Diabetes Accelerates Steatohepatitis in Mice: Liver Pathology and Single-Cell Gene Expression Signatures.

Glucose lowering independently reduces liver fibrosis in human nonalcoholic fatty liver disease. This study investigated the impact of diabetes on steatohepatitis and established a novel mouse model for diabetic steatohepatitis. Male C57BL/6J mice were fed a 60% high-fat diet (HFD) and injected with carbon tetrachloride (CCl4) and streptozotocin (STZ) to induce diabetes. The HFD+CCl4+STZ group showed more severe liver steatosis, hepatocyte ballooning, and regenerative nodules compared with other groups. Diabetes up-regulated inflammatory cytokine-associated genes and increased the M1/M2 macrophage ratios in the liver. Single-cell RNA sequencing analysis of nonparenchymal cells in the liver showed that diabetes reduced Kupffer cells and increased bone marrow-derived recruited inflammatory macrophages, such as Ly6Chi-RM. Diabetes globally reduced liver sinusoidal endothelial cells (LSECs). Furthermore, genes related to the receptor for advanced glycation end products (RAGE)/Toll-like receptor 4 (TLR4) were up-regulated in Ly6Chi-RM and LSECs in mice with diabetes, suggesting a possible role of RAGE/TLR4 signaling in the interaction between inflammatory macrophages and LSECs. This study established a novel diabetic steatohepatitis model using a combination of HFD, CCl4, and STZ. Diabetes exacerbated steatosis, hepatocyte ballooning, fibrosis, regenerative nodule formation, and the macrophage M1/M2 ratios triggered by HFD and CCl4. Single-cell RNA sequencing analysis indicated that diabetes activated inflammatory macrophages and impairs LSECs through the RAGE/TLR4 signaling pathway. These findings open avenues for discovering novel therapeutic targets for diabetic steatohepatitis.

Selenoprotein P deficiency protects against immobilization-induced muscle atrophy by suppressing atrophy-related E3 ubiquitin ligases.

The quality of skeletal muscle is maintained by a balance between protein biosynthesis and degradation. Disruption in this balance results in sarcopenia. However, its underlying mechanisms remain underinvestigated. Selenoprotein P (SeP; encoded by Selenop in mice) is a hepatokine that is upregulated in type 2 diabetes and aging and causes signal resistances via reductive stress. We created immobilized muscle atrophy model in Selenop knockout (KO) mice. Immobilization (IMM) significantly reduced cross-sectional areas and the size of skeletal muscle fibers, which were ameliorated in KO mice. IMM upregulated the genes encoding E3 ubiquitin ligases and their upstream FoxO1, FoxO3, and KLF15 transcription factors in the skeletal muscle, which were suppressed in KO mice. These findings suggest a possible involvement of SeP-mediated reductive stress in physical inactivity-mediated sarcopenia, which may be a therapeutic target against sarcopenia.NEW & NOTEWORTHY Selenoprotein P (SeP) is a hepatokine that is upregulated in type 2 diabetes and aging and causes signal resistances via reductive stress. Immobilization (IMM) significantly reduced skeletal muscle mass in mice, which was prevented in SeP knockout (KO) mice. IMM-induced Foxos/KLF15-atrogene upregulation was suppressed in the skeletal muscle of KO mice. These findings suggest that SeP-mediated reductive stress is involved in and may be a therapeutic target for physical inactivity-mediated muscle atrophy.

Relationship between the changes in hepatokine levels and metabolic effects after laparoscopic sleeve gastrectomy in severely obese patients.

PURPOSE: To clarify the relationships between the changes in hepatokines and weight loss, and between these changes and the metabolic effects, and the roles played by these changes, after laparoscopic sleeve gastrectomy (LSG). METHODS: We recruited 25 Japanese patients with severe obesity, who underwent LSG. We measured two hepatokines: selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2), at the baseline, and then 6 months and 1 year after LSG. Finally, we compared the changes in the hepatokines with the parameters of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). RESULTS: Changes in LECT2 were correlated with the percentage of total weight loss (ρ = - 0.499, P = 0.024) and the decrease in total fat area (ρ = 0.559, P = 0.003). The changes in SeP were correlated with those in hemoglobin A1c (ρ = 0.526, P = 0.043) and the insulinogenic index (ρ = 0.638, P = 0.010) in T2D patients. In patients with NASH, the LECT2 levels were correlated with liver steatosis (ρ = 0.601). CONCLUSIONS: SeP levels decrease in association with HbA1c reduction, whereas LECT2 levels are associated with reductions in fat mass and NASH scores after LSG. Hepatokines may be involved in the pathology of obesity and its complications.

