O-linked N-acetylglucosamine modification is essential for physiological adipose expansion induced by high-fat feeding.

Adipose tissues accumulate excess energy as fat and heavily influence metabolic homeostasis. O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), which involves the addition of N-acetylglucosamine to proteins by O-GlcNAc transferase (Ogt), modulates multiple cellular processes. However, little is known about the role of O-GlcNAcylation in adipose tissues during body weight gain due to overnutrition. Here, we report on O-GlcNAcylation in mice with high-fat diet (HFD)-induced obesity. Mice with knockout of Ogt in adipose tissue achieved using adiponectin promoter-driven Cre recombinase (Ogt-FKO) gained less body weight than control mice under HFD. Surprisingly, Ogt-FKO mice exhibited glucose intolerance and insulin resistance, despite their reduced body weight gain, as well as decreased expression of de novo lipogenesis genes and increased expression of inflammatory genes, resulting in fibrosis at 24 weeks of age. Primary cultured adipocytes derived from Ogt-FKO mice showed decreased lipid accumulation. Both primary cultured adipocytes and 3T3-L1 adipocytes treated with OGT inhibitor showed increased secretion of free fatty acids. Medium derived from these adipocytes stimulated inflammatory genes in RAW 264.7 macrophages, suggesting that cell-to-cell communication via free fatty acids might be a cause of adipose inflammation in Ogt-FKO mice. In conclusion, O-GlcNAcylation is important for healthy adipose expansion in mice. Glucose flux into adipose tissues may be a signal to store excess energy as fat.NEW & NOTEWORTHY We evaluated the role of O-GlcNAcylation in adipose tissue in diet-induced obesity using adipose tissue-specific Ogt knockout mice. We found that O-GlcNAcylation in adipose tissue is essential for healthy fat expansion and that Ogt-FKO mice exhibit severe fibrosis upon long-term overnutrition. O-GlcNAcylation in adipose tissue may regulate de novo lipogenesis and free fatty acid efflux to the degree of overnutrition. We believe that these results provide new insights into adipose tissue physiology and obesity research.

Microbiome potentiates endurance exercise through intestinal acetate production.

The intestinal microbiome produces short-chain fatty acids (SCFAs) from dietary fiber and has specific effects on other organs. During endurance exercise, fatty acids, glucose, and amino acids are major energy substrates. However, little is known about the role of SCFAs during exercise. To investigate this, mice were administered either multiple antibiotics or a low microbiome-accessible carbohydrate (LMC) diet, before endurance testing on a treadmill. Two-week antibiotic treatment significantly reduced endurance capacity versus the untreated group. In the cecum acetate, propionate, and butyrate became almost undetectable in the antibiotic-treated group, plasma SCFA concentrations were lower, and the microbiome was disrupted. Similarly, 6-wk LMC treatment significantly reduced exercise capacity, and fecal and plasma SCFA concentrations. Continuous acetate but not saline infusion in antibiotic-treated mice restored their exercise capacity (P < 0.05), suggesting that plasma acetate may be an important energy substrate during endurance exercise. In addition, running time was significantly improved in LMC-fed mice by fecal microbiome transplantation from others fed a high microbiome-accessible carbohydrate diet and administered a single portion of fermentable fiber (P < 0.05). In conclusion, the microbiome can contribute to endurance exercise by producing SCFAs. Our findings provide new insight into the effects of the microbiome on systemic metabolism.

Lack of O-GlcNAcylation enhances exercise-dependent glucose utilization potentially through AMP-activated protein kinase activation in skeletal muscle.

O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise.

Diverse metabolic effects of O-GlcNAcylation in the pancreas but limited effects in insulin-sensitive organs in mice.

