Dynamical network biomarker (DNB) theory has emerged as a powerful framework for detecting early warning signals of pre-disease states. Based on our previous work demonstrating its utility in adipose tissue of metabolic syndrome model mice, we conducted a comprehensive DNB analysis using RNA sequencing data across 13 organs and 14 to 16 time points in high-fat diet-fed mice. Our findings revealed organ-specific variation in the timing of early warning signals, suggesting heterogeneous inter-organ dynamics during the pre-disease state of metabolic syndrome. These results highlight the potential of DNB theory for elucidating systemic early-stage pathophysiology in complex metabolic disorders.

To identify pre-onset pathways of metabolic syndrome (MetS), we retrospectively analyzed Japanese health checkup data from 296 male MetS cases and 296 non-MetS controls matched by initial status (aged 49-64 years). Hierarchical clustering was applied to four MetS components-abdominal obesity (OB), hypertension (HT), dyslipidemia (DLP), and hyperglycemia (HG)-using longitudinal status from the 3 years (years -3 to -1) before onset (year 0). Six clusters were identified, showing shifts from early (year -3 to -1) to late (year -1 to 0) patterns. Cluster 1 (n = 97, 32.8%): early OB and HT; followed by DLP. Cluster 2 (n = 36, 12.2%): early OB and DLP; followed by HT. Cluster 3 (n = 48, 16.2%): early OB; followed by HT and HG. Cluster 4 (n = 44, 14.9%): early HT and DLP; followed by OB. Cluster 5 (n = 46, 15.5%): early HT; followed by OB, DLP, and HG. Cluster 6 (n = 25, 8.4%): early HT, DLP, and HG; followed by OB. Controls showed no clear changes. The waist circumference and triglyceride-glucose related indices were significantly higher at year 0 in the MetS group. These findings provide insight into the heterogeneous pathways leading to MetS. This clustering approach offers a flexible framework for capturing population-specific patterns in MetS development and may allow more targeted early intervention strategies. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported by JST Moonshot R&D Grant Number JPMJMS2021. The funder had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was granted by the Ethics Committee of Toyama University Hospital (approval number R2021070; approval date 19 August 2021). All participants provided written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The datasets generated during and/or analyzed in the current study are available from the corresponding author upon a substantiated request.

ABSTRACT Backgrounds Obesity and diabetes impair the ability of the muscle to regenerate, repair and remodel, resulting in a gradual decrease in muscle mass and function. However, the underlying mechanisms and effective therapeutic strategies remain poorly understood. M2 macrophages within skeletal muscle play an important role in tissue recovery following injury. This study aims to investigate the role of M2 macrophages derived transforming growth factor‐beta 1 (Tgf‐β1) in regulation of skeletal muscle function under diet‐induced obese conditions. Methods An CD206+ M2 macrophage‐specific Tgf‐β1 gene knockout (Tgf‐β1 KO) mouse model was generated by crossing CD206‐CreERT2 mice with Tgf‐β1f/f mice, followed by tamoxifen administration to induce Tgf‐β1 gene deletion. Mice were then placed on a high‐fat diet (HFD) for 12 weeks to develop obesity‐induced skeletal muscle dysfunction. The study used multiple physiological and molecular analyses, including exercise tolerance, hanging time, grip strength, glucose and insulin tolerance tests, western blotting, RT‐qPCR and others. Results The present findings demonstrated improved exercise performance, as evidenced by increased running distance twice (p = 0.0008) in Tgf‐β1 KO mice. Deletion of CD206+ M2 macrophage‐specific Tgf‐β1 stimulates fibro‐adipogenic progenitors (FAPs), inducing Follistatin (Fst) expression by 1.70‐fold (p = 0.04) and follistatin‐like protein 1 (Fstl1) by 2.60‐fold (p = 0.01) in tibialis anterior (TA), thus enhancing myogenesis. The Tgf‐β1 KO mice showed increased muscle fibre type I (Myh7 by 2.40‐fold, p = 0.005), muscle fibre type IIa (Myh2 by 1.50‐fold, p = 0.003), type IIx (Myh1 by 2.35‐fold, p = 0.002) in soleus and type II (Myh4 by 1.76‐fold, p = 0.02) in TA. In Tgf‐β1 KO mice, insulin‐stimulated Akt phosphorylation was significantly increased in adipose tissue by 2.14‐fold (p = 0.0003) and in the liver by 1.62‐fold (p = 0.01). Besides, the Tgf‐β1 KO mice showed increased circulating adiponectin by 1.23 fold (p = 0.003), thereby activating the AMPK/SIRT1/PGC‐1α pathway with the phosphorylation level of AMPKα increased by 1.5‐fold (p = 0.02), and PGC1α protein level increased by 2.2‐fold (p = 0.02) via increased AdipoR1 mRNA expression by 1.8‐fold in TA, p = 0.0001 and by 1.8‐fold in soleus, p = 0.01 in skeletal muscle, leading to improved mitochondrial function in skeletal muscle. Conclusions The CD206+ M2 macrophage‐specific Tgf‐β1 deletion ameliorates obesity‐induced muscle dysfunction, potentially via two distinct mechanisms. Firstly, it enhances myogenesis by promoting FAP‐mediated expression of Fst and Fstl1, thereby augmenting myogenesis‐related gene expression in skeletal muscle. Secondly, it also induces adipocytes to produce and secrete adiponectin into the bloodstream, thereby enhancing mitochondrial function via the AMPK/SIRT1/PGC‐1α pathway.