The prevalence of locomotive syndrome and its associated factors in patients with Type 2 diabetes mellitus.

OBJECTIVES: We investigated the prevalence of locomotive syndrome (LS) and related musculoskeletal diseases [osteoarthritis (OA), lumbar spondylosis, and spinal alignment] in Type 2 diabetes mellitus (DM) patients. METHODS: Clinical data were collected from 101 patients (55 males; 46 females) admitted to our hospital for diabetes education from October 2018 to April 2021. Patients underwent full-spine and whole-legs standing radiography and physical measurements (10-m walking and grip strength tests and three LS risk tests). RESULTS: The estimated prevalence of LS was 86.1% (Stage 1: 44.5%, Stage 2: 41.6%), lumbar spondylosis was 11.9%, and hip, knee, and ankle OA were 16.9%, 51.5%, and 12.9%, respectively. Multiple logistic regression analysis identified grip strength [odds ratio (OR) = 0.89, confidence interval (CI) = 0.83-0.94], diabetic retinopathy (OR = 5.85, CI = 1.64-20.78), knee OA (OR = 3.34, CI = 1.11-10.02), and a sagittal vertical axis >40 mm (OR = 3.42, CI = 1.13-10.39) as significantly associated risk factors for worsening LS in Type 2 DM patients. CONCLUSIONS: This study clarified the epidemiological indicators of LS and associated factors in DM patients. Exercise therapy and DM management are effective strategies to reduce the occurrence and progression of LS.

A retrospective cohort study on the association between poor sleep quality in junior high school students and high hemoglobin A1c level in early adults with higher body mass index values.

BACKGROUND: Few epidemiological studies have been performed to clarify the association between glucose metabolism disorders in early adults (20 years old) and physiological and environmental factors, including body mass index (BMI) in junior high school days. Therefore, we examined the association between hemoglobin A1c (HbA1c) level and body size (BMI) in early adulthood and lifestyles, including sleep habits and BMI in junior high school days in Shika town, a small town in Japan, by conducting a retrospective cohort study. METHODS: We examined the HbA1c levels and body size (BMI) of 99 early adults who turned 20 years old between 2016 and 2020 and were residing in Shika town, Ishikawa Prefecture. We obtained the information on lifestyles and living environment factors, including BMI, from a questionnaire survey conducted among the subjects during their junior high school days (13-15 years old) from 2009 to 2013. RESULTS: No correlations were observed between the HbA1c levels and the BMI values of the early adults. A two-way analysis of covariance (with the HbA1c levels and BMI values of the early adults as main factors) of the body size and lifestyle habits of the junior high school students revealed that "sleep quality in junior high school" was significantly poorer in the high HbA1c group than in the low HbA1c group in the early adults with high BMI values only. This result was also supported by the logistic regression analysis result. CONCLUSIONS: The present results indicate that poor sleep quality in junior high school was associated with the high HbA1c levels of the early adults with higher BMI values, which suggests that good sleep quality in junior high school prevents the development of hyperglycemia. However, the present study did not find any relationship between early-adult BMI and HbA1c level.

Thyrotropin-secreting pituitary adenomas induce left atrial enlargement with subclinical atrial fibrillation: an echocardiographic study.

