A pilot study of diabetic retinopathy in a porcine model of maturity onset diabetes of the young type 3 (MODY3).

Diabetic retinopathy (DR) is a leading cause of blindness in the working population. Because novel therapeutic intervention require testing, there is an urgent need for reliable animal models that faithfully replicate DR. Pig eyes have many similarities to human eyes anatomically and physiologically. Thus, attempts have been made to establish porcine models of DR by surgical, pharmaceutical or genetical induction of insulin deficiency, and dietary intervention. A previous study reported a transgenic pig model of maturity onset diabetes of the young type 3 (MODY3) developed signs of severe DR such as hemorrhage and proliferative tissue at the surface of the retina. However, the course of development of DR has not been studied in detail in this model. The purpose of this study was to investigate the early phase of DR in a MODY3. MODY3 and wild-type (WT) pigs underwent fundus photography and fluorescein angiogram (FA) before they developed cataracts. Animals were euthanized at age 1, 4, 7, and 10 months. Whole-mount retina and 10-µm thick paraffinized sections were stained with isolectin B4, and vessel density was determined by MATLAB software. At 4 and 7 months, retinal arterioles were immediately cannulated, and vasomotor action was measured by incubation with bradykinin and sodium nitroprusside. In the MODY3 pigs, fasting blood sugar levels gradually increased up to 500 mg/dL. Vascular tortuosity and yellowish spindle-shaped lesions were confirmed in MODY3 pigs at the age of 7 months; however, no microaneurysms were detected on FA. Compared with age-matched WT pigs, MODY3 pigs showed a significant decrease in blood vessel density in the intermediate and deep vascular plexus at 4 and 7 months of age and a slight decrease in capillary density in the superficial vascular plexus at 7 months of age. In MODY3 pigs, electron microscopy revealed thickening of the capillary basement membrane and leukostasis in the major blood vessels at 10 months of age. Bradykinin-induced dilation of retinal arterioles was diminished in MODY3 pigs as early as 7 months of age. Within 1 year after birth, MODY3 pigs show all typical early vascular lesions of diabetes except for microaneurysm formation. This pilot study suggests that the MODY3 pigs may serve as a suitable DR model to test effects of newly developed compounds on DR.

Dilation of porcine retinal arterioles to nobiletin, a polymethoxyflavonoid: Roles of nitric oxide and voltage-dependent potassium channel.

We examined the effects of nobiletin, a polymethoxyflavonoid, on the retinal microvascular diameter to determine if they depend on the endothelium and/or smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded diametric responses to nobiletin. The retinal arterioles dilated in a nobiletin concentration-dependent (100 pM-10 µM) manner and decreased by 50% after endothelial removal. The nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME), reduced nobiletin-induced vasodilation comparable to denudation. Blockade of soluble guanylyl cyclase by 1H-[1,2,4] oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) produced a similar inhibitory effect as that by L-NAME. Nobiletin-induced vasodilation was also inhibited by the nonselective potassium channel inhibitor, tetraethylammonium (TEA), and the voltage-gated K (Kv) inhibitor, 4-aminopyridine. Co-administration of L-NAME and TEA almost eliminated nobiletin-induced vasodilation. Nobiletin elicits both endothelium-dependent and -independent dilation of retinal arterioles mediated by NO release and Kv channel activation, respectively.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

Auraptene Enhances AMP-Activated Protein Kinase Phosphorylation and Thereby Inhibits the Proliferation, Migration and Expression of Androgen Receptors and Prostate-Specific Antigens in Prostate Cancer Cells.