Follistatin is a potent regulator of various TGF-β superfamily members, including myostatin (MSTN) and activin A. Previous studies have shown that follistatin is crucial in enhancing myogenesis during acute muscle injury. The mechanism by which fibro-adipogenic progenitors (FAPs)-specific follistatin influences muscle homeostasis in obese mice remains unknown. Therefore, we investigated the physiological role of follistatin in PDGFRα-positive FAPs in the regulation of muscle homeostasis and exercise in obese mice. A PDGFRα-specific follistatin knockout (follistatin KO) mouse model was generated using PDGFRα-GFP-CreERT2 (PDGFRα-GCE) and follistatinflox/flox mice. These mice were fed a 60

Decreased nicotinamide adenine dinucleotide (oxidized form) (NAD+) levels are reportedly associated with several aging-related disorders. Thus, supplementation with NAD+ precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), exhibits beneficial effects against these disorders. However, the in vivo metabolic pathways of NMN and NR remain to be elucidated. In this study, we comprehensively analyzed the fate of orally and intravenously administered NMN and NR in mice using NAD+ metabolomics. We found that only a small portion of orally administered NMN and NR was directly absorbed from the small intestine and that most of them underwent gut microbiota-mediated deamidation and conversion to nicotinic acid (NA). Moreover, intravenously administered NMN and NR were rapidly degraded into nicotinamide and secreted to bile followed by deamidation to NA by gut microbiota. Thus, enterohepatic circulated NA is preferentially used in the liver. These findings showed that NMN and NR are indirectly converted to NAD+ via unexpected metabolic pathways.

AIMS/INTRODUCTION:Little is known about the effect of diabetologists on perioperative complication risk. Given that colorectal cancer surgery is known to carry a high complication risk among patients with diabetes, this study aimed to examine the association between the presence of diabetologists at the facility level and the risk of perioperative complications during colorectal cancer surgery. MATERIALS AND METHODS:In this retrospective cohort study, we sourced the number of board-certified diabetologists from the Japan Diabetes Society and combined it with Diagnosis Procedure Combination (DPC) data, encompassing receipt data from acute care facilities across Japan. We used a modified Poisson model to estimate the risk of perioperative complications associated with the presence of diabetologists by adjusting for potential confounding patient and facility factors. RESULTS:Analysis included 887 facilities, of which 299 (34%) had no diabetologists. A total of 24,714 patients with diabetes underwent colorectal cancer surgery between April 2018 and March 2019, with a median age of 73 years (interquartile range: 67-79 years) and 16,274 (66%) men. There were 3,165 (13%) perioperative complications. After adjustment, the risk of perioperative complications was 0.86 times (95% confidence interval: 0.77-0.96) lower in centers with at least one diabetologist than in those without a diabetologist. CONCLUSIONS:Our study suggests that the presence of a diabetologist may be important in reducing the risk of perioperative complications during colorectal cancer surgery in patients with diabetes. Future studies factoring in other facility factors and surgical types may be required to further validate our findings.