PURPOSE: Thyrotropin-secreting pituitary adenoma (TSHoma) is rare but occasionally causes cardiovascular complications such as atrial fibrillation (AF) due to hyperthyroidism. Graves' disease (GD) is a common hyperthyroid condition often associated with subclinical AF. Some reports have shown echocardiographic changes in patients with GD. We aimed to evaluate the preoperative cardiac function in patients with TSHomas and compared the results among patients with TSHomas and GD and control subjects. METHODS: Patients with TSHomas (n = 6) and GD (n = 20) were compared with control subjects with normal cardiac function (n = 20) based on echocardiographic findings. The average age, sex, and proportions of patients with a history of diabetes mellitus and hypertension were equal in each group, and the AF prevalence was matched in patients with TSHomas and GD. The values of left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), left ventricular ejection fraction (LVEF), and left atrial diameter (LAD) were used to assess cardiac function. RESULTS: In echocardiography, LAD showed a significant difference between patients with TSHomas and control subjects (p = 0.026). The mean LAD values were 36.9 ± 7.1, 38.2 ± 8.9, and 28.7 ± 3.9 mm for patients with TSHomas and GD and control subjects, respectively. There were no significant differences in other echocardiographic parameters among the groups. Before treatment, serum thyroid hormone levels (free triiodothyronine and thyroxin) were not significantly different among patients with TSHomas and GD. CONCLUSION: We found that patients with TSHomas or GD had enlarged LADs. This finding suggests that AF may be more hidden in patients with TSHomas than previously reported.

Distinct effects of carbohydrate ingestion timing on glucose fluctuation and energy metabolism in patients with type 2 diabetes: a randomized controlled study.

This randomized, open-label, and parallel-group study aimed to investigate the effects of altering the timing of carbohydrate intake at breakfast or dinner on blood glucose fluctuations and energy metabolism. A total of 43 participants with type 2 diabetes were assigned to either the breakfast or dinner group. Participants were provided an isocaloric carbohydrate-restricted diet constituting 10% carbohydrate only at breakfast or dinner for 2 days during the study. Glucose fluctuations were compared using a continuous glucose monitoring system (iPro2) and body composition, energy expenditure, blood biochemistry, and endocrine function changes. The carbohydrate restriction either at breakfast or dinner significantly decreased postprandial glucose excursion and mean 24-h blood glucose levels. The incremental blood glucose area under the curve (AUC) for 2 h (iAUC0-2h) at lunch significantly increased in the breakfast group, whereas no significant differences were observed in the iAUC0-2h between breakfast and lunch in the dinner group. Carbohydrate restriction reduced diet-induced thermogenesis at breakfast (intragroup comparison; 223 ± 117 to 109 ± 104 kcal, p = 0.002) but did not affect diet-induced thermogenesis at dinner. However, fasting plasma free fatty acids were comparable in both groups, prelunch free fatty acids increased significantly only in the breakfast group (0.20 ± 0.09 to 0.63 ± 0.19 mEq/L, p < 0.001). Carbohydrate restriction in the diet once daily decreases mean 24-h blood glucose levels and exerts unique metabolic effects depending on the timing.

A mosaic mutation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) in X-linked hypophosphatemic rickets with mild bone phenotypes.

X-linked hypophosphatemic rickets (XLH) is primarily characterized by renal phosphate wasting with hypophosphatemia, short stature, and bone deformity of the leg. Here we present a male case of XLH with relatively mild bone deformity caused by a mosaic mutation of the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). Polymerase chain reaction (PCR) direct sequencing revealed a novel in-frame deletion, NM-000444.6:c.671-685del p.Gln224-Ser228del, at exon 6 in PHEX as a mosaic pattern. This mutation was not found in any database and may result in a significant change in higher-order protein structure and function. TA cloning of the PCR product and clone sequencing estimated the mutation allele frequency at 21%. Literature review of the previously reported three cases with novel mosaic mutations in PHEX, together with the present case, suggests that the rates of the mutation allele correlate with phenotype severity to some extent. We initially treated him with nutritional vitamin D supplements and phosphate salts. However, to avoid the development of secondary/tertiary hyperparathyroidism, we had switched nutritional to active vitamin D supplementation with reduced phosphorus salts. The present report contributes to understanding the relationship between the mosaic rate, in addition to the mutation locus, of the PHEX gene, and clinical features of XLH.

