LECT2 deletion exacerbates liver steatosis and macrophage infiltration in a male mouse model of LPS-mediated NASH.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a protein initially isolated as a neutrophil chemotactic factor. We previously found that LECT2 is an obesity-associated hepatokine that senses liver fat and induces skeletal muscle insulin resistance. In addition, hepatocyte-derived LECT2 activates macrophage pro-inflammatory activity by reinforcing the LPS-induced c-Jun N-terminal kinase (JNK) signaling. Based on these findings, we examined the impact of LECT2 deletion on NAFLD/NASH caused by bacterial translocation. We created the bacterial translocation-mediated NAFLD/NASH model using LECT2 knockout mice (LECT2 KO) with 28 times a low-dose LPS injection under high-fat diet feeding conditions. LECT2 deletion exacerbated steatosis and significantly reduced p38 phosphorylation in the liver. In addition, LECT2 deletion increased macrophage infiltration with decreased M1/M2 ratios. LECT2 might contribute to protecting against lipid accumulation and macrophage activation in the liver under pathological conditions, which might be accomplished via p38 phosphorylation. This study provides novel aspects of LECT2 in the bacterial translocation-mediated NAFLD/NASH model.

Potential utility of l-carnitine for preventing liver tumors derived from metabolic dysfunction-associated steatohepatitis.

BACKGROUND: Recent reports have unveiled the potential utility of l-carnitine to alleviate metabolic dysfunction-associated steatohepatitis (MASH) by enhancing mitochondrial metabolic function. However, its efficacy at preventing the development of HCC has not been assessed fully. METHODS: l-carnitine (2 g/d) was administered to 11 patients with MASH for 10 weeks, and blood liver function tests were performed. Five patients received a serial liver biopsy, and liver histology and hepatic gene expression were evaluated using this tissue. An atherogenic plus high-fat diet MASH mouse model received long-term l-carnitine administration, and liver histology and liver tumor development were evaluated. RESULTS: Ten-week l-carnitine administration significantly improved serum alanine transaminase and aspartate transaminase levels along with a histological improvement in the NAFLD activity score, while steatosis and fibrosis were not improved. Gene expression profiling revealed a significant improvement in the inflammation and profibrotic gene signature as well as the recovery of lipid metabolism. Long-term l-carnitine administration to atherogenic plus high-fat diet MASH mice substantially improved liver histology (inflammation, steatosis, and fibrosis) and significantly reduced the incidence of liver tumors. l-carnitine directly reduced the expression of the MASH-associated and stress-induced transcriptional factor early growth response 1. Early growth response 1 activated the promoter activity of neural precursor cell expressed, developmentally downregulated protein 9 (NEDD9), an oncogenic protein. Thus, l-carnitine reduced the activation of the NEDD9, focal adhesion kinase 1, and AKT oncogenic signaling pathway. CONCLUSIONS: Short-term l-carnitine administration ameliorated MASH through its anti-inflammatory effects. Long-term l-carnitine administration potentially improved the steatosis and fibrosis of MASH and may eventually reduce the risk of HCC.

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.

Association between animal protein intake, oral frailty and calf circumference in middle-aged and older adults: a cross-sectional analysis from the Shika study.

OBJECTIVE: To investigate the relationship between oral frailty (OF), nutrient intake and calf circumference (CC) in middle-aged and older adults. DESIGN: Cross-sectional study. SETTING: Residents of four model districts of Shika town, Ishikawa Prefecture, Japan, using data from November 2017 to February 2018. PARTICIPANTS: One hundred and ninety-four residents aged ≥50 years in four model districts of Shika town. The OF total score ≥3 was defined as OF. Participants were divided into OF and non-OF groups and divided into the low-CC/kg and the high-CC/kg groups. OUTCOME MEASURES: The primary outcome is to use a two-way analysis of covariance to analyse the interaction between the two CC/kg groups and the two OF groups on nutrition intake. The secondary outcome is to use multiple regression analysis to investigate the nutrients significantly related to CC/kg when stratified by OF, with age, sex, body mass index, drinking status, smoking status and regular exercise as input covariates. RESULTS: A two-way analysis of covariance revealed a significant interaction between the two CC/kg groups and the two OF groups on animal protein intake (p=0.039). Multiple comparisons using the Bonferroni analysis revealed a significantly lower animal protein intake in the OF group than in the non-OF group with a low CC/kg (p=0.033) but not in the group with a high CC/kg. The multiple regression analysis stratified by OF revealed a positive correlation between animal protein intake and CC/kg (p=0.002). CONCLUSIONS: The present results revealed a significantly lower animal protein intake in the OF group than in the non-OF group in the low-CC/kg group, but no such difference was observed in the high-CC/kg group. Further longitudinal studies are needed to elucidate this relationship.

