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Dopamine receptor agonists are typically used to treat Parkinson’s disease and certain pituitary tumors, such as prolactinoma or a growth hormone‐producing tumor. A 53‐year‐old woman with a history of prolactinoma was referred to Kumamoto University Hospital (Kumamoto, Japan) with poorly controlled type 2 diabetes. Her glycated hemoglobin and serum prolactin levels were increased (8.8% and 160.3 ng/mL, respectively). Bromocriptine, a dopamine D2 receptor agonist, was administered to reduce her serum prolactin level. Because bromocriptine‐QR (quick release) has been approved for the treatment of type 2 diabetes mellitus in the USA, a continuous glucose monitoring system, FreeStyle Libre Pro, was utilized to examine the effect of bromocriptine on glycemic control. After the initial administration of bromocriptine, glucose levels were rapidly and dramatically ameliorated, and the time in range (70–180 mg/dL) improved from 90% between 1 week before and after the initial administration of bromocriptine., The areas of time above range (>180 mg/dL, shown in yellow) almost disappeared immediately after bromocriptine administration.

Nonalcoholic fatty liver disease (NAFLD) is often accompanied by metabolic disorders such as metabolic syndrome and type 2 diabetes (T2DM). Heat shock response (HSR) is one of the most important homeostatic abilities but is deteriorated by chronic metabolic insults. Heat shock (HS) with an appropriate mild electrical stimulation (MES) activates HSR and improves metabolic abnormalities including insulin resistance, hyperglycemia and inflammation in metabolic disorders. To analyze the effects of HS + MES treatment on NAFLD biomarkers, three cohorts including healthy men (two times/week, n = 10), patients with metabolic syndrome (four times/week, n = 40), and patients with T2DM (n = 100; four times/week (n = 40) and two, four, seven times/week (n = 20 each)) treated with HS + MES were retrospectively analyzed. The healthy subjects showed no significant alterations in NAFLD biomarkers after the treatment. In patients with metabolic syndrome, many of the NAFLD steatosis markers, including fatty liver index, NAFLD-liver fat score, liver/spleen ratio and hepatic steatosis index and NAFLD fibrosis marker, aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, were improved upon the treatment. In patients with T2DM, all investigated NAFLD steatosis markers were improved and NAFLD fibrosis markers such as the AST/ALT ratio, fibrosis-4 index and NAFLD-fibrosis score were improved upon the treatment. Thus, HS + MES, a physical intervention, may become a novel treatment strategy for NAFLD as well as metabolic disorders.

Class Ia phosphoinositide 3-kinases (PI3K) are critical mediators of insulin and growth factor action. We have demonstrated that the p85α regulatory subunit of PI3K modulates the unfolded protein response (UPR) by interacting with and regulating the nuclear translocation of XBP-1s, a transcription factor essential for the UPR. We now show that PI3K activity is required for full activation of the UPR. Pharmacological inhibition of PI3K in cells blunts the ER stress-dependent phosphorylation of IRE1α and PERK, decreases induction of ATF4, CHOP, and XBP-1 and upregulates UPR target genes. Cells expressing a human p85α mutant (R649W) previously shown to inhibit PI3K, exhibit decreased activation of IRE1α and PERK and reduced induction of CHOP and ATF4. Pharmacological inhibition of PI3K, overexpression of a mutant of p85α that lacks the ability to interact with the p110α catalytic subunit (∆p85α) or expression of mutant p85α (R649W) in vivo, decreased UPR-dependent induction of ER stress response genes. Acute tunicamycin treatment of R649W+/- mice revealed reduced induction of UPR target genes in adipose tissue, whereas chronic tunicamycin exposure caused sustained increases in UPR target genes in adipose tissue. Finally, R649W+/- cells exhibited a dramatic resistance to ER stress-dependent apoptosis. These data suggest that PI3K pathway dysfunction causes ER stress that may drive the pathogenesis of several diseases including Type 2 diabetes and various cancers.

