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A newly developed resistant starch (RS) rice line with double mutation of starch synthase IIIa and branching enzyme IIb (ss3a/be2b) exhibits a tenfold greater percentage RS value than the wild-type rice line. Currently, the effects of cooked rice with such high RS content on secretion and action of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are unclear. Therefore, we conducted a pilot study to assess postprandial responses of GLP-1 and GIP along with glucose and insulin and also gastric emptying after ingestion of the high-RS cooked rice with ss3a/be2b in healthy subjects.In a non-randomized crossover design, five healthy men ingested two test foods, control (low-RS) and high-RS cooked rice, with at least 1-week washout period between testing days. Plasma glucose, serum insulin, plasma total GLP-1, plasma total GIP, and also gastric emptying rate were measured after ingestion of each test food, and the incremental area under the curves (iAUC) was calculated for each biochemical parameter using the values from 0 to 180 min after ingestion.The high-RS cooked rice ingestion tended to reduce iAUC-glucose (p = 0.06) and significantly reduced iAUC-insulin (p 0.01) and iAUC-GLP-1 (p 0.05) but not iAUC-GIP (p = 0.21) relative to control cooked rice ingestion. In addition, the high-RS cooked rice ingestion did not affect gastric emptying.The present results indicate that the suppressive effects of the high-RS cooked rice ingestion on postprandial responses of glucose and insulin may be provided through attenuation in GLP-1 secretion along with its low digestibility into glucose. We suggest that the high-RS rice with ss3a/be2b may serve as a better carbohydrate source and also as a novel functional food for dietary interventions to improve postprandial hyperglycemia and hyperinsulinemia without both enhancing GLP-1 secretion and affecting gastric emptying in patients with diabetes.

White adipose tissues (WAT) play a central role in lipid storage and systemic energy, lipid, and glucose homeostasis. Understanding the intricacies of adipocyte formation could inform therapies for obesity and metabolic disorders. We have identified the POZ/BTB and AT Hook Containing Zinc Finger 1 (PATZ1) protein as an adipogenic transcription factor through an unbiased high-throughput cDNA screen for modulators of adipogenesis. PATZ1 is expressed by both human and mouse adipocyte precursor cells (APC) and adipocytes, and its expression negatively correlates with obesity. In cell models, PATZ1 expression promotes adipogenesis through a mechanism dependent on protein-protein interaction and DNA binding. Adipose-specific ablation of PATZ1 in mice leads to decreased lipid storage and fat mass and protection from diet-induced obesity. Genome-wide PATZ1 DNA binding analyses using ChIP-Seq suggest that PATZ1 facilitates adipogenesis through interactions with transcription factor machinery at the promoter regions of key adipogenic factors and histone modifiers. Global PATZ1 protein interaction studies using immunoprecipitation-mass spectrometry (IP-MS) suggest that General Transcription Factor 2I (GTF2I) binding to PATZ1 forms a repressive complex, and knockdown of GTF2I augments PATZ1 adipogenic function. Collectively these findings identify PATZ1 as a regulator of the adipocyte differentiation programs and advance our understanding of the complex transcriptional mechanisms underlying adipose tissue development and homeostasis.

Thermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a

Glucagon is detected in plasma even after total pancreatectomy, and it is debated whether this glucagon is derived from the gastrointestinal tract. Here, we applied sandwich enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography–high‐resolution mass spectrometry to measure plasma glucagon levels in one patient after partial pancreatectomy (one‐seventh of the pancreas remaining) and three patients after total pancreatectomy. Sandwich ELISA detected higher glucagon levels in pancreatectomy patients than in healthy individuals. In contrast, liquid chromatography–high‐resolution mass spectrometry showed that plasma glucagon levels in pancreatectomy patients were below the lower limit of quantification. Plasma glucagon measured by sandwich ELISA showed a striking correlation with plasma glicentin, suggesting cross‐reaction with this gastrointestinal glucagon‐related peptide. These results indicated that pancreatectomized patients falsely showed pseudo‐hyperglucagonemia when measured by glucagon sandwich ELISA., Pancreatectomized patients exhibit hypoglucagonemia and sandwich ELISA measurements of plasma glucagon in pancreatectomized patients are incorrect.