Citrus hassaku extract reportedly activates AMPK. Because this extract contains an abundance of auraptene, we investigated whether pure auraptene activates AMPK and inhibits proliferation using prostate cancer cell lines. Indeed, auraptene inhibited the proliferation and migration of LNCaP cells and induced phosphorylation of AMPK or its downstream ACC in LNCaP, PC3, and HEK-293 cells, but not in DU145 cells not expressing LKB1. In addition, the mTOR-S6K pathway, located downstream from activated AMPK, was also markedly suppressed by auraptene treatment. Importantly, it was shown that auraptene reduced androgen receptor (AR) and prostate-specific antigen (PSA) expressions at both the protein and the mRNA level. This auraptene-induced downregulation of PSA was partially but significantly reversed by treatment with AMPK siRNA or the AMPK inhibitor compound C, suggesting AMPK activation to, at least partially, be causative. Finally, in DU145 cells lacking the LKB1 gene, exogenously induced LKB1 expression restored AMPK phosphorylation by auraptene, indicating the essential role of LKB1. In summary, auraptene is a potent AMPK activator that acts by elevating the AMP/ATP ratio, thereby potentially suppressing prostate cancer progression, via at least three molecular mechanisms, including suppression of the mTOR-S6K pathway, reduced lipid synthesis, and AR downregulation caused by AMPK activation.

Synergistic deterioration of prognosis associated with decreased grip strength and hyporesponse to erythropoiesis-stimulating agents in patients undergoing hemodialysis.

INTRODUCTION: We examined the combined effect of erythropoietin (EPO) hyporesponsiveness and low handgrip strength (HGS) on the prognosis of patients undergoing hemodialysis (HD). METHODS: We recruited patients with chronic kidney disease (CKD) Stage 5, who were undergoing HD at our dialysis clinic between January 2015 and March 2015 (n = 182). Patients of ≥20 years of age and who had been undergoing HD for ≧3 months at enrollment were eligible for inclusion. Seven patients treated with epoetin-ß pegol were excluded. First, the erythropoietin resistance index (ERI) and HGS were measured. The patients were stratified by the ERI of 9.44 (U/kg/week/g/dL), and by the HGS of 28 kg for men and 18 kg for women. We then observed death and cardiovascular disease (CVD), composite endpoint (deaths or CVD) for a median of 2 years. RESULTS: A total of 175 patients (male, n = 122; female, n = 53; age, 34-92 years) were included in the analysis. During the observation period of 24 months, 57 events (14 deaths and 43 CVD) were observed. High ERI and low HGS were associated with a high incidence of endpoints compared to low ERI and high HGS. Among the four groups classified by ERI and HGS values, the highest risk group was the high ERI/low HGS group (HR: 4.20 95% CI 2.12-8.33). CONCLUSIONS: EPO hyporesponsiveness combined with low HGS were found to be significant predictors of a poor outcome, and the synergistic effects of the two factors had stronger predictive ability than either single factor.

The Effect of Sodium-Dependent Glucose Cotransporter 2 Inhibitor Tofogliflozin on Neurovascular Coupling in the Retina in Type 2 Diabetic Mice.

We investigated the effect of tofogliflozin, a sodium-dependent glucose cotransporter 2 inhibitor (SGLT2i), on retinal blood flow dysregulation, neural retinal dysfunction, and the impaired neurovascular coupling in type 2 diabetic mice. Tofogliflozin was added to mouse chow to deliver 5 mg/kg/day and 6-week-old mice were fed for 8 weeks. The longitudinal changes in the retinal neuronal function and blood flow responses to systemic hyperoxia and flicker stimulation were evaluated every 2 weeks in diabetic db/db mice that received tofogliflozin (n =6) or placebo (n = 6) from 8 to 14 weeks of age. We also evaluated glial activation and vascular endothelial growth factor (VEGF) expression by immunofluorescence. Tofogliflozin treatment caused a sustained decrease in blood glucose in db/db mice from 8 weeks of the treatment. In tofogliflozin-treated db/db mice, both responses improved from 8 to 14 weeks of age, compared with vehicle-treated diabetic mice. Subsequently, the electroretinography implicit time for the oscillatory potential was significantly improved in SGLT2i-treated db/db mice. The systemic tofogliflozin treatment prevented the activation of glial fibrillary acidic protein and VEGF protein expression, as detected by immunofluorescence. Our results suggest that glycemic control with tofogliflozin significantly improved the impaired retinal neurovascular coupling in type 2 diabetic mice with the inhibition of retinal glial activation.