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.

Reductions in liver enzymes are associated with anti-hyperglycaemic and anti-obesity effects of tofogliflozin in people with type 2 diabetes: Post-hoc analyses.

AIMS: How the pathology of type 2 diabetes (T2D), including hyperglycaemia and obesity, affects liver enzymes has not been clinically demonstrated. Thus, we compared time courses of gamma-glutamyltransferase (GGT) and alanine aminotransferase (ALT) with those of fasting plasma glucose (FPG) and body weight (BW) during treatment with the SGLT2 inhibitor tofogliflozin for T2D. MATERIALS AND METHODS: We post-hoc analysed preexisting data on 1046 people with T2D administered tofogliflozin or placebo for 24 weeks in four tofogliflozin studies. First, time courses of percent changes in variables during the intervention were analysed using a mixed effect model to explore the similarity of the time courses and to evaluate time-treatment interactions. Second, clinical factors related to the percent changes in GGT and ALT were clarified using multivariate analyses. RESULTS: GGT levels and FPG values rapidly and significantly decreased via tofogliflozin as early as week 4, with decreases maintained until week 24. Conversely, BW and ALT decreased progressively until week 24. Time courses of FPG (p = .365, time-treatment interaction) and GGT (p = .510) reductions were parallel between tofogliflozin and placebo from weeks 4 to 24, while BW and ALT reductions (p < .001, respectively) were not. Reductions in GGT at week 24 were associated with reductions in FPG and BW at week 24, whereas ALT reductions were only associated with reductions in BW. CONCLUSIONS: Reductions in GGT and ALT were associated with the anti-hyperglycaemic and anti-obesity effects of tofogliflozin, respectively, in people with T2D. Therefore, GGT and ALT may be surrogate markers for hyperglycaemia and obesity in T2D.

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.

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.

Distinct associations between dietary omega-3 and omega-6 fatty acids intake with chronic kidney disease in adults with and without diabetes: A cross-sectional study.

OBJECTIVE: We explored the relationship of dietary intake of fatty acids with chronic kidney disease (CKD) according to glycemic status in Japanese people. METHODS: A total of 1031 participants aged ≥40 y were included in this population-based, cross-sectional study. A validated self-administered diet history questionnaire was used to measure the dietary intakes of fat and fatty acids, including omega-3 and omega-6 polyunsaturated fatty acids. CKD was defined as estimated glomerular filtration rate < 60 mL/min/1.73 m2 and diabetes as the use of antidiabetic medication, fasting plasma glucose ≥ 126 mg/dL, or hemoglobin A1c of ≥6.5%. Urine biomarkers of kidney injury (liver-type fatty acid-binding protein, ß2-microglobulin, and albumin) were also examined. RESULTS: The mean age of the participants was 62.5 ± 11.2 y, and 482 (46.8%) of them were men. Overall, 177 (17.2%) participants had CKD. In the multivariable model, low omega-3 intake (odds ratio = 0.109; 95% CI, 0.019-0.645) and high omega-6-to-omega-3 ratio (odds ratio = 2.112; 95% CI, 1.167-3.822) were associated with CKD in participants with diabetes but not in those without. In selected participants with diabetes, a substantial trend of urinary liver-type fatty acid-binding protein and ß2-microglobulin level elevation along with an increase in the dietary ratio of omega-6 to omega-3 was observed. CONCLUSIONS: Low dietary omega-3 intake and high omega-6-to-omega-3 ratio were associated with CKD in middle-aged and older Japanese people with diabetes but not in those without diabetes. These results may provide insight into the more tailored approaches for dietary polyunsaturated fatty acids to prevent CKD.

