased vessel density, retinal hypoxia, and increased vascular endothelial growth factor (VEGF)-A/VEGF receptor system occurred at 4 weeks. SGLT2 inhibitors (luseogliflozin and ipragliflozin) had a significant protective effect on retinal vascular leakage and retinal thickness at a low dose that did not show glucose-lowering effects. Furthermore, both inhibitors at this dose attenuated microglia morphological changes and these early pathogenic alterations in DR. In vitro study showed both inhibitors attenuated the lipopolysaccharide-induced activation of primary microglia, along with morphological changes toward an inactive form, suggesting the direct inhibitory effect of SGLT2 inhibitors on microglia. In summary, SGLT2 inhibitors may directly prevent early pathogenic mechanisms, thereby potentially playing a role in preventing DR.

The Rab-GTPase-activating protein (RabGAP) TBC1D4 (AS160) represents a key component in the regulation of glucose transport into skeletal muscle and white adipose tissue (WAT) and is therefore crucial during the development of insulin resistance and type 2 diabetes. Increased daily activity has been shown to be associated with improved postprandial hyperglycemia in allele carriers of a loss-of-function variant in the human TBC1D4 gene. Using conventional Tbc1d4-deficient mice (D4KO) fed a high-fat diet, we show that moderate endurance exercise training leads to substantially improved glucose and insulin tolerance and enhanced expression levels of markers for mitochondrial activity and browning in WAT from D4KO animals. Importantly, in vivo and ex vivo analyses of glucose uptake revealed increased glucose clearance in interscapular brown adipose tissue and WAT from trained D4KO mice. Thus, chronic exercise is able to overcome the genetically induced insulin resistance caused by Tbc1d4 depletion. Gene variants in TBC1D4 may be relevant in future precision medicine as determinants of exercise response.

Insulin resistance is a risk factor for type 2 diabetes, and exercise can improve insulin sensitivity. However, following exercise, high circulating fatty acid (FA) levels might counteract this. We hypothesized that such inhibition would be reduced by forcibly increasing carbohydrate oxidation through pharmacological activation of the pyruvate dehydrogenase complex (PDC). Insulin-stimulated glucose uptake was examined with a crossover design in healthy young men (n = 8) in a previously exercised and a rested leg during a hyperinsulinemic-euglycemic clamp 5 h after one-legged exercise with 1) infusion of saline, 2) infusion of intralipid imitating circulating FA levels during recovery from whole-body exercise, and 3) infusion of intralipid + oral PDC activator, dichloroacetate (DCA). Intralipid infusion reduced insulin-stimulated glucose uptake by 19% in the previously exercised leg, which was not observed in the contralateral rested leg. Interestingly, this effect of intralipid in the exercised leg was abolished by DCA, which increased muscle PDC activity (130%) and flux (acetylcarnitine 130%) and decreased inhibitory phosphorylation of PDC on Ser293 (∼40%) and Ser300 (∼80%). Novel insight is provided into the regulatory interaction between glucose and lipid metabolism during exercise recovery. Coupling exercise and PDC flux activation upregulated the capacity for both glucose transport (exercise) and oxidation (DCA), which seems necessary to fully stimulate insulin-stimulated glucose uptake during recovery.

In 2014, the American Diabetes Association instituted a novel funding paradigm to support diabetes research through its Pathway to Stop Diabetes program. This program took a multifaceted approach to providing key funding to diabetes researchers to advance a broad spectrum of research programs on all aspects of understanding, managing, and treating diabetes. Here, the personal perspective of a 2019 Pathway Accelerator awardee is offered, describing a research program seeking to advance a materials-centered approach to engineering glucose-responsive devices and new delivery tools for better therapeutic outcomes in treating diabetes. This is offered alongside a personal reflection on 5 years of support from the ADA Pathway Program.

Pluripotent stem cell-derived islets (SC-islets) have emerged as a new source for β-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences.

Prediabetes is a heterogenous metabolic state with various risks for development of type 2 diabetes (T2D). In this study, we used genetic data on 7,227 US Hispanic/Latino participants without diabetes from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) and 400,149 non-Hispanic White participants without diabetes from the UK Biobank (UKBB) to calculate five partitioned polygenetic risk scores (pPRSs) representing various pathways related to T2D. Consensus clustering was performed in participants with prediabetes in HCHS/SOL (n = 3,677) and UKBB (n = 16,284) separately based on these pPRSs. Six clusters of individuals with prediabetes with distinctive patterns of pPRSs and corresponding metabolic traits were identified in the HCHS/SOL, five of which were confirmed in the UKBB. Although baseline glycemic traits were similar across clusters, individuals in cluster 5 and cluster 6 showed an elevated risk of T2D during follow-up compared with cluster 1 (risk ratios [RRs] 1.29 [95% CI 1.08, 1.53] and 1.34 [1.13, 1.60], respectively). Inverse associations between a healthy lifestyle score and risk of T2D were observed across different clusters, with a suggestively stronger association observed in cluster 5 compared with cluster 1. Among individuals with a healthy lifestyle, those in cluster 5 had a similar risk of T2D compared with those in cluster 1 (RR 1.03 [0.91, 1.18]). This study identified genetic subtypes of prediabetes that differed in risk of progression to T2D and in benefits from a healthy lifestyle.

In 2014, the American Diabetes Association instituted a novel funding paradigm to support diabetes research through its Pathway to Stop Diabetes® Program. Pathway took a multifaceted approach to provide key funding to diabetes researchers in advancing a broad spectrum of research programs centered on all aspects of understanding, managing, and treating diabetes. Herein the personal perspective of a 2019 Pathway Accelerator awardee is offered, describing a research program seeking to advance a materials-centered approach to engineering glucose-responsive devices and new delivery tools for better therapeutic outcomes in treating diabetes. This is offered alongside a personal reflection on five years of support from the ADA Pathway Program.