Circulating Concentrations of Insulin Resistance-Associated Hepatokines, Selenoprotein P and Leukocyte Cell-Derived Chemotaxin 2, during an Oral Glucose Tolerance Test in Humans.

A hepatokine is a collective term for liver-derived secretory factors whose previously-unrecognized functions have been recently elucidated. We have rediscovered selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2) as hepatokines that are involved in the development of insulin resistance and hyperglycemia. The aim of this study was to determine whether and, if so, how oral glucose loading alters the two hepatokines in humans. We measured concentrations of serum SeP and plasma LECT2 during 75 g oral glucose tolerance test (OGTT) (n = 20) in people with various degrees of glucose tolerance. In OGTT, concentrations of both serum SeP and plasma LECT2 decreased at 120 min compared with the baseline values, irrespective of the severity of glucose intolerance. Decrement of serum SeP during OGTT showed no correlations to the clinical parameters associated with insulin resistance or insulin secretion. In multiple stepwise regression analyses, plasma cortisol was selected as the variable to explain the changes in plasma concentrations of LECT2. The current data reveal the acute inhibitory actions of oral intake of glucose on circulating SeP and LECT2 in humans, irrespective of the severity of glucose intolerance. This study suggests that circulating SeP is regulated by the unknown clinical factors other than insulin and glucose during OGTT.

Eicosapentaenoic acid down-regulates expression of the selenoprotein P gene by inhibiting SREBP-1c protein independently of the AMP-activated protein kinase pathway in H4IIEC3 hepatocytes.

Selenoprotein P (encoded by SELENOP in humans, Selenop in rat), a liver-derived secretory protein, induces resistance to insulin and vascular endothelial growth factor (VEGF) in type 2 diabetes. Suppression of selenoprotein P may provide a novel therapeutic approach to treating type 2 diabetes; however, few drugs inhibiting SELENOP expression in hepatocytes have been identified. The present findings demonstrate that eicosapentaenoic acid (EPA) suppresses SELENOP expression by inactivating sterol regulatory element-binding protein-1c (SREBP-1c, encoded by Srebf1 in rat) in H4IIEC3 hepatocytes. Treatment with EPA caused concentration- and time-dependent reduction in SELENOP promoter activity. EPA activated AMP-activated protein kinase (AMPK); however, the inhibitory effect of EPA on SELENOP promoter activity was not canceled with an AMPK inhibitor compound C and dominant-negative AMPK transfection. Deletion mutant promoter assays and computational analysis of transcription factor-binding sites conserved among the species resulted in identification of a sterol regulatory element (SRE)-like site in the SELENOP promoter. A chromatin immunoprecipitation (ChIP) assay revealed that EPA decreases binding of SREBP-1c to the SELENOP promoter. Knockdown of Srebf1 resulted in a significant down-regulation of Selenop expression. Conversely, SREBP-1c overexpression inhibited the suppressive effect of EPA. These data provide a novel mechanism of action for EPA involving improvement of systemic insulin sensitivity through the regulation of selenoprotein P production independently of the AMPK pathway and suggest an additional approach to developing anti-diabetic drugs.

Vanishing of ruptured adrenal mass with takotsubo cardiomyopathy.

A 45-year-old male suddenly experienced left-flank abdominal pain. Echocardiography revealed akinesis of the 'takotsubo cardiomyopathy' type. He experienced a sudden haemodynamic collapse (blood pressure, 324/154 mmHg; pulse rate, 180 beats/min) during emergency cardiac catheterisation. An abdominal computed tomography (CT) revealed expansion of a soft tissue mass 64 × 33 mm in dimension in the left adrenal region, with accumulation of fluid surrounding the left pararenal space. Three days after the attack, his urinary catecholamine concentrations were slightly elevated. We suspected the patient as having a pheochromocytoma followed by acute haemorrhagic rupture, based on signatures of adrenal mass, 'takotsubo cardiomyopathy', and the hypertensive crisis. Over the next few weeks, he recovered well as an outpatient, and his blood pressure remained around 110/60 mmHg without medication. Three weeks after the attack, an abdominal CT showed shrinkage of the ruptured adrenal mass (to a diameter of 30 mm) and absorption of the retroperitoneal hematoma. On day 190 after the attack, abdominal CT did not detect any left adrenal mass. This is the first report of the case showing a complete vanishing of ruptured adrenal mass with takotsubo cardiomyopathy. Although surgical approaches for ruptured adrenal mass involve either emergency or elective surgery, the patients did not need even the elective surgery. Accumulation of the similar cases may unravel clinical factors predicting self-limiting of the ruptured adrenal mass to avoid unnecessary risky surgery.