OBJECTIVE:A post hoc analysis of a subset of participants with visceral fat area (VFA) measurements in the STEP 6 trial was conducted to examine both the correlation between VFA and clinical parameters and the effect of semaglutide on VFA in key subgroups. METHODS:Participants were Japanese adults aged ≥ 20 years with obesity disease, randomized to once-weekly subcutaneous semaglutide 2.4 mg, semaglutide 1.7 mg, or placebo, plus lifestyle recommendations, for 68 weeks. Correlation between baseline VFA (and change in VFA from baseline to week 68) and clinical parameters (body weight, body mass index [BMI], waist circumference, hepatic parameters, glycated hemoglobin, blood pressure, lipids, high-sensitivity C-reactive protein, and plasminogen activator inhibitor-1 [PAI-1]) was evaluated. Percentage change in VFA between semaglutide and placebo was compared across subgroups. RESULTS:Among 180 participants (semaglutide 2.4 mg, n = 89; semaglutide 1.7 mg, n = 46; placebo, n = 45), mean VFA was 170.0 cm2 across subgroups. A positive correlation (Pearson's correlation coefficient [r] ≥0.3) was observed between baseline VFA and body weight (r = 0.415), BMI (r = 0.374), and both JASSO and WHO criterion waist circumference (r = 0.458 and r = 0.555). Correlation between changes in VFA and body weight, waist circumference, high-density and very low-density lipoprotein cholesterol, triglycerides, PAI-1, aspartate aminotransferase, and alanine transaminase were observed in ≥ 1 treatment arm. Semaglutide 2.4 mg and 1.7 mg reduced VFA compared with placebo in all subgroups. CONCLUSIONS:VFA partially correlated with clinical parameters in Japanese adults with obesity disease. Subcutaneous semaglutide was an efficacious treatment option for the reduction of VFA, regardless of clinical characteristics. TRIAL REGISTRY NAME:CT.gov TRIAL REGISTRATION IDENTIFICATION NUMBER: NCT03811574.

The objective of this cross-sectional study was to clarify the relationship between the type of first-degree family history of diabetes (FHD) and the presence and age at onset of diabetes (AOD) in the Japanese general population. Using anonymized processed data collected from community-based health checkups, we classified 10,691 subjects into 5 groups according to the type of FHD as follows: (1) no FHD; (2) diabetes only in a sibling (sFHD); (3) diabetes only in the mother (mFHD); (4) diabetes only in the father (pFHD); and (5) diabetes in ≥ 2 family members, e.g., one parent plus a sibling or both parents (FHD in ≥ 2 family members). Results of multivariate logistic regression analysis performed using the no FHD group as reference revealed a significant association between a positive FHD and the presence of diabetes (odds ratio: sFHD, 3.67; mFHD, 3.70; pFHD, 2.88; FHD in ≥ 2 family members, 6.35; P < 0.0001 for all). Moreover, the AOD was significantly younger in all the four groups with FHD than in the group without FHD (P < 0.01), being the youngest in the group of FHD in ≥ 2 family members. Our results revealed that the degree of associations between a positive FHD and the presence of diabetes and AOD differ according to the type of FHD. In particular, FHD in ≥ 2 family members appears to be especially strongly associated with a high risk of diabetes and a younger AOD.

Obesity impairs muscle function through effects on lipid metabolism, systemic inflammation, and insulin resistance, leading to muscle loss and reduced regeneration. Tofogliflozin (Tofo), a sodium-glucose cotransporter 2 inhibitor (SGLT2i), exclusively inhibits SGLT2 and is used to treat hyperglycemia in patients with diabetes. The mechanism by which Tofo promotes myogenic potential in an injury model remains elusive. This study investigated Tofo’s role in skeletal muscle repair in diet-induced obesity. C57BL/6 J male mice were fed a high-fat diet (HFD) with or without Tofo for 12 weeks. Cardiotoxin (CTX) was used to induce acute injury. We showed that Tofo administration during HFD alleviates obesity-induced disruption in glucose metabolism and upregulates Pax7 and MyoG expression in skeletal muscle, thereby promoting myogenesis following acute injury. Tofo activates fibro-adipogenic progenitors (FAPs) in skeletal muscle, leading to upregulated follistatin (Fst) expression and boosting the recovery process after acute injury. Mechanistically, Tofo prevented the obesity-induced decline in AMPK phosphorylation, rescued the impairment of lipid metabolism, and improved skeletal muscle function, which led to increased exercise tolerance, activation of FAPs, facilitation of skeletal muscle repair, and reduction of fibrosis.