Insulin and IGF1 signaling initiate Tyr phosphorylation of IR/IGF1R and adaptor IRSs, leading to Ser/Thr phosphorylation of PI3K/AKT and MAPK/ERK pathways essential for the maintenance of brown and white adipose tissues (BAT and WAT). Lack of insulin-signaling in adipose tissues caused metabolic disease in mice with hyperglycemia, hyperlipidemia, and fatty liver, but the mice recovered adipose tissues by regenerating adipocytes. We addressed the mechanism of how adipose tissues regenerate using adipocyte-specific and inducible IR/IGF1R knockout mice (Ai-DKO). We identified a reduction of protein phosphatase protector, α4, which can regulate Ser/Thr phosphorylation of down-stream insulin signaling. shRNA-mediated α4 knockdown (KD) in BAT affected not only insulin-stimulated down-stream Ser/Thr phosphorylation of AKT (S473) but also affected up-stream Tyr phosphorylation of IRβ (Y1162/1163) and IRS1 (Y612). Proteomics analysis of α4-binding molecule identified Y-box protein 1 (YBX1), which functions as a transcription factor for Tyr phosphatase PTP1B. We observed increased PTP1B expression in α4-KO preadipocytes. As the impact of α4 in vivo, inducible adipocyte-specific α4 KO (Ai-α4 KO) mice with tamoxifen-inducible Cre-ERT2 transgene showed cold intolerance, diabetes, ectopic lipid accumulation in the liver, and pancreatic islet hyperplasia, with an increase of C18: 0-ceramide in WAT and BAT. RNA-seq showed a marked reduction of genes associated with mitochondrial fatty acid oxidation and the increases in inflammatory cytokine pathways in Ai-α4 KO WAT and BAT, implicating the cause of fat tissue loss was the increased adipocyte apoptosis. The α4 to PTP1B loop is critical for fat tissue maintenance by regulating IR tyrosine phosphorylation. Disclosure M. Sakaguchi: None. S. Okagawa: None. Y. Okubo: None. K. Fukuda: None. M. Igata: None. J. Kawashima: None. T. Kondo: None. E. Araki: Other Relationship; Self; Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Mitsubishi Tanabe Pharma Corporation, MSD K. K., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Pharma Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K. K., Kowa Company, Ltd. Funding Japan Society for the Promotion of Science; Merck Sharp & Dohme Corp; Takeda Foundation

The gastrointestinal tract is considered an important endocrine organ for controlling glucose homeostasis via the production of incretins. A 21-year-old man emergently underwent total colectomy due to severe ulcerative colitis, and overt diabetes became evident. Weekly administration of a glucagon-like peptide (GLP)-1 receptor agonist (RA) dramatically improved his glucose control. Levels of GLP-1 or gastric inhibitory polypeptide (GIP) were low at the baseline in the duodenum and serum of the patient. After 11 months of GLP-1RA treatment, his HbA1c worsened again, and intensive insulin therapy was necessary to control his glucose levels. Our report may explain the significance of residual incretin for maintaining the pancreatic β-cell function.

The insulin and insulin-like growth factor-1 (IGF-1) receptors are important for the growth and development of embryonic tissues. To directly define their roles in the maintenance of pluripotency and differentiation of stem cells, we knocked out both receptors in induced pluripotent stem cells (iPSCs). iPSCs lacking both insulin and IGF-1 receptors (double knockout, DKO) exhibited preserved pluripotency potential despite decreased expression of transcription factors Lin28a and Tbx3 compared to control iPSCs. While embryoid body and teratoma assays revealed an intact ability of DKO iPSCs to form all three germ layers, the latter were composed of primitive neuroectodermal tumor-like cells in the DKO group. RNA-seq analyses of control vs DKO iPSCs revealed differential regulation of pluripotency, developmental, E2F1, and apoptosis pathways. Signaling analyses pointed to downregulation of the AKT/mTOR pathway and upregulation of the STAT3 pathway in DKO iPSCs in the basal state and following stimulation with insulin/IGF-1. Directed differentiation toward the three lineages was dysregulated in DKO iPSCs, with significant downregulation of key markers (Cebpα, Fas, Pparγ, and Fsp27) in adipocytes and transcription factors (Ngn3, Isl1, Pax6, and Neurod1) in pancreatic endocrine progenitors. Furthermore, differentiated pancreatic endocrine progenitor cells from DKO iPSCs showed increased apoptosis. We conclude that insulin and insulin-like growth factor-1 receptors are indispensable for normal lineage development and perturbations in the function and signaling of these receptors leads to upregulation of alternative compensatory pathways to maintain pluripotency., Highlights • Insulin and IGF-1 receptor signaling regulate the expression of pluripotency genes Lin28 and Tbx3. • The STAT3 pathway is upregulated in DKO iPSCs. • RNA-seq analyses revealed key developmental and apoptosis pathways regulated by insulin and IGF-1 receptors. • Lineage development was dysregulated in DKO iPSCs with downregulation of key mesoderm and endodermal markers.

Objective Accumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties. Methods We performed RNA sequencing on human subcutaneous and visceral preadipocytes and array gene expression profiling in murine subcutaneous and visceral preadipocytes. We conducted several insulin signaling and inflammatory response experiments on different cellular states of MME expression. Results MME in white preadipocytes is expressed at a higher level in subcutaneous compared to visceral WAT and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in subcutaneous preadipocytes increased the inflammatory response to substance P and amyloid β aggregates. This associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor. Conclusion MME is a novel regulator of the insulin receptor in adipose tissue. Given the clinical significance of both chronic inflammation and insulin sensitivity in metabolic disease, these results show a potentially new target to increase insulin sensitivity and decrease inflammatory susceptibility., Graphical abstract Image 1, Highlights • Preadipocytes show depot-specific gene expression differences, one of which is the membrane metallo-endopeptidase MME. • MME/Neprilysin perturbations affect insulin signaling and inflammatory response in preadipocytes. • MME knockdown differentially affects insulin receptor subunit expression and/or turnover.