Rapid response of the steatosis-sensing hepatokine LECT2 during diet-induced weight cycling in mice.

Dieting often leads to body weight cycling involving repeated weight loss and regain. However, little information is available regarding rapid-response serum markers of overnutrition that predict body weight alterations during weight cycling. Here, we report the rapid response of serum leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine that induces insulin resistance in skeletal muscle, during diet-induced weight cycling in mice. A switch from a high-fat diet (HFD) to a regular diet (RD) in obese mice gradually decreased body weight but rapidly decreased serum LECT2 levels within 10 days. In contrast, a switch from a RD to a HFD rapidly elevated serum LECT2 levels. Serum LECT2 levels showed a positive correlation with liver triglyceride contents but not with adipose tissue weight. This study demonstrates the rapid response of LECT2 preceding body weight alterations during weight cycling in mice and suggests that measurement of serum LECT2 may be clinically useful in the management of obesity.

Development of a Sol Particle Homogeneous Immunoassay for Measuring Full-Length Selenoprotein P in Human Serum.

BACKGROUND: Selenoprotein P (SeP), a selenium-rich extracellular glycoprotein, is the primary selenoprotein in the plasma. SeP plays an important role in the maintenance of selenium levels in the peripheral tissues. We developed a new sol particle homogeneous immunoassay (SPIA) for measuring full-length SeP (FL-SeP) levels in the human serum. METHODS: We used colloidal gold particles coated with two types of anti-SeP monoclonal antibodies, one recognizing the N-terminal side domain of SeP and the other recognizing the C-terminal side domain. RESULTS: The assay range was 0.2-9 mg/l, and the linearity was excellent. The within-day and between-day coefficients of variation ranged from 0.73% to 2.24% and 0.45% to 1.11%, respectively. Serum samples (n = 200) were examined using the newly developed assay system (employing a Model 7070 Hitachi automatic clinical analyzer) and the conventional enzyme-linked immunosorbent assay. These two methods were compared using the Passing-Bablok regression analysis; the resulting regression equation and correlation coefficient were y = 0.940x + 0.165 and r = 0.954, respectively. CONCLUSIONS: Our new SPIA assay is a fully automated homogeneous immunoassay that can be used in conjunction with various commercial analyzers. The assay was sensitive, precise, and suitable for clinical measurement of the FL-SeP in the human serum.

Characteristics of hepatic fatty acid compositions in patients with nonalcoholic steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and lipid metabolism. Recent studies have suggested that the quality of fat accumulated in the liver is associated with the development of nonalcoholic steatohepatitis (NASH). In this study, we investigated the fatty acid composition in liver tissue and its association with the pathology in NAFLD patients. METHODS: One hundred and three patients diagnosed with NAFLD [simple steatosis (SS): 63, NASH: 40] were examined and their hepatic fatty acids were measured using gas chromatography. In addition, relationships between the composition and composition ratios of various fatty acids and patient backgrounds, laboratory test values, histology of the liver, and expression of fat metabolism-related enzymes were investigated. RESULTS: The C16:1n7 content, the C16:1n7/C16:0 and C18:1n9/C18:0 ratios were increased and the C18:0/C16:0 ratio was decreased in the NASH group. The C18:0/C16:0 and C18:1n9/C18:0 ratios were associated with the steatosis score in liver tissue, and the C16:1n7/C16:0 ratio was associated with the lobular inflammation score. The expressions levels of genes: SCD1, ELOVL6, SREBP1c, FAS and PPARγ were enhanced in the NASH group. In multivariate analysis, the C18:0/C16:0 ratio was the most important factor that was correlated with the steatosis score. In contrast, the C16:1n7/C16:0 ratio was correlated with lobular inflammation. CONCLUSION: The fatty acid composition in liver tissue and expression of genes related to fatty acid metabolism were different between the SS and NASH groups, suggesting that the acceleration of fatty acid metabolism is deeply involved in pathogenesis of NASH.