Quantitative sonographic assessment of quadriceps muscle thickness for fall injury prediction in patients undergoing maintenance hemodialysis: an observational cohort study.

BACKGROUND: Accidental fall risk is high in patients undergoing maintenance hemodialysis. Falls are associated with fatal injury, comorbidities, and mortality. Risk assessment should be a primary component of fall prevention. This study investigated whether quadriceps muscle thickness measured using ultrasonography can predict fall injury among dialysis patients. METHODS: Using an observational cohort study design, 180 ambulatory hemodialysis patients were recruited from 2015 to 2016 from four dialysis clinics. The sum of the maximum quadriceps muscle thickness on both sides and the average of the maximum thigh circumference and handgrip strength after hemodialysis were calculated. Patients were stratified according to tertiles of quadriceps muscle thickness. Fall injury was surveyed according to the patient's self-report during the one-year period. RESULTS: Among the 180 hemodialysis patients, 44 (24.4%) had fall injuries during the 12-month follow-up period. When the quadriceps muscle thickness levels were stratified into sex-specific tertiles, patients in the lowest tertile were more likely to have a higher incidence of fall injury than those in the higher two tertiles (0.52 vs. 0.19 and 0.17 fall injuries/person-year). After adjusting for covariates, lower quadriceps muscle thickness was found to be an independent predictor of fall injury (hazard ratio [95% confidence interval], 2.33 [1.22-4.52], P < 0.05). Receiver operating characteristic curves were constructed to determine the optimal cutoffs of quadriceps muscle thickness, thigh circumference, and handgrip strength that best predicted fall injury (quadriceps muscle thickness, 3.37 cm and 3.54 cm in men and women; thigh circumference, 44.6 cm and 37.2 cm in men and women; and handgrip strength, 23.3 kg and 16.5 kg in men and women). Using these cutoff values, the areas under the curve were 0.662 (95% CI, 0.576-0.738), 0.625 (95% CI, 0.545-0.699), and 0.701 (95% CI, 0.617-0.774), for quadriceps muscle thickness, thigh circumference, and handgrip strength, respectively. Quadriceps muscle thickness was a more precise predictor of fall injury than thigh circumference and had similar diagnostic performance as handgrip strength tests in dialysis patients. CONCLUSIONS: Quadriceps muscle thickness can be measured easily at the bedside using ultrasonography and is a precise predictor of fall injury in patients undergoing maintenance hemodialysis.

Carnosic Acid and Carnosol Activate AMPK, Suppress Expressions of Gluconeogenic and Lipogenic Genes, and Inhibit Proliferation of HepG2 Cells.

Carnosic acid (CA), carnosol (CL) and rosmarinic acid (RA), components of the herb rosemary, reportedly exert favorable metabolic actions. This study showed that both CA and CL, but not RA, induce significant phosphorylation of AMP-dependent kinase (AMPK) and its downstream acetyl-CoA carboxylase 1 (ACC1) in HepG2 hepatoma cells. Glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1), rate-limiting enzymes of hepatic gluconeogenesis, are upregulated by forskolin stimulation, and this upregulation was suppressed when incubated with CA or CL. Similarly, a forskolin-induced increase in CRE transcriptional activity involved in G6PC and PCK1 regulations was also stymied when incubated with CA or CL. In addition, mRNA levels of ACC1, fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) were significantly reduced when incubated with CA or CL. Finally, it was shown that CA and CL suppressed cell proliferation and reduced cell viability, possibly as a result of AMPK activation. These findings raise the possibility that CA and CL exert a protective effect against diabetes and fatty liver disease, as well as subsequent cases of hepatoma.

Treatment of two mitochondrial disease patients with a combination of febuxostat and inosine that enhances cellular ATP.