A major target of insulin signaling is the FoxO family of Forkhead transcription factors, which translocate from the nucleus to the cytoplasm following insulin-stimulated phosphorylation. Here we show that the Forkhead transcription factors FoxK1 and FoxK2 are also downstream targets of insulin action, but that following insulin stimulation, they translocate from the cytoplasm to nucleus, reciprocal to the translocation of FoxO1. FoxK1/FoxK2 translocation to the nucleus is dependent on the Akt-mTOR pathway, while its localization to the cytoplasm in the basal state is dependent on GSK3. Knockdown of FoxK1 and FoxK2 in liver cells results in upregulation of genes related to apoptosis and down-regulation of genes involved in cell cycle and lipid metabolism. This is associated with decreased cell proliferation and altered mitochondrial fatty acid metabolism. Thus, FoxK1/K2 are reciprocally regulated to FoxO1 following insulin stimulation and play a critical role in the control of apoptosis, metabolism and mitochondrial function., Insulin signaling represses Forkhead transcription factor FoxO activity, which contributes to organismal metabolism. Here, the authors use proteomics to identify positively regulated insulin signaling targets FoxK1/K2 and demonstrate their role in lipid metabolism and mitochondrial regulation.

Exercise improves health and well-being across diverse organ systems, and elucidating mechanisms underlying the beneficial effects of exercise can lead to new therapies. Here, we show that transforming growth factor-β2 (TGF-β2) is secreted from adipose tissue in response to exercise and improves glucose tolerance in mice. We identify TGF-β2 as an exercise-induced adipokine in a gene expression analysis of human subcutaneous adipose tissue biopsies after exercise training. In mice, exercise training increases TGF-β2 in scWAT, serum, and its secretion from fat explants. Transplanting scWAT from exercise-trained wild type mice, but not from adipose tissue-specific Tgfb2-/- mice, into sedentary mice improves glucose tolerance. TGF-β2 treatment reverses the detrimental metabolic effects of high fat feeding in mice. Lactate, a metabolite released from muscle during exercise, stimulates TGF-β2 expression in human adipocytes. Administration of the lactate-lowering agent dichloroacetate during exercise training in mice decreases circulating TGF-β2 levels and reduces exercise-stimulated improvements in glucose tolerance. Thus, exercise training improves systemic metabolism through inter-organ communication with fat via a lactate-TGF-β2-signaling cycle.

Heat shock protein 72 (HSP72) is a major inducible molecule in the heat shock response that enhances intracellular stress tolerance. Decreased expression of HSP72 is observed in type 2 diabetes, which may contribute to the development of insulin resistance and chronic inflammation. We used HSP72 knockout (HSP72-KO) mice to investigate the impact of HSP72 on glucose metabolism and endoplasmic reticulum (ER) stress, particularly in the liver. Under a high-fat diet (HFD) condition, HSP72-KO mice showed glucose intolerance, insulin resistance, impaired insulin secretion, and enhanced hepatic gluconeogenic activity. Furthermore, activity of the c-Jun NH2-terminal kinase (JNK) was increased and insulin signaling suppressed in the liver. Liver-specific expression of HSP72 by lentivirus (lenti) in HFD-fed HSP72-KO mice ameliorated insulin resistance and hepatic gluconeogenic activity. Furthermore, increased adipocyte size and hepatic steatosis induced by the HFD were suppressed in HSP72-KO lenti-HSP72 mice. Increased JNK activity and ER stress upon HFD were suppressed in the liver as well as the white adipose tissue of HSP72-KO lenti-HSP72 mice. Thus, HSP72 KO caused a deterioration in glucose metabolism, hepatic gluconeogenic activity, and β-cell function. Moreover, liver-specific recovery of HSP72 restored glucose homeostasis. Therefore, hepatic HSP72 may play a critical role in the pathogenesis of type 2 diabetes.