East Asians are known to develop diabetes mellitus at a lower body weight than Caucasians, potentially because of the different mechanisms underlying disease development. This study aimed to evaluate the variation in weight transition leading to diabetes onset in two subtypes of individuals (obese and non-obese) in a Japanese population. We conducted a retrospective, observational, longitudinal cohort study using health checkup data from 9, 260 participants in Japan. Individuals who developed diabetes within three years of the start of the observation period were excluded. Among the participants, 61.4% were men, and 259 developed diabetes. In the obesity group (body mass index [BMI] ≥25 kg/m2), the average BMI increased prior to the diabetes onset and subsequently decreased. Conversely, in the non-obesity group (BMI <25 kg/m2), the average BMI decreased and then stabilized before the onset of diabetes. Notably, a greater number of participants in the non-obesity group exhibited a BMI change of ≤-0.15 kg/m2 per year compared with those with a BMI change of ≥0.15 kg/m2 per year before diabetes onset (p = 0.003). Our findings indicate that body weight loss precedes the onset of diabetes in the non-obesity group. We recommend that non-obese individuals with elevated blood glucose levels who do not meet the criteria for diabetes should be considered a high-risk group for diabetes development. Therefore, it is imperative to identify these individuals and provide lifestyle guidance that does not focus on weight loss to prevent the onset of diabetes.

Aims:To clarify the pathways from a healthy state to the diabetes onset via pre-disease states, we applied energy landscape analysis (ELA) to Specific Health Checkup data in Japan. Methods:This retrospective and observational cohort study analyzed data from 4,928 males aged 56.0 ± 3.2 years, including 242 individuals with diabetes, over a period of 5.26 ± 3.21 years. A total of 22,326 records were examined using six features: hemoglobin A1c, plasma glucose, high-density lipoprotein-cholesterol, body mass index (BMI), uric acid, and alanine aminotransferase. ELA was also applied to subdata from the 242 individuals with diabetes. Results:ELA revealed three stable states: healthy, intermediate, and unhealthy (pre-diabetes) states. The intermediate state was characterized by obesity. Obese individuals with BMI ≥ 25 kg/m2 (n = 1,460) preferred a pathway via the intermediate state, whereas non-obese individuals with BMI < 25 kg/m2 (n = 3,468) preferred to transit directly to the unhealthy state. There was a significant difference between the preferences of the two groups (p = 0.0085, chi-squared test). Two distinct pathways were also observed for obese and non-obese individuals with diabetes. Conclusions:We demonstrated that ELA could indicate different pathways of diabetes development in obese and non-obese individuals in a data-driven manner. These insights could inform more targeted diabetes prevention measures, such as reducing visceral fat in obese individuals and protecting beta-cells in non-obese individuals.

Regulatory T cells (Tregs) mediate tissue homeostasis and repair. The function of the interleukin-7 receptor α (IL-7Rα) in nonlymphoid tissue Tregs is still unknown, although low expression of IL-7Rα is a widely accepted marker for Tregs. Here, we show that IL-33R (ST2)–expressing Tregs in the visceral adipose tissue (VAT) express the IL-7Rα at high levels. Treg-specific IL-7Rα–deficient mice exhibited reduced adipose ST2+ Tregs and impaired glucose tolerance, whereas IL-7Rα was dispensable for Tregs in lymphoid tissues. Mice deficient in thymic stromal lymphopoietin (TSLP), an additional ligand for IL-7Rα, displayed a modest decrease in adipose ST2+ Tregs and a reduced accumulation of adipose eosinophils, accompanied by slightly impaired glucose tolerance. In the VAT, mesothelial cells expressed IL-7, whereas adipose stem cells and folate receptor β–expressing tissue-resident macrophages expressed TSLP. Thus, this study indicates the significance of IL-7Rα signaling in the maintenance of VAT Tregs and glucose homeostasis, revealing a novel role for IL-7 and TSLP in immunometabolism.