Since mitochondria are energy-generating micro-organisms, most of the disorders in patients with mitochondrial diseases (mt-disease) are considered secondary to defects in ATP synthesis, although some other factors such as reactive oxygen species may be involved. A simultaneous oral administration of febuxostat and inosine was reported to elevate both hypoxanthine and ATP levels in peripheral blood. Based on those results, we attempted co-administration of febuxostat and inosine in two patients with mitochondrial disease: one patient with mitochondrial cardiomyopathy and the other patient with mitochondrial diabetes. In the former case, brain natriuretic peptide (BNP), which is a specific marker for heart failure, was decreased by 31%, and in the latter case, the insulinogenic index increased 3.1 times, suggesting the favorable action of the treatment. Considering that there is no effective treatment available for this disorder, the present therapy may be quite useful for the management of patients with mitochondrial diseases.

Characteristics and Early Hypoglycemic Medications of Patients at Risk of Progression to Type 2 Diabetes in Japan: A Retrospective Cohort Study of Health Checkup and Claims Data.

Medication therapy management by tracking patients with risk of progression to type 2 diabetes has not been investigated in Japan. We aimed to assess the characteristics of these patients and their early medications. Claims (n = 190507) and health checkup data (n = 106984) between April 2005 and March 2015 in Japan were selected. We selected patients aged ≥40 years with fasting plasma glucose levels of 100-125 mg/dL or glycated hemoglobin A1c values of 5.7-6.4%. The early-medication group comprised patients who received hypoglycemic medications within 6 months after their first clinic visit, while the no-medication group comprised patients who did not receive any hypoglycemic medications. Main outcome measures were characteristics and early hypoglycemic medications of patients at risk of progression to type 2 diabetes. Of 5676 individuals, hypoglycemic medications were initiated in 276 (5%). The early-medication group had a higher proportion of individuals with a body mass index ≥25 kg/m2 and current smokers and drinkers than the no-medication group. Approximately 83% of patients in the early-medication group were prescribed a single hypoglycemic medication, and since 2010, dipeptidyl peptidase-4 inhibitors were prescribed to one-third of these patients. In our population, early hypoglycemic medication was initiated within 6 months of the first clinic visit, indicating that initiation took place earlier than recommended by current guidelines. Early hypoglycemic medications, especially dipeptidyl peptidase-4 inhibitors with low risks of hypoglycemia, might be prescribed based on patient characteristics. Further epidemiological studies are needed to confirm the suitability of early hypoglycemic medication.

Association of gait speed and grip strength with risk of cardiovascular events in patients on haemodialysis: a prospective study.

BACKGROUND: Gait speed (GS) and handgrip strength (HGS), both factors associated with frailty and sarcopenia, are reportedly associated with CV events in the general population. However, little is known about the impact of these factors on the outcome of patients on dialysis. This study aimed to evaluate whether evaluation of GS and HGS could be associated the onset of fatal/non-fatal cardiovascular (CV) events in patients on haemodialysis (HD). METHODS: One-hundred-eighty-two patients with end-stage renal disease (ESRD) undergoing HD at four dialysis clinics in April 2015 provided written informed consent to participate in the study. We excluded patients who had physical disability, were unable to walk without help, or had recently experienced CV events. Usual GS over a 4-m walk and HGS were measured at baseline, and 173 patients (men, 124; women, 49) were divided into sex-specific quartiles according to GS and HGS and were followed-up for fatal/non-fatal CV events for a median of 2 years. We examined the association of GS and HGS with CV events and determined cut-off values using Cox regression analysis adjusted for age, sex, HD duration, history of CVD, and diabetes. RESULTS: During the follow-up period, 46 CV events occurred. Both physical performance factors were significantly associated with CV events. Low GS (< 0.82 m/s for men and 0.81 m/s for women) and weak HGS (< 29.0 kg for men and 19.7 kg for women) were associated with CV events. For low vs. high GS, the hazard ratio (HR) for CV events was 2.29 [95% confidence interval (CI): 1.20-4.33; P = 0.01], and for low vs. high HGS, the HR was 2.15 [95% CI: 1.00-5.04; P < 0.05]. These HRs remained significant after adjusting for confounding factors, such as sex, age, dialysis vintage, history of CV disease, and diabetes. CONCLUSIONS: Slow GS and weak HGS in patients on HD were suggested to be independent predictors of fatal/non-fatal CV events.