Chronic intestinal pseudo-obstruction (CIPO) is a rare disorder of intestinal dysmotility characterized by chronic symptoms, including vomiting and abdominal pain, associated with bowel obstruction without any mechanical obstructive causes. We herein report a case of mitochondrial diseases with recurrent duodenal obstruction that was initially diagnosed as superior mesenteric artery syndrome (SMAS) for a few years but was later diagnosed as CIPO. Since CIPO is known to be associated with mitochondrial diseases, it should be considered in the differential diagnosis of patients with mitochondrial diseases presenting with recurrent intestinal obstruction.

To ensure that experiences and lessons learned from the unprecedented 2011 Great East Japan Earthquake are used to improve future disaster planning, the Japan Diabetes Society (JDS) launched the “Research and Survey Committee for Establishing Disaster Diabetes Care Systems Based on Relevant Findings from the Great East Japan Earthquake” under the supervision of the Chairman of the JDS. The Committee conducted a questionnaire survey among patients with diabetes, physicians, disaster medical assistance teams (DMATs), nurses, pharmacists, and nutritionists in disaster areas about the events they saw happening, the situations they found difficult to handle, and the needs that they felt required to be met during the 2011 Great East Japan Earthquake. A total of 3,481 completed questionnaires were received. Based on these and other experiences and lessons reported following the 2011 Great East Japan Earthquake and the 2004 Niigata‐Chuetsu Earthquakes, the current “Manual for Disaster Diabetes Care” has been developed by the members of the Committee and other invited authors from relevant specialties. To our knowledge, the current Manual is the world's first to focus on emergency diabetes care, with this digest English version translated from the Japanese original. It is sincerely hoped that patients with diabetes and healthcare providers around the world will find this manual helpful in promoting disaster preparedness and implementing disaster relief.

Background Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. Methods We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. Results We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. Conclusion This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

Aims Monogenic diabetes is clinically heterogeneous and differs from common forms of diabetes (type 1 and 2). We aimed to investigate the clinical usefulness of a comprehensive genetic testing system, comprised of targeted next-generation sequencing (NGS) with phenotype-driven bioinformatics analysis in patients with monogenic diabetes, which uses patient genotypic and phenotypic data to prioritize potentially causal variants. Methods We performed targeted NGS of 383 genes associated with monogenic diabetes or common forms of diabetes in 13 Japanese patients with suspected (n = 10) or previously diagnosed (n = 3) monogenic diabetes or severe insulin resistance. We performed in silico structural analysis and phenotype-driven bioinformatics analysis of candidate variants from NGS data. Results Among the patients suspected having monogenic diabetes or insulin resistance, we diagnosed 3 patients as subtypes of monogenic diabetes due to disease-associated variants of INSR, LMNA, and HNF1B. Additionally, in 3 other patients, we detected rare variants with potential phenotypic effects. Notably, we identified a novel missense variant in TBC1D4 and an MC4R variant, which together may cause a mixed phenotype of severe insulin resistance. Conclusions This comprehensive approach could assist in the early diagnosis of patients with monogenic diabetes and facilitate the provision of tailored therapy.

BACKGROUND: Beige adipose tissue is associated with improved glucose homeostasis in mice. Adipose tissue contains β3-adrenergic receptors (β3-ARs), and this study was intended to determine whether the treatment of obese, insulin-resistant humans with the β3-AR agonist mirabegron, which stimulates beige adipose formation in subcutaneous white adipose tissue (SC WAT), would induce other beneficial changes in fat and muscle and improve metabolic homeostasis. METHODS: Before and after β3-AR agonist treatment, oral glucose tolerance tests and euglycemic clamps were performed, and histochemical analysis and gene expression profiling were performed on fat and muscle biopsies. PET-CT scans quantified brown adipose tissue volume and activity, and we conducted in vitro studies with primary cultures of differentiated human adipocytes and muscle. RESULTS: The clinical effects of mirabegron treatment included improved oral glucose tolerance (P < 0.01), reduced hemoglobin A1c levels (P = 0.01), and improved insulin sensitivity (P = 0.03) and β cell function (P = 0.01). In SC WAT, mirabegron treatment stimulated lipolysis, reduced fibrotic gene expression, and increased alternatively activated macrophages. Subjects with the most SC WAT beiging showed the greatest improvement in β cell function. In skeletal muscle, mirabegron reduced triglycerides, increased the expression of PPARγ coactivator 1 α (PGC1A) (P < 0.05), and increased type I fibers (P < 0.01). Conditioned media from adipocytes treated with mirabegron stimulated muscle fiber PGC1A expression in vitro (P < 0.001). CONCLUSION: Mirabegron treatment substantially improved multiple measures of glucose homeostasis in obese, insulin-resistant humans. Since β cells and skeletal muscle do not express β3-ARs, these data suggest that the beiging of SC WAT by mirabegron reduces adipose tissue dysfunction, which enhances muscle oxidative capacity and improves β cell function. TRIAL REGISTRATION: Clinicaltrials.gov NCT02919176. FUNDING: NIH: DK112282, P30GM127211, DK 71349, and Clinical and Translational science Awards (CTSA) grant UL1TR001998.