Nutrigenetic Interaction Between Apolipoprotein C3 Polymorphism and Fat Intake in People with Nonalcoholic Fatty Liver Disease.

Background: Recent genome-wide association studies have revealed that nonalcoholic fatty liver disease (NAFLD) is correlated with genetic polymorphisms. However, the effects of genetic variation on nutritional metabolism and NAFLD are complex and further studies are still needed. Objectives: This study aimed to assess the nutritional characteristics interacting with the correlation between genetic predisposition and NAFLD. Methods: We assessed the 2013-2017 health examination data of 1191 adults aged ≥40 y living in Shika town, Ishikawa Prefecture, Japan. Adults with moderate or heavy alcohol consumption and hepatitis were excluded, and 464 participants who underwent genetic analyses were included in the study. Abdominal echography was performed to diagnose fatty liver condition, and dietary intake and nutritional balance were evaluated using the brief self-administered diet history questionnaire. NAFLD-related gene polymorphisms were identified using Japonica Array v2 (Toshiba). Results: Among the 31 single nucleotide polymorphisms, only the polymorphism T-455C in the apolipoprotein C3 (APOC3) gene (rs2854116) was significantly associated with fatty liver condition. The condition was more common in participants with heterozygotes of the APOC3 gene (rs2854116) than in those with the TT and CC genotypes. Significant interactions were observed between NAFLD and the intake of fat, vegetable fat, MUFAs, PUFAs, cholesterol, n-3 FAs, and n-6 FAs. Moreover, participants with NAFLD who presented with the TT genotype had a significantly higher fat intake than those without NAFLD. Conclusions: The polymorphism T-455C in the APOC3 gene (rs2854116) and fat intake are associated with the NAFLD risk in Japanese adults. Participants with a fatty liver who presented with the TT genotype of rs2854116 had a higher fat intake. Such nutrigenetic interaction can deepen our understanding of the NAFLD pathology. Moreover, in clinical settings, the correlation between genetic factors and nutrition intake should be considered in personalized nutritional interventions against NAFLD. Curr Dev Nutr 2023;xx:xx.The study was registered in the University Hospital Medical Information Network Clinical Trials Registry as UMIN 000024915.

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.

PAC1 Deficiency Protects Obese Male Mice From Immobilization-Induced Muscle Atrophy by Suppressing FoxO-Atrogene Axis.

Muscle atrophy is the cause and consequence of obesity. Proteasome dysfunction mediates obesity-induced endoplasmic reticulum (ER) stress and insulin resistance in the liver and adipose tissues. However, obesity-associated regulation of proteasome function and its role in the skeletal muscles remains underinvestigated. Here, we established skeletal muscle-specific 20S proteasome assembly chaperone-1 (PAC1) knockout (mPAC1KO) mice. A high-fat diet (HFD) activated proteasome function by ∼8-fold in the skeletal muscles, which was reduced by 50% in mPAC1KO mice. mPAC1KO induced unfolded protein responses in the skeletal muscles, which were reduced by HFD. Although the skeletal muscle mass and functions were not different between the genotypes, genes involved in the ubiquitin proteasome complex, immune response, endoplasmic stress, and myogenesis were coordinately upregulated in the skeletal muscles of mPAC1KO mice. Therefore, we introduced an immobilization-induced muscle atrophy model in obesity by combining HFD and immobilization. mPAC1KO downregulated atrogin-1 and MuRF1, together with their upstream Foxo1 and Klf15, and protected against disused skeletal muscle mass reduction. In conclusion, obesity elevates proteasome functions in the skeletal muscles. PAC1 deficiency protects mice from immobilization-induced muscle atrophy in obesity. These findings suggest obesity-induced proteasome activation as a possible therapeutic target for immobilization-induced muscle atrophy.

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.

Association between Bone Mineral Density and Oral Frailty on Renal Function: Findings from the Shika Study.