Insulin and IGF1 signaling through the PI3K/AKT and MAPK/ERK pathways are essential for maintenance of brown and white adipose tissues (BAT and WAT), as shown by our previous results using adipocyte-specific and inducible IR/IGF1R knockout mice (Ai-DKO). Here, through a research to screen the genes essential for adipose tissues maintenance in WAT and BAT using Ai-DKO mice, we identified a reduction of α4, a protein phosphatase protector which regulate phosphorylation state, in Ai-DKO BAT. shRNA-mediated α4 knockdown (KD) altered insulin-stimulated phosphorylation status in BAT; decreased IRβ (Y1162/1163), IRS1 (Y612) and Akt (S473), with a mild change in ERK1/2 (T202/Y204) but increased ribosomal S6 protein (S235/236), suggesting α4 reduces S6 phosphorylation state via a unique pathway. Next, to investigate the impact of α4 in vivo, we created inducible adipocyte-specific α4 KO (Ai-α4 KO) mice with tamoxifen-inducible Cre-ERT2 transgene. Once induced α4 KO in adipocytes, the mice revealed extensive losses of adipocytes in SC-WAT and BAT depots with increased apoptotic cell death. Ai-α4 KO mice showed cold intolerance, severe diabetes, ectopic lipid accumulation in the liver, and pancreatic islet hyperplasia. RNA-seq showed a marked reduction of genes associated with mitochondrial fatty acid oxidation and increases with inflammatory cytokine pathways in Ai-α4 KO SC-WAT and BAT. WAT and BAT mass can recover after a few months in Ai-α4KO. Interestingly, lipidomics analysis displayed that the regenerated adipocytes showed unique lipid content in the Ai-α4KO. Thus, our model demonstrates that the tuning of phosphorylation status for insulin-dependent signaling by α4 is critical for maintaining and regeneration of white and brown adipose tissues. Disclosure M. Sakaguchi: None. S. Okagawa: None. Y. Okubo: None. S. Kitano: None. M. Igata: None. T. Kondo: None. E. Araki: Advisory Panel; Self; Abbott. Speaker’s Bureau; Self; ARKRAY, Astellas Pharma Inc., AstraZeneca, Eli Lilly Japan K.K., Merck & Co., Inc., Novo Nordisk Inc., WebMD LLC. Other Relationship; Self; Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science; Merck Sharp & Dohme Foundation; Takeda Foundation; Boehringer Ingelheim; Eli Lilly and Company

Insulin and IGF1 receptors (IR/IGF1R) control adipose tissue development. We previously demonstrated that mice with an inducible adipocyte-specific knockout of IR/IGFR (Ai-DKO) develop severe lipodystrophy, insulin resistance, glucose intolerance, hepatosteatosis, islet hyperplasia with hyperinsulinemia. This phenotype resolves over 10-30 days due to proliferation of preadipocytes (PAds) and regeneration of adipose tissue. Here, we sought to identify circulating factors that trigger PAds proliferation by treating primary wild type PAds with serum from either WT or Ai-DKO mice. To exclude the hyperglycemia effect, we also incubated cells with serum from WT and Ai-DKO mice treated with the SGLT2 inhibitor Remogliflozin (Remo). Serum from either, chow or Remo-treated Ai-DKO mice, induced primary PAds proliferation of approximately 30%. Serum proteomics analysis revealed that Cathepsin D and multiple apolipoproteins, including C2 and C3, were significantly upregulated in Ai-DKO serum (FC>1 and FDR Disclosure B. Brandao: None. M. Sakaguchi: None. T.M. Batista: None. W. Qian: None. C. Kahn: Advisory Panel; Self; ERX Pharmaceuticals, Kaleido Biosciences. Consultant; Self; AntriaBio, Flagship Pioneering, Sana-Cobalt. Funding National Institutes of Health (R01DK082659-11)

Complications of diabetes affect tissues throughout the body, including the central nervous system. Epidemiological studies show that diabetic patients have an increased risk of depression, anxiety, age-related cognitive decline, and Alzheimer’s disease. Mice lacking insulin receptor (IR) in the brain or on hypothalamic neurons display an array of metabolic abnormalities; however, the role of insulin action on astrocytes and neurobehaviors remains less well studied. Here, we demonstrate that astrocytes are a direct insulin target in the brain and that knockout of IR on astrocytes causes increased anxiety- and depressive-like behaviors in mice. This can be reproduced in part by deletion of IR on astrocytes in the nucleus accumbens. At a molecular level, loss of insulin signaling in astrocytes impaired tyrosine phosphorylation of Munc18c. This led to decreased exocytosis of ATP from astrocytes, resulting in decreased purinergic signaling on dopaminergic neurons. These reductions contributed to decreased dopamine release from brain slices. Central administration of ATP analogs could reverse depressive-like behaviors in mice with astrocyte IR knockout. Thus, astrocytic insulin signaling plays an important role in dopaminergic signaling, providing a potential mechanism by which astrocytic insulin action may contribute to increased rates of depression in people with diabetes, obesity, and other insulin-resistant states.

Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region., Despite being structurally similar, the insulin receptor (IR) and insulin growth factor I receptor (IGF1R) elicit distinct signalling pathways. Here the authors use receptor chimeras to unveil that IR and IGF1R signalling is related primarily to differences in their intracellular juxtamembrane region.

Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine.

Summary Insulin and IGF1 signaling are important for adipose tissue development and function; however, their role in mature adipocytes is unclear. Mice with a tamoxifen-inducible knockout of insulin and/or IGF1 receptors (IR/IGF1R) demonstrate a rapid loss of white and brown fat due to increased lipolysis and adipocyte apoptosis. This results in insulin resistance, glucose intolerance, hepatosteatosis, islet hyperplasia with hyperinsulinemia, and cold intolerance. This phenotype, however, resolves over 10–30 days due to a proliferation of preadipocytes and rapid regeneration of both brown and white adipocytes as identified by mTmG lineage tracing. This cycle can be repeated with a second round of receptor inactivation. Leptin administration prior to tamoxifen treatment blocks development of the metabolic syndrome without affecting adipocyte loss or regeneration. Thus, IR is critical in adipocyte maintenance, and this loss of adipose tissue stimulates regeneration of brown/white fat and reversal of metabolic syndrome associated with fat loss.