Xanthine Oxidase Inhibitor Febuxostat Exerts an Anti-Inflammatory Action and Protects against Diabetic Nephropathy Development in KK-Ay Obese Diabetic Mice.

Hyperuricemia has been recognized as a risk factor for insulin resistance as well as one of the factors leading to diabetic kidney disease (DKD). Since DKD is the most common cause of end-stage renal disease, we investigated whether febuxostat, a xanthine oxidase (XO) inhibitor, exerts a protective effect against the development of DKD. We used KK-Ay mice, an established obese diabetic rodent model. Eight-week-old KK-Ay mice were provided drinking water with or without febuxostat (15 µg/mL) for 12 weeks and then subjected to experimentation. Urine albumin secretion and degrees of glomerular injury judged by microscopic observations were markedly higher in KK-Ay than in control lean mice. These elevations were significantly normalized by febuxostat treatment. On the other hand, body weights and high serum glucose concentrations and glycated albumin levels of KK-Ay mice were not affected by febuxostat treatment, despite glucose tolerance and insulin tolerance tests having revealed febuxostat significantly improved insulin sensitivity and glucose tolerance. Interestingly, the IL-1ß, IL-6, MCP-1, and ICAM-1 mRNA levels, which were increased in KK-Ay mouse kidneys as compared with normal controls, were suppressed by febuxostat administration. These data indicate a protective effect of XO inhibitors against the development of DKD, and the underlying mechanism likely involves inflammation suppression which is independent of hyperglycemia amelioration.

The prolyl isomerase Pin1 increases ß-cell proliferation and enhances insulin secretion.

The prolyl isomerase Pin1 binds to the phosphorylated Ser/Thr-Pro motif of target proteins and enhances their cis-trans conversion. This report is the first to show that Pin1 expression in pancreatic ß cells is markedly elevated by high-fat diet feeding and in ob/ob mice. To elucidate the role of Pin1 in pancreatic ß cells, we generated ß-cell-specific Pin1 KO (ßPin1 KO) mice. These mutant mice showed exacerbation of glucose intolerance but had normal insulin sensitivity. We identified two independent factors underlying impaired insulin secretion in the ßPin1 KO mice. Pin1 enhanced pancreatic ß-cell proliferation, as indicated by a reduced ß-cell mass in ßPin1 KO mice compared with control mice. Moreover, a diet high in fat and sucrose failed to increase pancreatic ß-cell growth in the ßPin1 KO mice, an observation to which up-regulation of the cell cycle protein cyclin D appeared to contribute. The other role of Pin1 was to activate the insulin-secretory step: Pin1 KO ß cells showed impairments in glucose- and KCl-induced elevation of the intracellular Ca2+ concentration and insulin secretion. We also identified salt-inducible kinase 2 (SIK2) as a Pin1-binding protein that affected the regulation of Ca2+ influx and found Pin1 to enhance SIK2 kinase activity, resulting in a decrease in p35 protein, a negative regulator of Ca2+ influx. Taken together, our observations demonstrate critical roles of Pin1 in pancreatic ß cells and that Pin1 both promotes ß-cell proliferation and activates insulin secretion.

Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes.