AIMS/HYPOTHESIS: O-GlcNAcylation is characterised by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (OGT) and serves in sensing intracellular nutrients by modulating various cellular processes. Although it has been speculated that O-GlcNAcylation is associated with glucose metabolism, its exact role in whole body glucose metabolism has not been fully elucidated. Here, we investigated whether loss of O-GlcNAcylation globally and in specific organs affected glucose metabolism in mammals under physiological conditions. METHODS: Tamoxifen-inducible global Ogt-knockout (Ogt-KO) mice were generated by crossbreeding Ogt-flox mice with R26-Cre-ERT2 mice. Liver, skeletal muscle, adipose tissue and pancreatic beta cell-specific Ogt-KO mice were generated by crossbreeding Ogt-flox mice with Alb-Cre, Mlc1f-Cre, Adipoq-Cre and Pdx1 PB-CreER™ mice, respectively. Glucose metabolism was evaluated by i.p. glucose and insulin tolerance tests. RESULTS: Tamoxifen-inducible global Ogt-KO mice exhibited a lethal phenotype from 4 weeks post injection, suggesting that O-GlcNAcylation is essential for survival in adult mice. Tissue-specific Ogt deletion from insulin-sensitive organs, including liver, skeletal muscle and adipose tissue, had little impact on glucose metabolism under physiological conditions. However, pancreatic beta cell-specific Ogt-KO mice displayed transient hypoglycaemia (Ogt-flox 5.46 ± 0.41 vs Ogt-ßKO 3.88 ± 0.26 mmol/l) associated with about twofold higher insulin secretion and accelerated adiposity, followed by subsequent hyperglycaemia (Ogt-flox 6.34 ± 0.32 vs Ogt-ßKO 26.4 ± 2.37 mmol/l) with insulin depletion accompanied by beta cell apoptosis. CONCLUSIONS/INTERPRETATION: These findings suggest that O-GlcNAcylation has little effect on glucose metabolism in insulin-sensitive tissues but plays a crucial role in pancreatic beta cell function and survival under physiological conditions. Our results provide novel insight into O-GlcNAc biology and physiology in glucose metabolism.

Acetate derived from the intestinal tract has a critical role in maintaining skeletal muscle mass and strength in mice.

Acetate is a short-chain fatty acid (SCFA) that is produced by microbiota in the intestinal tract. It is an important nutrient for the intestinal epithelium, but also has a high plasma concentration and is used in the various tissues. Acetate is involved in endurance exercise, but its role in resistance exercise remains unclear. To investigate this, mice were administered either multiple antibiotics with and without oral acetate supplementation or fed a low-fiber diet. Antibiotic treatment for 2 weeks significantly reduced grip strength and the cross-sectional area (CSA) of muscle fiber compared with the control group. Intestinal concentrations of SCFAs were reduced in the antibiotic-treated group. Oral administration of acetate with antibiotics prevented antibiotic-induced weakness of skeletal muscle and reduced CSA of muscle fiber. Similarly, a low-fiber diet for 1 year significantly reduced the CSA of muscle fiber and fecal and plasma acetate concentrations. To investigate the role of acetate as an energy source, acetyl-CoA synthase 2 knockout mice were used. These mice had a shorter lifespan, reduced skeletal muscle mass and smaller CSA of muscle fiber than their wild type littermates. In conclusion, acetate derived from the intestinal microbiome can contribute to maintaining skeletal muscle performance.

O-GlcNAc modification in endothelial cells modulates adiposity via fat absorption from the intestine in mice.

Introduction: Endothelial cells have a crucial function in transporting and exchanging various nutrients. O-GlcNAcylation, mediated by O-GlcNAc transferase (OGT), involves the addition of N-acetylglucosamine to proteins and serves as an intracellular nutrient sensing mechanism. However, the role of O-GlcNAcylation in endothelial cells remains poorly understood. Objective: This study investigated the role of O-GlcNAcylation in endothelial cells. Methods: Endothelial-cell-specific Ogt -knockout mice (Ogt-ECKO) were generated by crossing Ogt-floxed mice (Ogt-flox) with VE-Cadherin Cre ERT2 mice. Ogt-ECKO mice and Ogt-flox control mice were subjected to a normal or high-fat diet, and their body weight, glucose metabolism, and lipid metabolism were examined. Results: Ogt-ECKO mice exhibited reduced body weight compared with Ogt-flox control mice under a high-fat diet. Lipid absorption was significantly impaired in Ogt-ECKO mice. Changes in the intercellular junctions of small intestinal lacteal endothelial cells from a button-like to a zipper-like configuration were observed. Furthermore, Ogt-ECKO mice showed decreased expression of VEGFR3. The administration of a nitric oxide donor restored lipid absorption and reversed the morphological alterations in Ogt-ECKO mice. Conclusions: These findings demonstrate the critical role of O-GlcNAcylation in endothelial cells in lipid absorption in the intestine through the modulation of lacteal junction morphology. These results provide novel insight into the metabolic regulatory mechanisms under physiological conditions and have implications for the development of new therapeutic strategies for obesity.

A Century-long Journey From the Discovery of Insulin to the Implantation of Stem Cell-derived Islets.

For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing ß cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing ß cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature ß-like cells in vitro that produce insulin but do not yet fully mimic healthy mature ß cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell-derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease.