Skeletal muscle insulin resistance, decreased phosphatidylinositol 3-kinase (PI3K) activation and altered mitochondrial function are hallmarks of type 2 diabetes. To determine the relationship between these abnormalities, we created mice with muscle-specific knockout of the p110α or p110β catalytic subunits of PI3K. We find that mice with muscle-specific knockout of p110α, but not p110β, display impaired insulin signaling and reduced muscle size due to enhanced proteasomal and autophagic activity. Despite insulin resistance and muscle atrophy, M-p110αKO mice show decreased serum myostatin, increased mitochondrial mass, increased mitochondrial fusion, and increased PGC1α expression, especially PCG1α2 and PCG1α3. This leads to enhanced mitochondrial oxidative capacity, increased muscle NADH content, and higher muscle free radical release measured in vivo using pMitoTimer reporter. Thus, p110α is the dominant catalytic isoform of PI3K in muscle in control of insulin sensitivity and muscle mass, and has a unique role in mitochondrial homeostasis in skeletal muscle., Diabetes is associated with decreased PI3K activation in skeletal muscle. Here, the authors show that p110a is the predominant PI3K subunit in muscle, and show that its ablation in muscle, but not ablation of p110beta, leads to insulin resistance, increased proteosomal and autophagic activity, and altered mitochondria homeostasis in mice.

Summary The role of leptin receptor (OB-R) signaling in linking pluripotency with growth and development and the consequences of dysfunctional leptin signaling on progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying the db/db mutation exhibit metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) show altered expression of proteins involved in embryonic development. An upregulation in expression of eukaryotic translation initiation factor 4e (Eif4e) and Stat3 binding to the Eif4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs toward the neuronal lineage showed defects. Gene editing to correct the point mutation in db/db iPSCs using CRISPR-Cas9, restored expression of neuronal markers and protein synthesis while reversing the metabolic defects. These data imply a direct role for OB-R in regulating metabolism in embryonic fibroblasts and key developmental pathways in iPSCs., Graphical Abstract, Highlights • Pluripotency markers are decreased in db/db iPSCs (lacking functional OB-R) • Mouse db/db iPSCs exhibit higher protein synthesis mediated by the Stat3/Eif4e axis • OB-R signaling regulates neuronal development markers—NOGGIN, NESTIN, GFAP • CRISPR correction reverses defects in db/db iPSCs, In this article Kulkarni and colleagues use CRISPR-Cas9 gene editing and pluripotent stem cells as a model system to report that leptin receptor regulates protein synthesis pathways, pluripotency markers, and early neural development in early stages of life.

To examine the efficacy and safety of once-daily insulin degludec/insulin aspart (IDegAsp) or once-daily second-generation basal insulin analogs (insulin degludec and insulin glargine 300 units/mL) in insulin-naive Japanese adults with type 2 diabetes in routine clinical practice. A 12-week multicenter, open-label, randomized, pilot study was performed in 52 subjects with type 2 diabetes treated with oral antidiabetic drugs (OADs). Subjects were randomized to once-daily IDegAsp (n = 26) or basal insulin (n = 26). The primary endpoint was percent change in HbA1c from baseline to week 12. Furthermore, it was analyzed post hoc in subgroups stratified by baseline HbA1c. During a follow-up period, percent change in HbA1c was not significantly different between the two groups (p = 0.161). Daily insulin doses and frequency of overall hypoglycemia were also similar in the two groups. In post hoc analyses, once-daily basal insulin was more effective than IDegAsp in subjects with HbA1c more than or equal to 8.5% (p < 0.05); however, in subjects with HbA1c less than 8.5%, once-daily IDegAsp showed a significant improvement in percent change in HbA1c at week 12, compared with basal insulin (p < 0.01). Although there was no apparent difference in the HbA1c-lowering effects between two groups, when compared in subjects with HbA1c less than 8.5%, once-daily IDegAsp showed a significant effect in comparison with once-daily basal insulin. These findings suggest that the baseline HbA1c level might provide the important information for choosing IDegAsp or basal insulin in patients insufficiently controlled with OADs. This trial was registered with UMIN (no. UMIN000035431).