Glial fibrillary acidic protein (GFAP) is expressed in peri-islet Schwann cells, as well as in glia cells, and has been reported to be an autoantigen candidate for type 1 diabetes mellitus (T1DM). We confirmed that the production of the autoantibodies GFAP and glutamic acid decarboxylase 65 (GAD65) was increased and inversely correlated with the concentration of secreted C peptide in female nonobese diabetic mice (T1DM model). Importantly, the development of T1DM in female nonobese diabetic mice at 30 wk of age was predicted by the positive GFAP autoantibody titer at 17 wk. The production of GFAP and GAD65 autoantibodies was also increased in KK-Ay mice [type 2 diabetes mellitus (T2DM) model]. In patients with diabetes mellitus, GFAP autoantibody levels were increased in patients with either T1DM or T2DM, and were significantly associated with GAD65 autoantibodies but not zinc transporter 8 autoantibodies. Furthermore, we identified a B-cell epitope of GFAP corresponding to the GFAP autoantibody in both mice and patients with diabetes. Thus, these results indicate that autoantibodies against GFAP could serve as a predictive marker for the development of overt autoimmune diabetes.-Pang, Z., Kushiyama, A., Sun, J., Kikuchi, T., Yamazaki, H., Iwamoto, Y., Koriyama, H., Yoshida, S., Shimamura, M., Higuchi, M., Kawano, T., Takami, Y., Rakugi, H., Morishita, R., Nakagumi, H. Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes.

Protective Effect of Sex Hormone-Binding Globulin against Metabolic Syndrome: In Vitro Evidence Showing Anti-Inflammatory and Lipolytic Effects on Adipocytes and Macrophages.

Sex hormone-binding globulin (SHBG) is a serum protein released mainly by the liver, and a low serum level correlates with a risk for metabolic syndrome including diabetes, obesity, and cardiovascular events. However, the underlying molecular mechanism(s) linking SHBG and metabolic syndrome remains unknown. In this study, using adipocytes and macrophages, we focused on the in vitro effects of SHBG on inflammation as well as lipid metabolism. Incubation with 20 nM SHBG markedly suppressed lipopolysaccharide- (LPS-) induced inflammatory cytokines, such as MCP-1, TNFα, and IL-6 in adipocytes and macrophages, along with phosphorylations of JNK and ERK. Anti-inflammatory effects were also observed in 3T3-L1 adipocytes cocultured with LPS-stimulated macrophages. In addition, SHBG treatment for 18 hrs or longer significantly induced the lipid degradation of differentiated 3T3-L1 cells, with alterations in its corresponding gene and protein levels. Notably, these effects of SHBG were not altered by coaddition of large amounts of testosterone or estradiol. In conclusion, SHBG suppresses inflammation and lipid accumulation in macrophages and adipocytes, which might be among the mechanisms underlying the protective effect of SHBG, that is, its actions which reduce the incidence of metabolic syndrome.

Roles of Gut-Derived Secretory Factors in the Pathogenesis of Non-Alcoholic Fatty Liver Disease and Their Possible Clinical Applications.

The rising prevalence of non-alcoholic fatty liver disease (NAFLD) parallels the global increase in the number of people diagnosed with obesity and metabolic syndrome. The gut-liver axis (GLA) plays an important role in the pathogenesis of NAFLD/non-alcoholic steatohepatitis (NASH). In this review, we discuss the clinical significance and underlying mechanisms of action of gut-derived secretory factors in NAFLD/NASH, focusing on recent human studies. Several studies have identified potential causal associations between gut-derived secretory factors and NAFLD/NASH, as well as the underlying mechanisms. The effects of gut-derived hormone-associated drugs, such as glucagon-like peptide-1 analog and recombinant variant of fibroblast growth factor 19, and other new treatment strategies for NAFLD/NASH have also been reported. A growing body of evidence highlights the role of GLA in the pathogenesis of NAFLD/NASH. Larger and longitudinal studies as well as translational research are expected to provide additional insights into the role of gut-derived secretory factors in the pathogenesis of NAFLD/NASH, possibly providing novel markers and therapeutic targets in patients with NAFLD/NASH.

The Xanthine Oxidase Inhibitor Febuxostat Suppresses the Progression of IgA Nephropathy, Possibly via Its Anti-Inflammatory and Anti-Fibrotic Effects in the gddY Mouse Model.