The effects of ezetimibe on non-alcoholic fatty liver disease and glucose metabolism: a randomised controlled trial.

AIMS/HYPOTHESIS: The cholesterol absorption inhibitor ezetimibe has been shown to ameliorate non-alcoholic fatty liver disease (NAFLD) pathology in a single-armed clinical study and in experimental animal models. In this study, we investigated the efficacy of ezetimibe on NAFLD pathology in an open-label randomised controlled clinical trial. METHODS: We had planned to enrol 80 patients in the trial, as we had estimated that, with this sample size, the study would have 90% power. The study intervention and enrolment were discontinued because of the higher proportion of adverse events (significant elevation in HbA(1c)) in the ezetimibe group than in the control group. Thirty-two patients with NAFLD were enrolled and randomised (allocation by computer program). Ezetimibe (10 mg/day) was given to 17 patients with NAFLD for 6 months. The primary endpoint was change in serum aminotransferase level. Secondary outcomes were change in liver histology (12 control and 16 ezetimibe patients), insulin sensitivity including a hyperinsulinaemic-euglycaemic clamp study (ten control and 13 ezetimibe patients) and hepatic fatty acid composition (six control and nine ezetimibe patients). Hepatic gene expression profiling was completed in 15 patients using an Affymetrix gene chip. Patients and the physician in charge knew to which group the patient had been allocated, but people carrying out measurements or examinations were blinded to group. RESULTS: Serum total cholesterol was significantly decreased in the ezetimibe group. The fibrosis stage and ballooning score were also significantly improved with ezetimibe treatment. However, ezetimibe treatment significantly increased HbA1c and was associated with a significant increase in hepatic long-chain fatty acids. Hepatic gene expression analysis showed coordinate downregulation of genes involved in skeletal muscle development and cell adhesion molecules in the ezetimibe treatment group, suggesting a suppression of stellate cell development into myofibroblasts. Genes involved in the L-carnitine pathway were coordinately downregulated by ezetimibe treatment and those in the steroid metabolism pathway upregulated, suggestive of impaired oxidation of long-chain fatty acids. CONCLUSIONS/INTERPRETATION: Ezetimibe improved hepatic fibrosis but increased hepatic long-chain fatty acids and HbA1c in patients with NAFLD. These findings shed light on previously unrecognised actions of ezetimibe that should be examined further in future studies. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN) Clinical Trials Registry UMIN000005250. FUNDING: The study was funded by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and research grants from MSD.

Selenoprotein P as a diabetes-associated hepatokine that impairs angiogenesis by inducing VEGF resistance in vascular endothelial cells.

AIMS/HYPOTHESIS: Impaired angiogenesis induced by vascular endothelial growth factor (VEGF) resistance is a hallmark of vascular complications in type 2 diabetes; however, its molecular mechanism is not fully understood. We have previously identified selenoprotein P (SeP, encoded by the SEPP1 gene in humans) as a liver-derived secretory protein that induces insulin resistance. Levels of serum SeP and hepatic expression of SEPP1 are elevated in type 2 diabetes. Here, we investigated the effects of SeP on VEGF signalling and angiogenesis. METHODS: We assessed the action of glucose on Sepp1 expression in cultured hepatocytes. We examined the actions of SeP on VEGF signalling and VEGF-induced angiogenesis in HUVECs. We assessed wound healing in mice with hepatic SeP overexpression or SeP deletion. The blood flow recovery after ischaemia was also examined by using hindlimb ischaemia model with Sepp1-heterozygous-knockout mice. RESULTS: Treatment with glucose increased gene expression and transcriptional activity for Sepp1 in H4IIEC hepatocytes. Physiological concentrations of SeP inhibited VEGF-stimulated cell proliferation, tubule formation and migration in HUVECs. SeP suppressed VEGF-induced reactive oxygen species (ROS) generation and phosphorylation of VEGF receptor 2 (VEGFR2) and extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs. Wound closure was impaired in the mice overexpressing Sepp1, whereas it was improved in SeP (-/-)mice. SeP (+/-)mice showed an increase in blood flow recovery and vascular endothelial cells after hindlimb ischaemia. CONCLUSIONS/INTERPRETATION: The hepatokine SeP may be a novel therapeutic target for impaired angiogenesis in type 2 diabetes.