Insulin and IGF1 signaling regulate function of brown and white adipose tissues. Recently we created adipocyte-specific inducible IR/IGF1R knockout mouse (Ai-DKO) and demonstrated the critical role of insulin signaling in maintenance of BAT and WAT. To further investigate downstream targets of these hormones regulating adipose tissue mass in adult animals, we compared gene expression patterns in WAT and BAT by using Ai-DKO mice, and found α4 expression was significantly reduced in Ai-DKO BAT but not in WAT compared with those of control. Knockdown of α4 in brown preadipocytecell lines using shRNA did not affect insulin-stimulated phosphorylation of IR, IRS1 or Akt; however, phosphorylation of ribosomal S6 protein was significantly increased in α4 KD cells after insulin stimulation. To investigate the impact of α4in mature adipocytesin vivo, we created inducible adipocyte-specific knockout of α4 gene (Ai-α4 KO) using floxed mice and mice carrying a tamoxifen-inducible Cre-ERT2transgene on the adiponectin promoter. Within 7 days, Ai-α4KO displayed an apparent decrease in SC-WAT and BAT mass, but not a significant effect on PG-WAT mass. Ai-α4 KO revealed extensive losses of adipocytes in SC-WAT and BAT depots with a significant increase in stromovascular cells in histological analysis with elevation of F4/80, TNF-α and IL-6, while no apparent changes were observed in PG-WAT. This appears to be due to a rapid development of an apoptotic process with increased TUNEL and cleaved caspase-3.Interestingly, Ai-α4KO showed marked reduction of BAT-associated functional gene UCP1 as well as PRDM16, Tfam and PGC1α mRNA compared to control. Consistent with these changes in mRNA and the decrease in BAT mass, Ai-α4 KO mice showed marked cold intolerance, glucose intolerance and insulin resistance. Thus, α4 is one of the components in control of insulin signaling and preferentially BAT mass, and has a unique role in regulating anti-apoptotic effect in brown adipocytes. Disclosure M. Sakaguchi: None. S. Okagawa: None. S. Kitano: None. T. Kondo: None. E. Araki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi. Funding Japan Society for the Promotion of Science; Takeda Foundation; MSD Foundation; Kenzou Suzuki Foundation; Ono Foundation; Motida Foundation

Exercise improves health and well-being across diverse organ systems, and elucidating mechanisms underlying the beneficial effects of exercise can lead to new therapies. Here, we show that transforming growth factor-β2 (TGF-β2) is secreted from adipose tissue in response to exercise and improves glucose tolerance in mice. We identify TGF-β2 as an exercise-induced adipokine in a gene expression analysis of human subcutaneous adipose tissue biopsies after exercise training. In mice, exercise training increases TGF-β2 in scWAT, serum, and its secretion from fat explants. Transplanting scWAT from exercise-trained wild type mice, but not from adipose tissue-specific Tgfb2-/- mice, into sedentary mice improves glucose tolerance. TGF-β2 treatment reverses the detrimental metabolic effects of high fat feeding in mice. Lactate, a metabolite released from muscle during exercise, stimulates TGF-β2 expression in human adipocytes. Administration of the lactate-lowering agent dichloroacetate during exercise training in mice decreases circulating TGF-β2 levels and reduces exercise-stimulated improvements in glucose tolerance. Thus, exercise training improves systemic metabolism through inter-organ communication with fat via a lactate-TGF-β2-signaling cycle.

Because the renin-angiotensin-aldosterone system influences glucose homeostasis, the mineralocorticoid receptor (MR) signal in pancreatic islets may regulate insulin response upon glucose load. Glucagon-like peptide-1 (GLP-1) production is stimulated by interleukin-6 (IL-6) in pancreatic &alpha, cells. To determine how glucose homeostasis is regulated by interactions of MR, IL-6 and GLP-1 in islets, we performed glucose tolerance and histological analysis of islets in primary aldosteronism (PA) model rodents and conducted in vitro experiments using &alpha, cell lines. We measured active GLP-1 concentration in primary aldosteronism (PA) patients before and after the administration of MR antagonist eplerenone. In PA model rodents, aldosterone decreased insulin-secretion and the islet/pancreas area ratio and eplerenone added on aldosterone (E+A) restored those with induction of IL-6 in &alpha, cells. In &alpha, cells treated with E+A, IL-6 and GLP-1 concentrations were increased, and anti-apoptotic signals were enhanced. The E+A-treatment also significantly increased MR and IL-6 mRNA and these upregulations were blunted by MR silencing using small interfering RNA (siRNA). Transcriptional activation of the IL-6 gene promoter by E+A-treatment required an intact MR binding element in the promoter. Active GLP-1 concentration was significantly increased in PA patients after eplerenone treatment. MR signal in &alpha, cells may stimulate IL-6 production and increase GLP-1 secretion, thus protecting pancreatic &beta, cells and improving glucose homeostasis.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is central to the action of insulin and many growth factors. Heterozygous mutations in the gene encoding the p85α regulatory subunit of PI3K (PIK3R1) have been identified in patients with SHORT syndrome — a disorder characterized by short stature, partial lipodystrophy, and insulin resistance. Here, we evaluated whether SHORT syndrome–associated PIK3R1 mutations account for the pathophysiology that underlies the abnormalities by generating knockin mice that are heterozygous for the Pik3r1Arg649Trp mutation, which is homologous to the mutation found in the majority of affected individuals. Similar to the patients, mutant mice exhibited a reduction in body weight and length, partial lipodystrophy, and systemic insulin resistance. These derangements were associated with a reduced capacity of insulin and other growth factors to activate PI3K in liver, muscle, and fat; marked insulin resistance in liver and fat of mutation-harboring animals; and insulin resistance in vitro in cells derived from these mice. In addition, mutant mice displayed defective insulin secretion and GLP-1 action on islets in vivo and in vitro. These data demonstrate the ability of this heterozygous mutation to alter PI3K activity in vivo and the central role of PI3K in insulin/growth factor action, adipocyte function, and glucose metabolism.