The association between oral frailty (OFr) and body action has been investigated, but its association with systemic function remains unclear. Therefore, this cross-sectional study examined the association between OFr with decreased bone mineral density (BMD) and renal function in residents of Shika town, Ishikawa Prefecture, Japan aged ≥40 years. This study included 400 inhabitants. The OFr total score was assessed using three oral domains in the Kihon Checklist (a self-reported comprehensive health checklist), the number of teeth, and brushing frequency per day. Measurements were the estimated glomerular filtration rate (eGFR) and the osteo-sono assessment index (OSI). Using a two-way analysis of covariance (p = 0.002), significantly lower OSI was indicated in the eGFR < 60 and OFr group than in the eGFR of < 60 and non-OFr group after adjusting for age, body mass index, and drinking and smoking status as confounding factors. Multiple logistic regression analysis confirmed this relationship (p = 0.006). Therefore, lower BMD seems to be associated with lower renal function only when accompanied by OFr. Further longitudinal studies are needed to confirm these results.

Cystatin C-based eGFR predicts cardiovascular disease in patients with overweight/obesity and hyperglycemia.

Background: Although many clinical parameters have been identified as predictors for cardiovascular disease (CVD) development in the general population, the accurate predictor for CVD in patients with obesity is still unknown. Objective: The study aimed to explore an additional risk factor and predictor for CVD in patients with overweight/obesity considering the interaction of obesity-related pathophysiology. Methods: The Japan Obesity and Metabolic Syndrome study, a multicenter prospective study, enrolled 787 outpatients, of which 318 eligible patients were analyzed. Patients with fasting plasma glucose (FPG) levels ≥6.11 and < 6.11 mmol/L were considered to have high FPG (HFPG) and normal FPG (NFPG), respectively. Thirty-six patients who developed CVD during the 5 years follow-up were assigned to the CVD group. Results: Cox's proportional hazards model revealed no significant association between CVD and cystatin C-based estimated glomerular filtration rate (eGFRcys) or creatinine-based eGFR (eGFRcr) in the NFPG group. In the HFPG group, lower eGFRcys, but not eGFRcr, was significantly associated with CVD development. A generalized linear mixed model demonstrated greater reduction in eGFRcys levels over time with HFPG than with NFPG. Although the CVD group showed gradual reduction in eGFRcys levels, the non-CVD group-matched using propensity scores-did not show a decline in eGFRcys levels. Conclusions: Lower eGFRcys levels may be more accurate than eGFRcr in predicting CVD development in patients with overweight/obesity and hyperglycemia. Furthermore, eGFRcys reduction over time is associated with CVD development. Clinical Trial Registry Number: UMIN000000559.

Insulin Suppresses Ubiquitination via the Deubiquitinating Enzyme Ubiquitin-Specific Protease 14, Independent of Proteasome Activity in H4IIEC3 Hepatocytes.

Ubiquitin-proteasome dysfunction contributes to obesity-related metabolic disorders, such as diabetes and fatty liver disease. However, the regulation of ubiquitin-proteasome activity by insulin remains to be elucidated. Here, we show that prolonged insulin stimulation activates proteasome function even though it reduces the ubiquitinated proteins in H4IIEC3 hepatocytes. Looking for a pathway by which insulin inhibits ubiquitination, we found that hepatic expression of ubiquitin-specific protease 14 (USP14) was upregulated in the liver of patients with insulin resistance. Indeed, the USP14-specific inhibitor IU1 canceled the insulin-mediated reduction of ubiquitinated proteins. Furthermore, insulin-induced endoplasmic reticulum (ER) stress, which was canceled by IU1, suggesting that USP14 activity is involved in insulin-induced ER stress. Co-stimulation with insulin and IU1 for 2 hours upregulated the nuclear translocation of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), upregulated the expression of the lipogenic gene, fatty acid synthase (Fasn), and repressed the gluconeogenic genes. In conclusion, insulin activates proteasome function even though it inhibits protein ubiquitination by activating USP14 in hepatocytes. USP14 activation by insulin inhibits mature SREBP-1c while upregulating ER stress and the expression of genes involved in gluconeogenesis. Further understanding mechanisms underlying the USP14 activation and its pleiotropic effects may lead to therapeutic development for obesity-associated metabolic disorders, such as diabetes and fatty liver disease. SIGNIFICANCE STATEMENT: This study shows that insulin stimulation inhibits ubiquitination by activating USP14, independent of its effect on proteasome activity in hepatocytes. USP14 also downregulates the nuclear translocation of the lipogenic transcription factor SREBP-1c and upregulates the expression of genes involved in gluconeogenesis. Since USP14 is upregulated in the liver of insulin-resistant patients, understanding mechanisms underlying the USP14 activation and its pleiotropic effects will help develop treatments for metabolic disorders such as diabetes and fatty liver.