A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3'-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional control of adipose development and function.

Atypical diabetes is considered to be different from the common forms of diabetes (types 1 and 2); however, it may share pathogenetic and phenotypic features with the common forms of diabetes. Targeted next-generation sequencing focused on genes causing monogenic diabetes has been reported to be useful for diagnosis of a few subtypes of atypical diabetes caused by genetic defects, such as MODY. In this study, considering the clinically heterogeneous nature of atypical diabetes, we developed a comprehensive gene panel containing 383 monogenic diabetes causing genes and genes associated with common forms of diabetes, and conducted targeted next-generation sequencing in 3 patients with previously confirmed and 10 patients with suspected atypical diabetes. In addition, phenotype-driven bioinformatics analysis using genetic and phenotypic information was performed to identify causal variants from sequencing data. A total of 11 mutations including 2 gross deletions were identified, which led to diagnosis of four previously undiagnosed patients and identification of potentially pathogenic variants of interest in other three patients. Of note, a novel missense mutation of Rab GAP domain of TBC1D4 was identified in a diabetic patient with severe insulin resistance. The TBC1D4 locus has been reported to be associated with type 2 diabetes and insulin resistance. In silico structural analysis suggested that the mutation would cause pathogenesis by functional and structural modification of TBC1D4, insulin-dependent phosphorylation of which is necessary for GLUT4 translocation mediated glucose uptake in skeletal muscle and adipose tissues. The results from this study indicate that comprehensive genetic screening analyzing genes associated with common and rare subtypes of diabetes can identify causal variants efficiently, thereby aiding early diagnosis of atypical diabetes. Disclosure J. Hosoe: None. H. Kadowaki: Research Support; Spouse/Partner; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Spouse/Partner; Astellas Pharma Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. F. Miya: None. M. Takakura: None. H. Waki: Research Support; Self; Astellas Pharma Inc., Kyowa Hakko Kirin Co., Ltd., MSD K.K., Nippon Boehringer Ingelheim Co Ltd, Novo Nordisk Foundation, Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, The Cell Science Research Foundation. T. Sasako: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk Inc. T. Kato: None. T. Tsunoda: None. N. Shojima: None. T. Yamauchi: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Takeda Pharmaceutical Company Limited. T. Kadowaki: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