Study Protocol for the Pleiotropic Effects of Sodium-Glucose Cotransporter 2 Inhibitor on Organ-Specific Sympathetic Nerve Activity and Insulin Sensitivity in Participants with Type 2 Diabetes.

INTRODUCTION: Hyperinsulinemia and hyperglycemia are associated with exaggerated systemic sympathetic nerve activity (SNA) in patients with type 2 diabetes. Sodium-glucose cotransporter 2 (SGLT2) inhibitors lower insulin levels, whereas sulfonylureas increase insulin levels. We will test whether these two classes of antidiabetic agents have different effects on SNA. METHODS: The present study is an ongoing, 24-week, one-center (only Kanazawa University Hospital), open-label, randomized, parallel trial (jRCTs 041200035). Participants with type 2 diabetes with multiple atherosclerosis risk factors are randomly assigned in a 1:1 manner to receive 2.5 mg luseogliflozin or 0.5 mg glimepiride once daily. The sample size was calculated to be 14 in each group, with a significance level of 0.05 and a power of 0.80. The design required 40 evaluable study participants. Our primary endpoint will be the change in muscle SNA (MSNA). The secondary endpoints included organ-specific insulin sensitivity measured by a hyperinsulinemic-euglycemic clamp study using an artificial pancreas combined with a stable isotope-labeled glucose infusion, bioelectrical impedance analysis, and organ-specific (cardiac, renal, and hepatic) 123I-meta-iodobenzylguanidine (MIBG) innervation imaging. PLANNED OUTCOMES: Study recruitment started in April 2020 and will end in June 2024, with 40 participants randomized into the two groups. The treatment follow-up of the participants is currently ongoing and is due to finish by March 2025. TRIAL REGISTRATION: The study protocol has been approved by the Certified Review Board, Kanazawa University, Ishikawa, Japan, in accordance with the guidelines stipulated in the Declaration of Helsinki (CRB4180005, 2019-001). This trial is registered with the Japan Registry of Clinical Trials, jRCTs 041200035.

The declining insulinogenic index correlates with inflammation and metabolic dysregulation in non-obese individuals assessed by blood gene expression.

AIMS: Diabetes onset is difficult to predict. Since decreased insulinogenic index (IGI) is observed in prediabetes, and blood gene expression correlates with insulin secretion, candidate biomarkers can be identified. METHODS: We collected blood from 96 participants (54 males, 42 females) in 2008 (age: 52.5 years) and 2016 for clinical and gene expression analyses. IGI was derived from values of insulin and glucose at fasting and at 30 min post-OGTT. Two subgroups were identified based on IGI variation: "Minor change in IGI" group with absolute value variation between -0.05 and +0.05, and "Decrease in IGI" group with a variation between -20 and -0.05. RESULTS: Following the comparison of "Minor change in IGI" and "Decrease in IGI" groups at time 0 (2008), we identified 77 genes correlating with declining IGI, related to response to lipid, carbohydrate, and hormone metabolism, response to stress and DNA metabolic processes. Over the eight years, genes correlating to declining IGI were related to inflammation, metabolic and hormonal dysregulation. Individuals with minor change in IGI, instead, featured homeostatic and regenerative responses. CONCLUSIONS: By blood gene expression analysis of non-obese individuals, we identified potential gene biomarkers correlating to declining IGI, associated to a pathophysiology of inflammation and metabolic dysregulation.

Circulating selenoprotein P levels predict glucose-lowering and insulinotropic effects of metformin, but not alogliptin: A post-hoc analysis.