Recent clinical studies have demonstrated the protective effect of xanthine oxidase (XO) inhibitors against chronic kidney diseases, although the underlying molecular mechanisms remain unclear. However, to date, neither clinical nor basic research has been carried out to elucidate the efficacy of XO inhibitor administration for IgA nephropathy. We thus investigated whether febuxostat, an XO inhibitor, exerts a protective effect against the development of IgA nephropathy, using gddY mice as an IgA nephropathy rodent model. Eight-week-old gddY mice were provided drinking water with (15 µg/mL) or without febuxostat for nine weeks and then subjected to experimentation. Elevated serum creatinine and degrees of glomerular sclerosis and fibrosis, judged by microscopic observations, were significantly milder in the febuxostat-treated than in the untreated gddY mice, while body weights and serum IgA concentrations did not differ between the two groups. In addition, elevated mRNA levels of inflammatory cytokines such as TNFα, MCP-1, IL-1ß, and IL-6, collagen isoforms and chemokines in the gddY mouse kidneys were clearly normalized by the administration of febuxostat. These data suggest a protective effect of XO inhibitors against the development of IgA nephropathy, possibly via suppression of inflammation and its resultant fibrotic changes, without affecting the serum IgA concentration.

Reduced SHARPIN and LUBAC Formation May Contribute to CCl4- or Acetaminophen-Induced Liver Cirrhosis in Mice.

Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl4) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.

The SGLT2 Inhibitor Luseogliflozin Rapidly Normalizes Aortic mRNA Levels of Inflammation-Related but Not Lipid-Metabolism-Related Genes and Suppresses Atherosclerosis in Diabetic ApoE KO Mice.

Recent clinical studies have revealed the treatment of diabetic patients with sodium glucose co-transporter2 (SGLT2) inhibitors to reduce the incidence of cardiovascular events. Using nicotinamide and streptozotocin (NA/STZ) -treated ApoE KO mice, we investigated the effects of short-term (seven days) treatment with the SGLT2 inhibitor luseogliflozin on mRNA levels related to atherosclerosis in the aorta, as well as examining the long-term (six months) effects on atherosclerosis development. Eight-week-old ApoE KO mice were treated with NA/STZ to induce diabetes mellitus, and then divided into two groups, either untreated, or treated with luseogliflozin. Seven days after the initiation of luseogliflozin administration, atherosclerosis-related mRNA levels in the aorta were compared among four groups; i.e., wild type C57/BL6J, native ApoE KO, and NA/STZ-treated ApoE KO mice, with or without luseogliflozin. Short-term luseogliflozin treatment normalized the expression of inflammation-related genes such as F4/80, TNFα, IL-1ß, IL-6, ICAM-1, PECAM-1, MMP2 and MMP9 in the NA/STZ-treated ApoE KO mice, which showed marked elevations as compared with untreated ApoE KO mice. In contrast, lipid metabolism-related genes were generally unaffected by luseogliflozin treatment. Furthermore, after six-month treatment with luseogliflozin, in contrast to the severe and widely distributed atherosclerotic changes in the aortas of NA/STZ-treated ApoE KO mice, luseogliflozin treatment markedly attenuated the progression of atherosclerosis, without affecting serum lipid parameters such as high density lipoprotein, low density lipoprotein and triglyceride levels. Given that luseogliflozin normalized the aortic mRNA levels of inflammation-related, but not lipid-related, genes soon after the initiation of treatment, it is not unreasonable to speculate that the anti-atherosclerotic effect of this SGLT2 inhibitor emerges rapidly, possibly via the prevention of inflammation rather than of hyperlipidemia.

Prolyl isomerase Pin1 negatively regulates AMP-activated protein kinase (AMPK) by associating with the CBS domain in the γ subunit.

AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the ß subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr(211)-Pro-containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, whereas Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1, and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPKα subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes, such as PGC-1α, which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation in the muscles of Pin1 KO mice as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK.