Heterogeneous circulating miRNA profiles of PBMAH

ObjectivePrimary bilateral macronodular adrenal hyperplasia (PBMAH), a rare cause of Cushing syndrome, is often diagnosed as a bilateral adrenal incidentaloma with subclinical cortisol production. Circulating microRNAs (miRNAs) are a characteristic of adrenocortical adenomas, but miRNA expression in PBMAH has not been investigated. We aimed to evaluate the circulating miRNA expression in patients with PBMAH and compare them with those in patients with non-functioning adrenocortical adenoma (NFA) and cortisol-producing adrenocortical adenoma (CPA).MethodsmiRNA profiling of plasma samples from four, five, and five patients with NFA, CPA, and PBMAH, respectively, was performed. Selected miRNA expressions were validated using quantitative RT-PCR.ResultsPBMAH samples showed distinct miRNA expression signatures on hierarchical clustering while NFA and CPA samples were separately clustered. PBMAH was distinguished from the adenoma group of NFA and CPA by 135 differentially expressed miRNAs. Hsa-miR-1180-3p, hsa-miR-4732-5p, and hsa-let-7b-5p were differentially expressed between PBMAH and adenoma (P = 0.019, 0.006, and 0.003, respectively). Furthermore, PBMAH could be classified into two subtypes based on miRNA profiling: subtype 1 with a similar profile to those of adenoma and subtype 2 with a distinct profile. Hsa-miR-631, hsa-miR-513b-5p, hsa-miR-6805-5p, and hsa-miR-548av-5p/548k were differentially expressed between PBMAH subtype 2 and adenoma (P = 0.027, 0.027, 0.027, and 1.53E-04, respectively), but not between PBMAH, as a whole, and adenoma.ConclusionCirculating miRNA signature was identified specific for PBMAH. The existence of subtype-based miRNA profiles may be associated with the pathophysiological heterogeneity of PBMAH

Ipragliflozin Improves Hepatic Steatosis in Obese Mice and Liver Dysfunction in Type 2 Diabetic Patients Irrespective of Body Weight Reduction.

Type 2 diabetes mellitus (T2DM) is associated with a high incidence of non-alcoholic fatty liver disease (NAFLD) related to obesity and insulin resistance. Currently, medical interventions for NAFLD have focused on diet control and exercise to reduce body weight, and there is a requirement for effective pharmacological therapies. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote the urinary excretion of glucose by blocking its reabsorption in renal proximal tubules. SGLT2 inhibitors lower blood glucose independent of insulin action and are expected to reduce body weight because of urinary calorie loss. Here we show that an SGLT2 inhibitor ipragliflozin improves hepatic steatosis in high-fat diet-induced and leptin-deficient (ob/ob) obese mice irrespective of body weight reduction. In the obese mice, ipragliflozin-induced hyperphagia occurred to increase energy intake, attenuating body weight reduction with increased epididymal fat mass. There is an inverse correlation between weights of liver and epididymal fat in ipragliflozin-treated obese mice, suggesting that ipragliflozin treatment promotes normotopic fat accumulation in the epididymal fat and prevents ectopic fat accumulation in the liver. Despite increased adiposity, ipragliflozin ameliorates obesity-associated inflammation and insulin resistance in epididymal fat. Clinically, ipragliflozin improves liver dysfunction in patients with T2DM irrespective of body weight reduction. These findings provide new insight into the effects of SGLT2 inhibitors on energy homeostasis and fat accumulation and indicate their potential therapeutic efficacy in T2DM-associated hepatic steatosis

Increased serum extracellular vesicle miR-144-3p and miR-486a-3p in a mouse model of adipose tissue regeneration promote hepatocyte proliferation by targeting Txnip.

Adipose-derived stem cells are expected to be applied to regenerative medicine for various incurable diseases including liver cirrhosis. Although microRNAs contained in extracellular vesicles (EV-miRNAs) have been implicated in their regenerative effects, the precise mechanism has not been fully elucidated. Tamoxifen-inducible adipocyte-specific insulin receptor knockout (iFIRKO) mice are known to exhibit acute adipose tissue regeneration with increased numbers of adipose stem and progenitor cells (ASPCs). Because adipose tissue is the major source of circulating EV-miRNAs, we investigated alterations in serum EV-miRNAs in iFIRKO mice. A comprehensive analysis using miRNA sequencing on serum EVs revealed that most EV-miRNAs were decreased due to the loss of mature adipocytes, but there were 19 EV-miRNAs that were increased in the serum of iFIRKO mice. Among them, miR-144-3p and miR-486a-3p were found to be increased in the liver as well as serum EVs. While the expression levels of pri-miR-144-3p and pri-miR-486a-3p were not increased in the liver, they were elevated in the adipose tissue, suggesting that these miRNAs may be delivered from ASPCs increased in the adipose tissue to the liver via EVs. Increased hepatocyte proliferation was observed in the liver of iFIRKO mice, and we found that both miR-144-3p and miR-486a-3p have a function to promote hepatocyte proliferation by suppressing Txnip expression as a target gene. miR-144-3p and miR-486a-3p can be candidate therapeutic tools for conditions requiring hepatocyte proliferation, such as liver cirrhosis, and our current study suggests that examining EV-miRNAs secreted in vivo may lead to the discovery of miRNAs involved in regenerative medicine that have not been identified by in vitro analysis

Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH

Abstract Sodium glucose cotransporter 2 (SGLT2) inhibitors, an antidiabetic drug, promotes urinary excretion of glucose by blocking its reabsorption in the renal proximal tubules. It is unclear whether SGLT2 inhibition could attenuate nonalcoholic steatohepatitis (NASH) and NASH-associated hepatocellular carcinoma. We examined the preventive effects of an SGLT2 inhibitor canagliflozin (CANA) in Western diet (WD)-fed melanocortin 4 receptor-deficient (MC4R-KO) mice, a mouse model of human NASH. An eight-week CANA treatment attenuated hepatic steatosis in WD-fed MC4R-KO mice, with increased epididymal fat mass without inflammatory changes. CANA treatment for 20 weeks inhibited the development of hepatic fibrosis in WD-fed MC4R-KO mice. After one year of CANA treatment, the number of liver tumors was significantly reduced in WD-fed MC4R-KO mice. In adipose tissue, CANA suppressed the ratio of oxidative to reduced forms of glutathiones (GSSG/GSH) in WD-fed MC4R-KO mice. Treatment with GSH significantly attenuated the H2O2-induced upregulation of genes related to NADPH oxidase in 3T3-L1 adipocytes, and that of Il6, Tgfb, and Pdgfb in RAW264.7 cells. This study provides evidence that SGLT2 inhibitors represent the unique class of drugs that can attenuate or delay the onset of NASH and eventually hepatocellular carcinoma, at least partly, through “healthy adipose expansion”

Metabolic characterization and hepatic steatosis of HFD-fed WT mice treated with or without ipragliflozin.

<p>Eight-week-old WT mice were fed a SD or a HFD for 16 weeks. Mice were given with the vehicle or 10mg/kg of ipragliflozin during last 4 weeks. (A) Blood glucose during ipragliflozin treatment. (B) Urine glucose concentration after 3 weeks of ipragliflozin treatment. (C) Food intake and (D) body weight during ipragliflozin treatment. Weights of the (E) liver and (F) epididymal fat after 4 weeks of ipragliflozin treatment. (G) Correlation of the liver weight with the epididymal fat weight in ipragliflozin-treated mice. (H) Hematoxylin and eosin (HE) staining and (I) triglyceride (TG) content of the liver. (J) Serum ALT levels. Veh, vehicle; Ipra, ipragliflozin. Original magnification, × 200. Scale bars, 100 μm. # <i>p</i> < 0.05, ## <i>p</i> < 0.01 vs SD/Veh; * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs HFD/Veh. <i>n</i> = 6–8.</p

Effect of ipragliflozin on liver gene expression in HFD-fed WT mice.

<p>Gene expression levels in the liver associated with (A) lipogenesis, β-oxidation, gluconeogenesis, and (B) inflammation after 4 weeks of the vehicle or ipragliflozin treatment. # <i>p</i> < 0.05, ## <i>p</i> < 0.01 vs SD/Veh; * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs HFD/Veh. <i>n</i> = 6–8.</p

Effect of ipragliflozin on insulin signaling in HFD-fed WT mice.

<p>Representative immunoblots and quantification of phosphorylated Akt (S473) in the (A) liver, (B) epididymal fat, and (C) skeletal muscle after 4 weeks of the vehicle or ipragliflozin treatment. After 16 h of fasting, mice were injected with insulin (5U/kg) or phosphate buffered saline to portal vein, and the liver was collected 3 minutes later. Ins, insulin. # <i>p</i> < 0.05, ## <i>p</i> < 0.01 vs SD/Veh; * <i>p</i> < 0.05 vs HFD/Veh. <i>n</i> = 2–6.</p

Metabolic parameters of SD- or HFD-fed WT mice treated with or without ipragliflozin for 4 weeks.

<p>Metabolic parameters of SD- or HFD-fed WT mice treated with or without ipragliflozin for 4 weeks.</p