AIMS/INTRODUCTION: Selenoprotein P (SeP; encoded by SEPP1 in humans) is a hepatokine that causes impaired insulin secretion and insulin resistance. Metformin downregulates SELENOP promoter activity through an adenosine monophosphate-activated kinase-forkhead box protein O3a pathway in hepatocytes. This study aimed to test our hypothesis that circulating SeP levels are associated with the glucose-lowering effect of metformin in humans. MATERIALS AND METHODS: A total of 84 participants with poorly controlled type 2 diabetes were randomly assigned to receive metformin (1,000 mg, twice daily) or a dipeptidyl peptidase-4 inhibitor, alogliptin (25 mg, once daily) for 12 weeks. We tested metformin and alogliptin on SeP levels and factors associated therewith as a post-hoc analysis. RESULTS: Both metformin and aloglipitin did not change the SeP levels. Although metformin significantly increased the insulin secretory index secretory units of islets in transplantation only in participants with higher baseline SeP (>3.87), both agents similarly reduced fasting plasma glucose and glycated hemoglobin. SeP levels at baseline were correlated negatively with changes in SeP (r = -0.484, P = 0.004) and fasting plasma glucose (r = -0.433, P = 0.011), and positively with changes in C-peptide immunoreactivity (r = 0.420, P = 0.017) and secretory units of islets in transplantation (r = 0.388, P = 0.028) in the metformin, but not alogliptin, group. CONCLUSIONS: Higher baseline levels of SeP significantly predicted metformin-mediated, but not alogliptin-mediated, glucose-lowering and insulinotropic effects. Serum SeP levels might be a novel biomarker for predicting the outcomes of metformin therapy, which might be helpful in tailoring diabetes medication.

Trajectories of Liver Fibrosis and Gene Expression Profiles in Nonalcoholic Fatty Liver Disease Associated With Diabetes.

Understanding the mechanisms linking steatosis to fibrosis is needed to establish a promising therapy against nonalcoholic fatty liver disease (NAFLD). The aim of this study was to clarify clinical features and hepatic gene expression signatures that predict and contribute to liver fibrosis development during the long-term real-world histological course of NAFLD in subjects with and without diabetes. A pathologist scored 342 serial liver biopsy samples from 118 subjects clinically diagnosed with NAFLD during a 3.8-year (SD 3.45 years, maximum 15 years) course of clinical treatment. At the initial biopsy, 26 subjects had simple fatty liver, and 92 had nonalcoholic steatohepatitis (NASH). In the trend analysis, the fibrosis-4 index (P < 0.001) and its components at baseline predicted the future fibrosis progression. In the generalized linear mixed model, an increase in HbA1c, but not BMI, was significantly associated with fibrosis progression (standardized coefficient 0.17 [95% CI 0.009-0.326]; P = 0.038) for subjects with NAFLD and diabetes. In gene set enrichment analyses, the pathways involved in zone 3 hepatocytes, central liver sinusoidal endothelial cells (LSECs), stellate cells, and plasma cells were coordinately altered in association with fibrosis progression and HbA1c elevation. Therefore, in subjects with NAFLD and diabetes, HbA1c elevation was significantly associated with liver fibrosis progression, independent of weight gain, which may be a valuable therapeutic target to prevent the pathological progression of NASH. Gene expression profiles suggest that diabetes-induced hypoxia and oxidative stress injure LSECs in zone 3 hepatocytes, which may mediate inflammation and stellate cell activation, leading to liver fibrosis. ARTICLE HIGHLIGHTS: It remains uncertain how diabetes and obesity contribute to histological courses of nonalcoholic fatty liver disease (NAFLD). Clinical features and gene expression signatures that predict or are associated with future liver fibrosis development were assessed in a serial liver biopsy study of subjects with NAFLD. An increase in HbA1c, but not BMI, was associated with liver fibrosis progression in the generalized linear mixed model. Considering hepatic gene set enrichment analyses, diabetes may enhance liver fibrosis via injuring central liver sinusoidal endothelial cells that mediate inflammation and stellate cell activation during NAFLD development.