The transcription factor nuclear factor I-A (NFIA) is a regulator of brown adipocyte differentiation. Here we show that the C-terminal 17 amino acid residues of NFIA (which we call pro#3 domain) are required for the transcriptional activity of NFIA. Full-length NFIA—but not deletion mutant lacking pro#3 domain—rescued impaired expression of PPARγ, the master transcriptional regulator of adipogenesis and impaired adipocyte differentiation in NFIA-knockout cells. Mechanistically, the ability of NFIA to penetrate chromatin and bind to the crucial Pparg enhancer is mediated through pro#3 domain. However, the deletion mutant still binds to Myod1 enhancer to repress expression of MyoD, the master transcriptional regulator of myogenesis as well as proximally transcribed non-coding RNA called DRReRNA, via competition with KLF5 in terms of enhancer binding, leading to suppression of myogenic gene program. Therefore, the negative effect of NFIA on the myogenic gene program is, at least partly, independent of the positive effect on PPARγ expression and its downstream adipogenic gene program. These results uncover multiple ways of action of NFIA to ensure optimal regulation of brown and beige adipocyte differentiation., Author summary Obesity and its complications including type 2 diabetes are growing concerns worldwide. While white adipocytes generally store energy in the form of lipid, classical brown and cold- or β-adrenergic stimulation-induced beige adipocytes dissipate chemical energy in the form of heat through uncoupling protein-1 (Ucp1). Since the re-discovery of human brown and beige adipocytes, it has been considered a promising target for the treatment of obesity. During mesenchymal development, not only activation of brown/beige adipocyte gene program but also repression of muscle gene program is required to achieve thermogenic adipocyte differentiation. Previously, we identified the transcription factor nuclear factor I-A (NFIA) as a regulator of brown adipocyte differentiation. Here we show that the C-terminal 17 amino acid residues of NFIA, which we call pro#3 domain, is required for activation of adipocyte differentiation. However, the deletion mutant which lacks this domain is still able to suppress muscle gene program by repressing the expression of Myod1, which encode the master transcriptional regulator of myogenesis, MyoD. We demonstrate that NFIA activates adipogenesis and also “actively” suppresses myogenesis through distinct molecular pathways to ensure brown and beige adipocyte differentiation.

Brown fat dissipates energy as heat by means of the uncoupling protein-1 (UCP1) on the mitochondrial inner membrane. Brown fat activity is inversely correlated with body mass index in humans, and pilot studies have shown that interventions such as chronic cold exposure successfully recruit human brown fat and increase systemic energy expenditure. Therefore, stimulating development and/or function of brown fat would be a novel strategy for the treatment of obesity and its complications. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of the brown-fat-specific open chromatin regions. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. In human brown fat, expression of NFIA and the brown-fat-specific genes including UCP1 is positively correlated. Mechanistically, NFIA co-localizes with the master transcriptional regulator of adipogenesis, PPARgamma, selectively at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARgamma, leading to increased chromatin accessibility and active transcription. Most recently, we performed mass spectrometry analysis of NFIA protein complex to identify and characterize the crucial partner of NFIA-dependent brown-fat-specific chromatin regulation. Collectively, these results indicate that NFIA is a key transcription factor that activates the cell-type-specific enhancers and facilitates the binding of PPARgamma for controlling the brown fat gene program. Disclosure Y. Hiraike: Speaker's Bureau; Self; Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S, Mitsubishi Tanabe Pharma Corporation. H. Waki: Research Support; Self; MSD K.K., Novartis Pharma K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi K.K., Ono Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda PHarmaceutical Co., Ltd.. S. Tsutsumi: None. H. Aburatani: None. T. Yamauchi: Research Support; Self; Novo Nordisk Inc., Daiichi Sankyo Company, Limited, Sanofi, Takeda, Tanabe-Mitsubishi. T. Kadowaki: Consultant; Self; Novo Nordisk A/S, AstraZeneca, Merck Sharp & Dohme Corp.. Research Support; Self; Kissei Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Sanofi, Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Astellas Pharma, Daiichi Sankyo Company, Limited, Takeda, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Astellas Pharma, AstraZeneca, Merck Sharp & Dohme Corp., Ono Pharmaceutical Co., Ltd., Takeda, Eli Lilly and Company, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk A/S.