Background: Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD). Methods: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. Results: The speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. Conclusions: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture.

The generation of functional ß-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

Human A2-CAR T Cells Reject HLA-A2 + Human Islets Transplanted Into Mice Without Inducing Graft-versus-host Disease.

BACKGROUND: Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). METHODS: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4 + and CD8 + T cells and tested their ability to reject HLA-A2 + islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T-cell engraftment, islet function, and xGVHD were assessed longitudinally. RESULTS: The speed and consistency of A2-CAR T-cell-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of coinjected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, coinjection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2 + human islets within 1 wk and without xGVHD for 12 wk. CONCLUSIONS: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of islet-replacement therapies.

Metabolic changes induced by dapagliflozin, an SGLT2 inhibitor, in Japanese patients with type 2 diabetes treated by oral anti-diabetic agents: A randomized, clinical trial.

AIM: We aimed to determine whether SGLT2 inhibitor dapagliflozin treatment affects body composition and amino acid (AA) metabolism. METHODS: Fifty-two overweight patients treated by oral antidiabetic agents were randomly assigned to dapagliflozin (Dapa) or a standard treatment (Con) and followed for 24 weeks. The primary outcome was the change in body mass (BM) between baseline and week 24. Body composition, intrahepatic triglyceride (IHTG) content, and plasma AA concentrations were examined as secondary outcomes. RESULTS: The change in BM was significantly larger in the Dapa than in the Con group, with a difference in the mean change of -1.72 kg (95 %CI: -2.85, -0.59; P = 0.004) between the groups. Total fat mass was reduced by dapagliflozin treatment, but fat-free mass was maintained. IHTG content was significantly reduced in the Dapa than in the Con (P = 0.033). Changes in AAs showed small differences between the groups, but only serine concentrations were significantly reduced in the Dapa. Intra-group analysis showed that positive associations were observed between changes in branched chain AA concentrations and body composition only in the Dapa. CONCLUSIONS: Dapagliflozin treatment causes a reduction in BM mainly by reducing fat mass. AA metabolism shows subtle changes with dapagliflozin treatment.

Glycaemia and body weight are regulated by sodium-glucose cotransporter 1 (SGLT1) expression via O-GlcNAcylation in the intestine.

OBJECTIVE: The intestine is an important organ for nutrient metabolism via absorption and endocrine systems. Nutrients regulate O-GlcNAcylation, a post-translational modification of various proteins by O-GlcNAc transferase (OGT). We have previously shown that general OGT knockout induced severe weight loss and hypoglycaemia in mice, but little is known about how O-GlcNAcylation in the intestine modulates nutrient metabolism, especially glucose metabolism, through absorption. We aimed to reveal the roles of O-GlcNAcylation in glucose absorption by the small intestine and elucidate the mechanism by which O-GlcNAcylation regulates sodium-glucose cotransporter 1 (SGLT1) expression. METHODS: First, we fasted normal mice and examined the changes in glucose transporters and O-GlcNAcylation in the intestine. Then, we generated two lines of small intestine-specific OGT-deficient mice (congenital: Ogt-VKO, tamoxifen-inducible: Ogt-iVKO) and observed the changes in body weight and in glucose and lipid metabolism. Finally, we investigated Sglt1 gene regulation by O-GlcNAcylation using enteroendocrine STC-1 cells. RESULTS: Fasting decreased O-GlcNAcylation in the intestinal epithelium of normal mice. The Ogt-VKO mice showed significantly lower non-fasted blood glucose levels and were underweight compared with litter matched controls. Glycaemic excursion in the Ogt-VKO mice was significantly lower during the oral glucose tolerance test but comparable during the intraperitoneal glucose tolerance test. Furthermore, the Ogt-VKO mice exhibited lower Sglt1 expression in the small intestine compared with the control mice. We obtained similar results using the Ogt-iVKO mice only after tamoxifen administration. The oral d-xylose administration test revealed that the intestinal sugar absorption was diminished in the Ogt-iVKO mice and that GLP-1 secretion did not sufficiently increase after glucose gavage in the Ogt-iVKO mice. When using STC-1 cells, O-GlcNAcylation increased Sglt1 mRNA via a PKA/CREB-dependent pathway. CONCLUSION: Collectively, loss of O-GlcNAcylation in the intestine reduced glucose absorption via suppression of SGLT1 expression; this may lead to new treatments for malabsorption, obesity and diabetes.