Wound healing is a multifaceted and dynamic sequence of tissue repair and regeneration processes involving interrelated stages: inflammation, regeneration, and remodeling. Throughout these processes, macrophages change their phenotypes and interact with cells and extracellular components to facilitate healing. In particular, macrophages expressing the surface marker CD206 associate with inflammation resolution and tissue repair. However, how CD206 + macrophages contribute to these processes is insufficiently understood. Here, using a mouse model of CD206 + macrophage depletion and single-cell transcriptomics, we report that selective depletion of CD206 + macrophages results in modest but significant delays in wound healing, prolongs inflammation, and significantly reduces the number of Gpnmb hi fibroblasts in injured skin. Single-cell data suggest that CD206 + macrophages communicate with Gpnmb hi fibroblasts via multiple pathways. Notably, topical administration of PDGF-AA to wounds of CD206 + macrophage-depleted mice restores healing processes, identifying PDGF-A signaling from CD206 + macrophages to PDGFRA on fibroblasts as an important mechanism promoting wound healing. Collectively, these data demonstrate that CD206 + macrophages communicate with Gpnmb hi fibroblasts to activate their proliferation and extracellular matrix deposition in wound healing.

Adipose tissue (AT) metabolism involves coordinating various cells and cellular processes to regulate energy storage, release, and overall metabolic homeostasis. Therein, macrophage and its cytokine are important in controlling tissue homeostasis. Among cytokines, the role of transforming growth factor-β1 (Tgf-β1), a cytokine abundantly expressed in CD206+ M2-like macrophage and correlated with the expansion of AT and fibrosis, in AT metabolism, remains unknown. We used CD206CreERT2; Tgf-β1f/f mouse model in which the Tgf-β1 gene was conditionally deleted in CD206+ M2-like macrophages followed by tamoxifen administration, to investigate the role of the Tgf-β1 gene in glucose and insulin metabolism. Our data demonstrated that lack of CD206+ M2-like macrophages derived Tgf-β1 gene improved glucose metabolism and insulin sensitivity by enhancing adipogenesis via hyperplasia. The Tgf-β1 gene, specifically from CD206+ M2-like macrophages, deletion stimulated APs’ proliferation and differentiation, leading to the generation of smaller mature adipocytes, therefore enhancing insulin sensitivity and improving glucose metabolism under normal chow conditions. Our study brings a new perspective that Tgf-β1 gene deletion specific from CD206+ M2-like macrophage promotes adipocyte hyperplasia, improving glucose homeostasis and insulin sensitivity in the lean state.

Prevention of diabetic retinopathy is important to keep vision and quality of life. To examine the effects of an intensive multifactorial intervention and hypoglycemia on retinopathy in people with type 2 diabetes. J-DOIT3 (Japan Diabetes Optimal Integrated Treatment Study for 3 Major Risk Factors of Cardiovascular Diseases) is a multicenter, open-label, parallel-group randomized clinical trial that examined the efficacy of an intensified multifactorial intervention on cardiovascular outcomes and mortality in people with type 2 diabetes aged 45 to 69 years with hypertension and/or dyslipidemia. The study was conducted at 81 sites in Japan from June 2006 to March 2009, and data analysis was performed from September 2018 to August 2025. Participants were randomly assigned to intensive therapy for glucose, blood pressure, and lipids or conventional therapy and were followed up for a median duration of 8.5 years. This study is a secondary analysis of retinopathy events of the secondary outcomes, composed of onset of retinopathy, progression of retinopathy, and loss of vision likely due to retinopathy. Among 2540 total participants (5080 eyes) randomly assigned to intensive therapy or conventional therapy, mean (SD) age was 59.0 (6.3) years, and 965 participants (38.0%) were female. Intensive therapy was associated with a risk reduction in onset of retinopathy (hazard ratio [HR], 0.83; 95% CI, 0.70-0.98; P = .03) but not with progression of retinopathy (HR, 1.02; 95% CI, 0.70-1.49; P = .93). Hemoglobin A 1 c (HbA 1 c ) at 1 year after randomization was associated with onset (HR, 1.31; 95% CI, 1.13-1.51; P < .001), even after adjustment for baseline risk factors (ie, lower body mass index, longer duration of diabetes, higher fasting plasma glucose, higher blood pressure, and comorbid nephropathy), with no clear HbA 1 c threshold observed. Moreover, compared with those without a hypoglycemic episode during the intervention, the risk of onset was higher in those with 0.5 hypoglycemic episodes per year or fewer (HR, 1.25; 95% CI, 1.02-1.53) and even higher in those with more than 1 hypoglycemic episode per year (HR, 1.85; 95% CI, 1.39-2.47). This secondary analysis of the J-DOIT3 randomized clinical trial shows that in a randomized clinical trial setting, higher HbA 1 c and nonsevere hypoglycemia are associated with higher risk of onset of retinopathy in people with type 2 diabetes, even when good glycemic management, with a very low incidence of severe hypoglycemia, is achieved, suggesting the importance of strict glycemic management without any hypoglycemia. ClinicalTrials.gov Identifier: NCT00300976