Background: Near-infrared time-resolved spectroscopy is a non-invasive method that facilitates analysis of human brown adipose tissue (BAT) density. We measured BAT density in middle-aged patients who had type 2 diabetes by this method and examined the relationship using clinical parameters. Methods: We studied 33 Japanese middle-aged diabetic patients (30 male and 3 female, age 53.8±10.1 years, BMI 25.4±4.5, and HbA1c 7.2±1.1%). Tissue hemoglobin concentration (tHb) of the supraclavicular region was measured using near-infrared time-resolved spectroscopy, and normalized by subcutaneous fat thickness at the measurement sites. Results: The average tHb concentration was 51.1±17.7 μM and lower than 77.8 μM—the value equivalent to the detection threshold of BAT by standard FDG-PET in young adults. Univariate regression analysis showed that the tHb of the supraclavicular region was inversely correlated with BMI (r = −0.55, p=0.0009), but the tHb of the deltoid region was not (r = 0.04, p = 0.31). Other parameters showing significant correlations with BAT mass included systolic blood pressure (r = −0.47, p = 0.01) and insulin treatment (r=0.45, p=0.01). Multivariate regression analysis suggested that BMI (β=−0.42, p=0.005), systolic blood pressure (β=−0.29, p=0.042), and insulin treatment (β=0.34, p=0.015) independently account for BAT density. Conclusion: Our findings suggest that BAT density in middle-aged diabetic patients may relate to obesity more closely than previously thought. Disclosure H. Waki: Research Support; Self; MSD K.K., Novartis Pharma K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi K.K., Ono Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda PHarmaceutical Co., Ltd.. N. Watanabe: None. S. Fuse: None. Y. Kurosawa: None. T. Yamauchi: Research Support; Self; Novo Nordisk Inc., Daiichi Sankyo Company, Limited, Sanofi, Takeda, Tanabe-Mitsubishi. R. Kime: None. T. Hamaoka: None. T. Kadowaki: Consultant; Self; Novo Nordisk A/S, AstraZeneca, Merck Sharp & Dohme Corp.. Research Support; Self; Kissei Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Sanofi, Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Astellas Pharma, Daiichi Sankyo Company, Limited, Takeda, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Astellas Pharma, AstraZeneca, Merck Sharp & Dohme Corp., Ono Pharmaceutical Co., Ltd., Takeda, Eli Lilly and Company, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk A/S.

Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear., Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear. N6-Methyladenosine (m6A) posttranscriptional RNA modification, whose methylation and demethylation are performed by respective enzyme molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N6-adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like 3 (METTL3), and METTL14 positively controls adipogenesis by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro. Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistent with this, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through the WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders.

CDK5 Regulatory Subunit-Associated Protein 1-like 1 (CDKAL1) was identified as a susceptibility gene for type 2 diabetes and body mass index in genome-wide association studies. Although it was reported that CDKAL1 is a methylthiotransferase essential for tRNALys(UUU) and faithful translation of proinsulin generated in pancreatic β cells, the role of CDKAL1 in adipocytes has not been understood well. In this study, we found that CDKAL1 is expressed in adipose tissue and its expression is increased during differentiation. Stable overexpression of CDKAL1, however, inhibited adipocyte differentiation of 3T3-L1 cells, whereas knockdown of CDKAL1 promoted differentiation. CDKAL1 increased protein levels of β-catenin and its active unphosphorylated form in the nucleus, thereby promoting Wnt target gene expression, suggesting that CDKAL1 activated the Wnt/β-catenin pathway—a well-characterized inhibitory regulator of adipocyte differentiation. Mutant experiments show that conserved cysteine residues of Fe-S clusters of CDKAL1 are essential for its anti-adipogenic action. Our results identify CDKAL1 as novel negative regulator of adipocyte differentiation and provide insights into the link between CDKAL1 and metabolic diseases such as type 2 diabetes and obesity.

Brown fat dissipates energy as heat and protects against obesity. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of brown-fat-specific open chromatin regions. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat. These results indicate that NFIA activates the cell-type-specific enhancers and facilitates the binding of PPARγ for controlling the brown fat gene program.

Aims/Introduction Preventing dropout is crucial in managing diabetes. Accordingly, we investigated whether patients who had dropped out of diabetic care are suitable candidates for the use of mobile technologies – such as smartphone applications – to support self-management (mHealth), which might help prevent dropout. Materials and Methods We carried out a cross-sectional study in Tokyo, Japan. Patients aged 20 years or older who were clinically diagnosed as diabetic and who regularly visited the outpatient unit at the University of Tokyo Hospital were recruited between August 2014 and March 2015. Data were collected through face-to-face structured interviews, physical measurements and medical records. Participants were asked whether they were willing to use mHealth after being shown DialBetics – an mHealth application for diabetics – as an example, and about their history of dropout and previous mHealth experience. Data were analyzed by multivariate logistic regression models. Results Of 307 patients with type 1 and type 2 diabetes, 34 (11.1%) had previously dropped out from diabetic care. Multivariate analysis identified previous mHealth experience as a negative predictor of dropout (odds ratio 0.211, P = 0.023). Of those 34 patients, 27 (79.4%) expressed willingness to use mHealth, a significantly higher percentage than for those who had never dropped out (51.5%, P = 0.002). After adjusting for confounders, history of dropout remained a strong predictor of willingness (odds ratio 3.870, P = 0.004). Conclusions Patients who previously dropped out of diabetic care are suitable candidates for mHealth. Future studies must evaluate whether mHealth is effective for preventing repeated dropout and improving glycemic control among this population.