Ipragliflozin, a sodium-glucose cotransporter 2 inhibitor, reduces bodyweight and fat mass, but not muscle mass, in Japanese type 2 diabetes patients treated with insulin: A randomized clinical trial.

AIMS/INTRODUCTION: Sodium-glucose cotransporter 2 inhibitors reduce bodyweight (BW) by creating a negative energy balance. Previous reports have suggested that this BW reduction is mainly loss of body fat and that ~20% of the reduction is lean mass. However, the effects of sodium-glucose cotransporter 2 inhibitors on BW and body composition remain unclear. We examined these effects in Japanese patients with type 2 diabetes mellitus treated with insulin. MATERIALS AND METHODS: In this open-label, randomized controlled trial, 49 overweight patients (body mass index ≥23 kg/m2 ) with inadequate glycemic control (hemoglobin A1c >7.0%) receiving insulin treatment were randomly assigned to receive add-on ipragliflozin or no additional treatment (control group). Patients were followed for 24 weeks. The goal for all patients was to achieve glycated hemoglobin <7.0% without hypoglycemia. The primary end-point was a change in BW from baseline to week 24. Body composition was assessed with dual-energy X-ray absorptiometry and bioelectrical impedance analysis. RESULTS: BW change was significantly larger in the ipragliflozin group than in the control group (-2.78 vs -0.22 kg, P < 0.0001). Total fat mass was reduced evenly in the arms, lower limbs and trunk in the ipragliflozin group. Total muscle mass and bone mineral content were maintained, but muscle mass in the arms might have been affected by ipragliflozin treatment. CONCLUSIONS: Ipragliflozin treatment for 24 weeks resulted in reduced BW, mainly from fat mass loss. Muscle mass and bone mineral content were maintained. Further study is necessary to elucidate the long-term effects of ipragliflozin.

MiR-494-3p regulates mitochondrial biogenesis and thermogenesis through PGC1-α signalling in beige adipocytes.

Mitochondria are critical in heat generation in brown and beige adipocytes. Mitochondrial number and function are regulated in response to external stimuli, such as cold exposure and ß3 adrenergic receptor agonist. However, the molecular mechanisms regulating mitochondrial biogenesis during browning, especially by microRNAs, remain unknown. We investigated the role of miR-494-3p in mitochondrial biogenesis during adipogenesis and browning. Intermittent mild cold exposure of mice induced PPARγ coactivator1-α (PGC1-α) and mitochondrial TFAM, PDH, and ANT1/2 expression along with uncoupling protein-1 (Ucp1) in inguinal white adipose tissue (iWAT). miR-494-3p levels were significantly downregulated in iWAT upon cold exposure (p < 0.05). miR-494-3p overexpression substantially reduced PGC1-α expression and its downstream targets TFAM, PDH and MTCO1 in 3T3-L1 white and beige adipocytes (p < 0.05). miR-494-3p inhibition in 3T3-L1 white adipocytes resulted in increased PDH (p < 0.05). PGC1-α, TFAM and Ucp1 mRNA levels were robustly downregulated by miR-494-3p overexpression in 3T3-L1 beige adipocytes, along with strongly decreased oxygen consumption rate. PGC1-α and Ucp1 proteins were downregulated by miR-494-3p in primary beige cells (p < 0.05). Luciferase assays confirmed PGC1-α as a direct gene target of miR-494-3p. Our findings demonstrate that decreased miR-494-3p expression during browning regulates mitochondrial biogenesis and thermogenesis through PGC1-α.

Pivotal Role of O-GlcNAc Modification in Cold-Induced Thermogenesis by Brown Adipose Tissue Through Mitochondrial Biogenesis.

Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism. O-linked N-acetylglucosamine (O-GlcNAc) modification is characterized by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (Ogt), subsequently modulating various cellular processes. However, little is known about the role of O-GlcNAc modification in adipose tissues. Here, we report the critical role of O-GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and BAT using adiponectin promoter-driven Cre recombinase resulted in severe cold intolerance with decreased uncoupling protein 1 (Ucp1) expression. Furthermore, Ogt deletion led to decreased mitochondrial protein expression in conjunction with decreased peroxisome proliferator-activated receptor γ coactivator 1-α protein expression. This phenotype was further confirmed by deletion of Ogt in BAT using Ucp1 promoter-driven Cre recombinase, suggesting that O-GlcNAc modification in BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under fasting conditions. This effect was mitigated after normal diet consumption but not after consumption of a fatty acid-rich ketogenic diet lacking carbohydrates, suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, O-GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT physiological responses.