Introduction/Objective: Environmental factors such as lifestyle, diet, and associated changes in the gut microbiota are the most important determinants of obesity. Recently, we investigated the relationship among metabolic parameters, lifestyle habits, and fecal 16S rRNA seq data of Japanese healthy adults. These parameters are intricately and closely related, and we identified two microbes that can prevent metabolic syndrome (ADA 83rd scientific sessions). In this study, we further expanded this cohort study to examine lifestyle habits, nutrient intake, and microbial species associated with visceral obesity (VO). Methods: A total of 435 participants who underwent annual health check in 2019 were enrolled, provided data on metabolic parameters, and completed a lifestyle questionnaire including a dietary record of the previous three days. Fecal 16S rRNA seq data were obtained from 38 participants in 2022 and analyzed along with the changes in body weight from 2019. Results: The average age, body mass index, and HbA1c were 40.5±9.7, 24.0±4.1 kg/m2 and 5.5±0.37%, respectively. VO was clearly associated with metabolic parameters. There was a significant positive correlation between VO and lifestyle habits such as smoking, eating speed, irregular meal time, and frequency of fried food intake. Regarding nutrient composition, intake of resistant starch, β-carotene, maltose, and sorbitol were negatively correlated with waist circumference, while intake of caffeine and tannins were positively correlated. Furthermore, significant differences depending on weight changes were observed in the microbial species such as increased Bifidobacterium adolescentis and Prevotella stercorea in subjects who lost weight compared to those who gained weight. Conclusion: Lifestyle, diet intake, and characteristic changes in the microbiota are associated with VO in healthy adults prior to onset of metabolic syndrome. Disclosure Y. Watanabe: None. S. Fujisaka: None. H. Honoki: None. T. Kado: None. A. Nishimura: None. M. Bilal: None. M. Aslam: None. Y. Igarashi: None. Y. Morinaga: None. K. Tobe: None. Funding Japan Society for the Promotion of Science (JSPS) KAKENHI (20K08882, 21K20896 and 22K16424); AMED PRIME (JP18gm6010023h0001); Lotte Foundation; Yakult Bio-Science Foundation; First Bank of Toyama Scholarship Foundation; Japan Diabetes Foundation; Japan Society for the Study of Obesity (JASSO); Yamaguchi Endocrine Resarch Foundation and Hokugin Young Researcher Grant

Introduction & Objective: Autoreactive T cell responses in type 1 diabetes (T1D) may vary depending on race. In Japan, T1D is classified into three subtypes based on disease progression: acute onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). The aim of this study was to compare the features of islet antigen-specific CD4+ T cell (Th) responses among these subtypes and determine the Th epitope in Japanese AT1D patients. Methods: Blood mononuclear cells from 20 AT1D, 17 SP1D, and 18 FT1D patients, and 17 nondiabetics (NDs) were stimulated with GAD65, preproinsulin (PPI), IGRP, and ZnT8 peptide clusters (4-6 peptides/cluster) in the presence of IL-2. Subsequently, intracytoplasmic cytokine staining (ICS) was performed to analyze cytokine expression on specific T cells (cluster analysis). After identifying the feature of Th cell responses in each T1D subtype, another stimulation and ICS were performed using single peptides to identify Th cell epitopes (epitope analysis). Results: In the cluster analysis, the frequencies of GAD65- and PPI-specific Th1 cells were significantly higher in AT1D patients (P = 0.007 and P = 0.004, respectively) than in NDs, whereas GAD65- and IGRP-specific Th2 cells were more prevalent in SP1D patients (P = 0.010 and P = 0.030, respectively) than in NDs. Notably, FT1D patients displayed significantly less Tr1 cells specific for all four antigens. Epitope analysis, focusing on GAD65- and PPI-specific Th1 responses in AT1D patients, revealed that 3, 16, 3, and 3 out of 20 patients displayed positive Th1 responses (stimulation index > 3.0) under GAD65115-127, GAD65247-266, PPIC19-A3, and PPIC22-A5 stimulation, respectively. In addition, the frequencies of GAD65247-266-specific Th1 cells were significantly higher than those specific for other epitopes (P < 0.001). Conclusion: The phenotypes of islet antigen-specific Th cells were distinct among the three T1D subtypes. Furthermore, GAD65247-266 was identified as a major Th1 epitope in Japanese AT1D patients. Disclosure D. Chujo: Research Support; Kyowa Kirin Co., Ltd. K. Ajima: None. M. Matsushita: None. C. Tsutsumi: None. F. Haseda: None. A. Imagawa: None. T. Hanafusa: None. H. Kajio: None. M. Shimoda: None. K. Tobe: None. Funding Japan Society for the Promotion of Science (21K08525)