Insulin and IGF-1 are regulators of cell growth and metabolism. Despite considerable progress in understanding the molecular mechanisms underlying these effects, except for members of the FoxO family, many regulators of the transcriptional effects of these hormones remain poorly understood. To identify new components of IR/IGF1R signaling, we created brown preadipocyte lines in which both endogenous IR and IGF1R genes had been deleted and then reconstituted these cells with normal IR, IGF1R or chimeric receptors containing the extracellular domain of IR fused to intracellular domain of IGF1R and vice versa. Mass spectroscopic proteomic analysis revealed a number of proteins co-precipitated with each receptor construct, in both ligand stimulation-dependent and -independent manners. Among the proteins that associated with both receptors and chimeric receptors in a ligand-dependent manner was FoxK1. In contrast to FoxO1, the transcription factor FoxK1 was translocated from the cytoplasm to the nucleus after insulin stimulation, a pattern that is reciprocal to that of FoxO1 after insulin stimulation. We show that FoxK1 and FoxK2 are phosphorylated in two major domains and that translocation to the nucleus is dependent on the Akt-mTOR mediated phosphorylation, while their localization to the cytoplasm in the basal state is dependent on GSK3 phosphorylation. Knockdown of FoxK1 and FoxK2 in a mouse hepatocyte cell line causes marked alteration of the transcription of genes associated with apoptotic pathway and down-regulation of genes involved in control of cell cycle and metabolism. This resulted in decreased cell proliferation and altered mitochondrial fatty acid metabolism in these cells. Thus, insulin stimulates the reciprocal translocation of FoxKs and FoxO1 between the cytoplasm and nucleus turning off FoxO1-regulated genes and turning on FoxK-regulated genes resulting in an elaborate balance in insulin regulation of metabolism, growth and cell survival. Disclosure M. Sakaguchi: None. W. Cai: None. C. Wang: None. T.M. Batista: None. E. Araki: Speaker's Bureau; Self; Astellas Pharma US, Inc., MSD K.K., Kowa Pharmaceuticals America, Inc., Sanofi, Novo Nordisk Inc.. Research Support; Self; Astellas Pharma US, Inc., MSD K.K., Ono Pharmaceutical Co., Ltd., Shionogi & Co., Ltd., Takeda Pharmaceuticals U.S.A., Inc., Daiichi Sankyo Company, Limited, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Sanofi, Mitsubishi Tanabe Pharma Corporation, Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co. C. Kahn: Advisory Panel; Self; CohBar, ERX Therapeutics, AntriaBio, Inc.. Board Member; Self; Kaleio Biosciences.

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene expression and have been shown to participate in almost every cellular process. Several miRNAs have recently been implicated in glucose metabolism, but the roles of miRNAs in insulin-resistant conditions, such as obesity or type 2 diabetes, are largely unknown. Herein, we focused on miR-222, the expression of which was increased in the livers of high fat/high sucrose diet-fed mice injected with gold thioglucose (G+HFHSD). Overexpression of miR-222 in primary mouse hepatocytes attenuated Akt phosphorylation induced by insulin, indicating that miR-222 negatively regulates insulin signaling. As per in silico analysis, miR-222 potentially binds to the 3' untranslated region (3' UTR) of the IRS-1 gene, a key insulin signaling molecule. In fact, IRS-1 protein expression was decreased in the livers of G+HFHSD-fed mice. We further confirmed a direct interaction between miR-222 and the 3' UTR of IRS-1 via luciferase assays. Our findings suggest that up-regulation of miR-222 followed by reduction in IRS-1 expression may be a viable mechanism of insulin resistance in the liver.

The kidney develops through mutual interactions between the metanephric mesenchyme and the ureteric bud, the former of which contains nephron progenitors that give rise to glomeruli and renal tubules. Bone morphogenetic protein (BMP) signaling and its modifiers play important roles in many steps of kidney development. BMP4 inhibits ureteric bud attraction, and the BMP antagonist Gremlin is essential for the initial stage of ureteric budding. During mid-gestation, BMP7 maintains the nephron progenitors and, at the same time, sensitizes them to the ureteric bud-derived differentiation signal. Crossveinless2 is a pro-BMP factor, and its absence leads to kidney hypoplasia. After birth, when nephron progenitors have disappeared, Dullard, a phosphatase that inactivates BMP receptors, keeps BMP signaling at an appropriate level. Deletion of Dullard results in excessive BMP signaling and apoptosis of the postnatal nephrons. In this review I discuss the similarities and differences of BMP functions in kidney development, as well as in diseases.