Genomic DNA in eukaryotes forms a highly-organized structure called chromatin. Epigenetic regulation of genes involves DNA methylation and modifications of the histone tails such as acetylation and methylation, which lead to a given phenotype without a change in nucleotide sequence. Both genetic and environmental factors play important roles in the development of life-style diseases. Epigenetic regulation is implicated to contribute to the interplay between the environmental and genetic factors. Advance in DNA sequencing technologies provides novel insights into transcriptional and epigenetic regulation of the genes and mechanisms by which genomic polymorphism causes diseases. We will overview recent progress in the epigenetic studies on life-style diseases.

Both genetic and environmental factors play critical roles in the development of diabetes. Epidemiological evidence and data from clinical studies suggest the persistence of a "metabolic memory" of past exposures to environmental factors or glycemic control. Epigenetic mechanisms are regarded as one of the likeliest candidates underlying these phenomena. On the other hand, owing to the recent elucidation of mechanisms that erase epigenetic marks, it has gradually become recognized that epigenetic regulation is a more dynamic process than previously thought. A technological breakthrough in epigenome research in the past decade was the development of high-throughput sequencing. This new technology lets us investigate the epigenome in a global and comprehensive manner, and provides previously unrecognized findings and insights. This review presents an overview of the recent progress in our understanding of epigenetic regulation in type 1 and type 2 diabetes research.

SummaryPPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program.

We studied the molecular mechanism of obesity-induced insulin resistance and adipogenesis. Plasma adiponectin and adiponectin receptor (AdipoR1) in muscle are down-regulated in obesity. Analysis of muscle-specific AdipoR1 knockout mice revealed the pivotal role of adiponectin/AdipoR1 in the regulation of mitochondrial biogenesis via AMPK- and SIRT1-mediated PGC-1α activation as well as Ca(2+)-dependent up-regulation of PGC-1α expression. Reduced adiponectin/AdipoR1 signals in muscle in obesity appear to cause PGC-1α inactivation as well as down-regulation and consequently impaired mitochondrial biogenesis and insulin resistance. In the epigenetic analysis of adipogenesis, we demonstrated that adipocyte-specific formaldehyde-assisted isolation of regulatory elements (FAIRE) peaks are associated with genes up-regulated by adipogenesis, whereas preadipocyte-specific FAIRE peaks are associated with genes down-regulated by adipogenesis. Computational motif analyses of adipocyte-specific FAIRE peaks confirmed PPARγ and CCAAT-enhancer binding proteins (C/EBPs) on the top list, consistent with their crucial roles in adipogenic transcription, and also revealed NFIA and NFIB to be important regulators of proper adipocyte differentiation.

We previously identified the small molecule harmine as a regulator of peroxisome proliferator activated-receptor gamma (PPARgamma) and adipocyte differentiation. In an effort to identify signaling pathways mediating harmine's effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. Inhibitor of DNA biding 2 (Id2) was identified as a gene rapidly induced by harmine but not by PPARgamma agonists. Id2 is also induced in 3T3-L1 preadipocytes treated with dexamethasone, 3-isobutyl-1-methylxanthine, and insulin, suggesting that Id2 regulation is a common feature of the adipogenic program. Stable overexpression of Id2 in preadipocytes promotes expression of PPARgamma and enhances morphological differentiation and lipid accumulation. Conversely, small interfering RNA-mediated knockdown of Id2 antagonizes adipocyte differentiation. Mice lacking Id2 expression display reduced adiposity, and embryonic fibroblasts derived from these mice exhibit reduced PPARgamma expression and a diminished capacity for adipocyte differentiation. Finally, Id2 expression is elevated in adipose tissues of obese mice and humans. These results outline a role for Id2 in the modulation of PPARgamma expression and adipogenesis and underscore the utility of adipogenic small molecules as tools to dissect adipocyte biology.

PPARgamma is the master regulator of adipogenesis and the molecular target of the thiazolidinedione antidiabetic drugs. By screening for compounds that promote adipogenesis, we identified a small molecule that targets the PPARgamma pathway by a distinct mechanism. This molecule, harmine, is not a ligand for the receptor; rather, it acts as a cell-type-specific regulator of PPARgamma expression. Administration of harmine to diabetic mice mimics the effects of PPARgamma ligands on adipocyte gene expression and insulin sensitivity. Unlike thiazolidinediones, however, harmine does not cause significant weight gain or hepatic lipid accumulation. Molecular studies indicate that harmine controls PPARgamma expression through inhibition of the Wnt signaling pathway. This work validates phenotypic screening of adipocytes as a promising strategy for the identification of bioactive small molecules and suggests that regulators of PPARgamma expression may represent a complementary approach to PPARgamma ligands in the treatment of insulin resistance.

Adiponectin is an adipocyte-derived hormone and known to form several species of multimer, however, the precise components of each multimer have not been fully determined. We purified each multimer adiponectin selectively from human plasma and characterized them by affinity columns using anti-adiponectin, gelatin, or anti-albumin antibody and gel filtration. We found that adiponectin exists as four species of multimers in human plasma. According to their migrating mobility and N-terminal amino acid analysis, we defined them as a trimer, albumin-binding trimer, hexamer, and HMW. Low pH shifted HMW to hexamer, raising the possibility that HMW is a 12 mer or larger multimer. We also showed that HMW had the highest binding activity to the membrane fractions of C2C12 myocytes and activated AMPK most potently. Our results indicate that adiponectin forms diverse multimer species and at least some of the functional properties are dependent on a multimer status.

Obesity is a risk factor for type 2 diabetes, dyslipidemia, and cardiovascular disease. Dissection of the molecular mechanisms underlying obesity and its relationship to insulin resistance and the metabolic syndrome are essential for developing new strategies for prevention and treatment of these disorders. Both excess adipose tissue and lack of adipose tissue cause insulin resistance and dyslipidemia, suggesting that normal fat is required for the maintenance of systemic glucose and lipid homeostasis. Recent advances in obesity research have led to the recognition that adipose tissue is an active endocrine organ that secretes multiple bioactive factors termed adipokines. Secretion of adipokines provides a link between adipose tissue lipid accumulation and the metabolic function of other tissues such as liver and muscle. Dysregulation of adipokines is emerging as an important mechanism by which adipose tissue contributes to systemic insulin resistance and metabolic disease.

Hepatic glucose production is crucial for glucose homeostasis, and its dysregulation contributes to the pathogenesis of diabetes. Here, we show that members of the NR4A family of ligand-independent orphan nuclear receptors are downstream mediators of cAMP action in the hormonal control of gluconeogenesis. Hepatic expression of Nur77, Nurr1 and NOR1 is induced by the cAMP axis in response to glucagon and fasting in vivo and is increased in diabetic mice that exhibit elevated gluconeogenesis. Adenoviral expression of Nur77 induces genes involved in gluconeogenesis, stimulates glucose production both in vitro and in vivo, and raises blood glucose levels. Conversely, expression of an inhibitory mutant Nur77 receptor antagonizes gluconeogenic gene expression and lowers blood glucose levels in db/db mice. These results outline a previously unrecognized role for orphan nuclear receptors in the transcriptional control of glucose homeostasis.

Adiponectin is an adipocyte-derived hormone, which has been shown to play important roles in the regulation of glucose and lipid metabolism. Eight mutations in human adiponectin have been reported, some of which were significantly related to diabetes and hypoadiponectinemia, but the molecular mechanisms of decreased plasma levels and impaired action of adiponectin mutants were not clarified. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer. Herein, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Adiponectin in human or mouse serum and adiponectin expressed in NIH-3T3 or Escherichia coli formed a wide range of multimers from trimers to high molecular weight (HMW) multimers. A disulfide bond through an amino-terminal cysteine was required for the formation of multimers larger than a trimer. An amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90S mutants, which are associated with diabetes and hypoadiponectinemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponectinemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states.

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.