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.

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.

Cyclosporine A Downregulates Selenoprotein P Expression via a Signal Transducer and Activator of Transcription 3-Forkhead Box Protein O1 Pathway in Hepatocytes In Vitro.

Cyclosporine A (CsA) is an immunosuppressant applied worldwide for preventing graft rejection and autoimmune diseases. However, CsA elevates oxidative stress, which can lead to liver injuries. The present study aimed to clarify the mechanisms underlying the CsA-mediated oxidative stress. Among the redox proteins, CsA concentration-dependently downregulated Selenop-encoding selenoprotein P, a major circulating antioxidant protein reducing reactive oxygen species, in hepatocytes cell lines and primary hepatocytes. The luciferase assay identified the CsA-responsive element in the SELENOP promoter containing a putative binding site for forkhead box protein O (FoxO) 1. The CsA-mediated suppression on the SELENOP promoter was independent of the nuclear factor of activated T-cell, a classic target repressed by CsA. A chromatin immunoprecipitation assay showed that CsA suppressed the FoxO1 binding to the SELENOP promoter. Foxo1 knockdown significantly downregulated Selenop expression in H4IIEC3 cells. Furthermore, CsA downregulated FoxO1 by inactivating its upstream signal transducer and activator of transcription 3 (STAT3). Knockdown of Stat3 downregulated Foxo1 and Selenop expression in hepatocytes. These findings revealed a novel mechanism underlying CsA-induced oxidative stress by downregulating the STAT3-FoxO1-Selenop pathway in hepatocytes. SIGNIFICANCE STATEMENT: This study shows that Cyclosporine A (CsA) downregulates Selenop, an antioxidant protein, by suppressing the signal transducer and activator of transcription 3-forkhead box protein O1 pathway in hepatocytes, possibly one of the causations of CsA-induced oxidative stress in hepatocytes. The present study sheds light on the previously unrecognized CsA-redox axis.

Comparison of Tofogliflozin and Glimepiride Effects on Nonalcoholic Fatty Liver Disease in Participants With Type 2 Diabetes: A Randomized, 48-Week, Open-Label, Active-Controlled Trial.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is a liver phenotype of type 2 diabetes and obesity. Currently, the efficacy of sodium-glucose cotransporter 2 (SGLT2) inhibitors and sulfonylureas in liver pathology and hepatic gene expression profiles for type 2 diabetes with NAFLD are unknown. RESEARCH DESIGN AND METHODS: We conducted a 48 week, randomized, open-label, parallel-group trial involving participants with biopsy-confirmed NAFLD. A total of 40 participants were randomly assigned to receive once daily 20 mg tofogliflozin or 0.5 mg glimepiride. The primary outcome was the percentage of participants with at least an improvement in all individual scores for histological categories of steatosis, hepatocellular ballooning, lobular inflammation, and fibrosis by at least 1 point. The secondary end points were the changes in liver enzymes, metabolic markers, and hepatic gene expression profiles. RESULTS: Fibrosis scores improved in the tofogliflozin group (60%, P = 0.001), whereas the change from baseline did not differ significantly between the groups (P = 0.172). The histological variables of steatosis (65%, P = 0.001), hepatocellular ballooning (55%, P = 0.002), and lobular inflammation (50%, P = 0.003) were improved in the tofogliflozin group, whereas only hepatocellular ballooning was improved in the glimepiride group (25%, P = 0.025). Hepatic gene expression profiling revealed histology-associated signatures in energy metabolism, inflammation, and fibrosis that were reversed with tofogliflozin. CONCLUSIONS: Tofogliflozin and, to a lesser degree, glimepiride led to liver histological and metabolic improvement in participants with type 2 diabetes and NAFLD, with no significant difference between the agents. The hepatic expression of the genes involved in energy metabolism, inflammation, and fibrosis was well correlated with liver histological changes and rescued by tofogliflozin. We need further confirmation through long-term larger-scale clinical trials of SGLT2 inhibitors.