Interactions of diet, gut microbiota, and host genetics play important roles in the development of obesity and insulin resistance. Here, we have investigated the molecular links between gut microbiota, insulin resistance, and glucose metabolism in 3 inbred mouse strains with differing susceptibilities to metabolic syndrome using diet and antibiotic treatment. Antibiotic treatment altered intestinal microbiota, decreased tissue inflammation, improved insulin signaling in basal and stimulated states, and improved glucose metabolism in obesity- and diabetes-prone C57BL/6J mice on a high-fat diet (HFD). Many of these changes were reproduced by the transfer of gut microbiota from antibiotic-treated donors to germ-free or germ-depleted mice. These physiological changes closely correlated with changes in serum bile acids and levels of the antiinflammatory bile acid receptor Takeda G protein–coupled receptor 5 (TGR5) and were partially recapitulated by treatment with a TGR5 agonist. In contrast, antibiotic treatment of HFD-fed, obesity-resistant 129S1 and obesity-prone 129S6 mice did not improve metabolism, despite changes in microbiota and bile acids. These mice also failed to show a reduction in inflammatory gene expression in response to the TGR5 agonist. Thus, changes in bile acid and inflammatory signaling, insulin resistance, and glucose metabolism driven by an HFD can be modified by antibiotic-induced changes in gut microbiota; however, these effects depend on important interactions with the host’s genetic background and inflammatory potential.

The kidney is formed by a reciprocally inductive interaction between two precursor tissues, the metanephric mesenchyme and the ureteric bud. This interaction can be divided into three processes: attraction of the ureteric bud toward the mesenchyme, maintenance of the mesenchyme in an undifferentiated state versus transition to an epithelial state, and further differentiation into multiple epithelial lineages, such as glomeruli and renal tubules. In this review we describe our recent findings related to each process. A mesenchymal nuclear zinc finger protein, Sall1, controls ureteric bud attraction by regulating a novel kinesin, Kif26b. The Sall1 gene is highly expressed in multipotent nephron progenitors in the mesenchyme, and these cells can partially reconstitute a three-dimensional structure in organ cultures following Wnt4 stimulation. While Notch2 is required for further differentiation of proximal nephron structures, ectopic Notch2 activation in the embryonic kidney depletes nephron progenitors, suggesting that Notch2 stabilizes--rather than dictates--nephron fate by shutting down the maintenance of undifferentiated progenitor cells.

Nature 542, 450–455 (2017); doi:10.1038/nature21365 In this Article, in Fig. 4f–h, the x-axis labels ‘WT + exoKO’ and ‘ADicerKO + exoKO’ were inadvertently reversed, and in Fig. 5c, f, the y-axis labels should have been labelled ‘miR-302f 3′UTR total flux’ rather than ‘Fgf21 3′UTR total flux’.

Most kidney nephron components, including glomeruli and renal tubules, derive from the metanephric mesenchyme. The overall differentiation into each component finishes at birth, but the molecular events linking the perinatal and adult kidneys remain elusive. Dullard was cloned from Xenopus kidneys, and encodes a phosphatase that negatively regulates BMP signalling. Here we report that Dullard deletion in the murine metanephric mesenchyme leads to failure of nephron maintenance after birth, resulting in lethality before adulthood. The nephron components are lost by massive apoptosis within 3 weeks after birth, leading to formation of a large hollow with a thin-layered cortex and medulla. Phosphorylated Smad1/5/8 is upregulated in the mutant nephrons, probably through cell-autonomous inhibitory effects of Dullard on BMP signalling. Importantly, administration of the BMP receptor kinase inhibitor LDN-193189 partially rescued the defects caused by Dullard deletion. Thus, Dullard keeps BMP signalling at an appropriate level, which is required for nephron maintenance in the postnatal period.

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development.

The kidney develops through reciprocal interactions between two precursor tissues: the metanephric mesenchyme and the ureteric bud. We previously demonstrated that the zinc finger protein Sall1 is essential for ureteric bud attraction toward the mesenchyme. Here, we show that Kif26b , a kinesin family gene, is a downstream target of Sall1 and that disruption of this gene causes kidney agenesis because of impaired ureteric bud attraction. In the Kif26b -null metanephros, compact adhesion between mesenchymal cells adjacent to the ureteric buds and the polarized distribution of integrin α8 were impaired, resulting in failed maintenance of Gdnf , a critical ureteric bud attractant. Overexpression of Kif26b in vitro caused increased cell adhesion through interactions with nonmuscle myosin. Thus, Kif26b is essential for kidney development because it regulates the adhesion of mesenchymal cells in contact with ureteric buds.