Introduction & Objective: To appreciate the pathophysiological changes in the transcriptome and cell type landscape during obesity, single-nucleus (sn)RNA-seq overcomes the difficulty of maintaining intact transcripts. Sedimentation of dissociated live cells is used to concentrate adipocytes (Ads) and stromal cells. The current study compared the single cell level transcriptome in libraries with and without the fractionation. Methods: C57BL/6J mice were fed an HFD for 12 weeks starting at 8 weeks of age. Dissected epididymal fat from the same donor was divided into three conditions: 1) nuclei from unfractionated frozen fat (UF), 2) nuclei from the Ad fraction (AF) after enzymatic digestion and centrifugation, and 3) cells from the stromal fraction corresponding to the AF. As a dietary control, nuclei were prepared from the UF of an age-matched normal chow-fed mouse. Single cell/nucleus transcriptome libraries were sequenced using short-read NGS. Results: A total of 32,949 cells were recovered by snRNA-seq. Fractionation lost recovery of certain cell types, including epithelial, mesothelial, and B cells. Fractionation barely enriched Ads in the AF at 12.4%, comparable to the Ads (11.2%) in the UF. Macrophages (Mφs), rather than Ads, accounted for 54.6% of the AF. The Mφs were clustered into six subtypes by Seurat independent of dietary conditions or fractionation. However, certain cytokines were more highly expressed in Mφs from fractions than those from UF. In support of this, functional annotation-driven unsupervised clustering, ASURAT, mapped three Mφ subtypes in the libraries from fractions that differed from those from the UF, where there were four other subtypes. Conclusion: The current study suggests that the conventional fractionation could bias the transcriptome and a nuclear library from unfractionated frozen fat would represent a naïve comprehensive transcriptome that better reflects the pathophysiology of obesity, which should be important for understanding cell function. Disclosure Y. Onogi: None. T. Wada: None. S. Fujisaka: None. T. Sasaoka: None. K. Iida: None. Y. Okada: None. M. Kadowaki: None. S. Saito: None. K. Tobe: None. Funding JST Moonshot R&D (JPMJMS2021)

Diabetic striatopathy is a rare condition with a prevalence of less than one in 100,000. Herein, we report a case of diabetic striatopathy exacerbated by hyperglycemia and hypoglycemia, with repeated follow-up with multiple imaging studies. This case suggested that putamen neuronal loss and dysfunction, gliosis, and ischemia are associated with diabetic striatopathy pathophysiology. In addition, striatal hyperintensity on T1-weighted MRI images was more pronounced after symptom remission when evaluated several times over a short period. Therefore, clinicians should be aware that even if MRI findings are normal in the very early stages of the onset of diabetic striatopathy, repeating MRIs at intervals may reveal typical findings.

Disruption of the gut microbiota causes metabolic dysfunction, and intervention in the gut microbiota has the potential to improve host glucose metabolism. Akkermanisa muciniphila is an intestinal bacterium involved in anti-obesity and insulin resistance. Developing interventions to increase A. muciniphla would be useful for new treatment strategies. In this study, we screened herbal drug extracts that promoted the growth of A. muciniphila. Among the 123 herbal drugs, five herbal drug extracts significantly increased A. muciniphila DNA levels compared with that in controls. In particular, Dioscoreae rhizoma extract increased the growth of A. muciniphila in the intestines of mice fed a high-fat diet and improved obesity. It significantly reduced body weight gain, improved glucose tolerance even when the administration was initiated after the induction of dietary obesity. These results suggest that herbal drug extracts, such as Dioscoreae rhizome, that increase A. muciniphila could be a new therapeutic strategy for metabolic syndrome.