Single-Nucleus Analysis Reveals Tumor Heterogeneity of Aldosterone-Producing Adenoma.

BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells. Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. METHODS: We conducted single-nucleus RNA sequencing of APAs with KCNJ5 mutation and nonfunctional adenomas obtained from 3 and 2 patients, respectively. RESULTS: The single-nucleus RNA sequencing revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of nonfunctional adenoma and APA. In addition, we extracted 2 cell fates in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in 1 of these fates, whereas those related to the regulation of glycolysis were upregulated in the other fate. Furthermore, the total RNA reads in the nucleus were higher in hormonally activated clusters, indicating a marked activation of transcription per cell. CONCLUSIONS: The single-nucleus RNA sequencing revealed intratumoral heterogeneity of APA with KCNJ5 mutation. The observation of 2 cell fates in KCNJ5-mutated APAs provides the postulation that a heterogeneous process of cellular differentiation was implicated in the pathophysiological mechanisms underlying APA tumors.

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.

Long-term effects of primary aldosteronism treatment on patients with primary aldosteronism and chronic kidney disease.

OBJECTIVE: Primary aldosteronism (PA) is a major cause of secondary hypertension and is associated with chronic renal injury. The glomerular filtration rate (GFR) in PA rapidly decreases after the removal of glomerular hyperfiltration due to aldosterone excess by adrenalectomy (ADX) or mineralocorticoid receptor antagonist (MRA) treatment and is stable in the long term. However, the effects of these treatments on the long-term renal function of PA patients with chronic kidney disease (CKD) is not well understood. DESIGN AND PATIENTS: In this single-center, retrospective study, acute and chronic changes in the estimated GFR (eGFR) were examined in 107 patients with PA, including 49 patients with post-treatment CKD　defined as eGFR < 60 ml/min/1.73 m2 . RESULTS: The reduction in eGFR observed 1 month after ADX in the CKD group (N = 31) was -20.1 ± 8.2 ml/min/1.73 m2 . Multivariate analysis showed that pre-treatment eGFR and plasma aldosterone concentration were independent predictive factors of the acute reduction in eGFR after ADX. The reduction of eGFR observed 1 month after MRA administration in the post-treatment CKD group (N = 18) was -9.2 ± 5.9 ml/min/1.73 m2 . Multivariate analysis showed that the duration of hypertension and pre-treatment eGFR were independent predictive factors of the acute reduction in eGFR after ADX administration. In 20 patients with CKD (N = 12 ADX and N = 8 MRA) followed for more than 5 years post-treatment, there was no further significant decline in eGFR over a follow-up period of 7 (6, 8) years nor any difference between the two treatment modalities. CONCLUSIONS: Our study suggests that treatment of PA in stage 3 CKD is safe and useful in preventing renal injury.

Heterogeneous circulating miRNA profiles of PBMAH.

Objective: Primary 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). Methods: miRNA 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. Results: PBMAH 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. Conclusion: Circulating miRNA signature was identified specific for PBMAH. The existence of subtype-based miRNA profiles may be associated with the pathophysiological heterogeneity of PBMAH.

A case of ezetimibe-effective hypercholesterolemia with a novel heterozygous variant in ABCG5.

Sitosterolemia is caused by homozygous or compound heterozygous gene mutations in either ATP-binding cassette subfamily G member 5 (ABCG5) or 8 (ABCG8). Since ABCG5 and ABCG8 play pivotal roles in the excretion of neutral sterols into feces and bile, patients with sitosterolemia present elevated levels of serum plant sterols and in some cases also hypercholesterolemia. A 48-year-old woman was referred to our hospital for hypercholesterolemia. She had been misdiagnosed with familial hypercholesterolemia at the age of 20 and her serum low-density lipoprotein cholesterol (LDL-C) levels had remained about 200-300 mg/dL at the former clinic. Although the treatment of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors was ineffective, her serum LDL-C levels were normalized by ezetimibe, a cholesterol transporter inhibitor. We noticed that her serum sitosterol and campesterol levels were relatively high. Targeted analysis sequencing identified a novel heterozygous ABCG5 variant (c.203A>T; p.Ile68Asn) in the patient, whereas no mutations were found in low-density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9), or Niemann-Pick C1-like intracellular cholesterol transporter 1 (NPC1L1). While sitosterolemia is a rare disease, a recent study has reported that the incidence of loss-of-function mutation in the ABCG5 or ABCG8 gene is higher than we thought at 1 in 220 individuals. The present case suggests that serum plant sterol levels should be examined and ezetimibe treatment should be considered in patients with hypercholesterolemia who are resistant to HMG-CoA reductase inhibitors.

A reduced M1-like/M2-like ratio of macrophages in healthy adipose tissue expansion during SGLT2 inhibition.

The adipose tissue includes various stromal cells, such as preadipocytes, endothelial cells, fibroblasts, and immune cells, which are involved in adipose tissue functions. We previously reported that, in obese mice, the sodium-glucose cotransporter 2 inhibitor ipragliflozin (Ipra) promoted the expansion of the epididymal adipose tissue (Epi) with increase of serum ketone body concentration. The Ipra-induced adipose tissue expansion did not deteriorate adipose inflammation, or systemic glucose/lipid metabolism, referred to as "healthy adipose tissue expansion." Here we found that Ipra promoted healthy adipose tissue expansion with a reduced ratio of pro-inflammatory M1-like adipose tissue macrophages (ATMs) to anti-inflammatory M2-like ATMs. Ipra downregulated the gene expression of interleukin (IL)-15 (Il15) in stromal cells of Epi. IL-15 inhibited lipogenesis in 3T3-L1 cells associated with downregulation of the lipogenic gene. Ketone body ß-hydroxybutyrate suppressed Il15 gene induction in M1-polarized cultured macrophages, and a ketogenic diet reproduced the adipose tissue expansion without deteriorating systemic glucose metabolism in mice. Our data indicate that the phenotypic switch of ATMs could mediate healthy adipose tissue expansion by treatment with Ipra, and it may offer new insights into the pathophysiological mechanisms of adipose tissue expansion.

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

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".

Roles for Cell-Cell Adhesion and Contact in Obesity-Induced Hepatic Myeloid Cell Accumulation and Glucose Intolerance.

Obesity promotes infiltration of inflammatory cells into various tissues, leading to parenchymal and stromal cell interaction and development of cellular and organ dysfunction. Liver sinusoidal endothelial cells (LSECs) are the first cells that contact portal blood cells and substances in the liver, but their functions in the development of obesity-associated glucose metabolism remain unclear. Here, we find that LSECs are involved in obesity-associated accumulation of myeloid cells via VLA-4-dependent cell-cell adhesion. VLA-4 blockade in mice fed a high-fat diet attenuated myeloid cell accumulation in the liver to improve hepatic inflammation and systemic glucose intolerance. Ex vivo studies further show that cell-cell contact between intrahepatic leukocytes and parenchymal hepatocytes induces gluconeogenesis via a Notch-dependent pathway. These findings suggest that cell-cell interaction between parenchymal and stromal cells regulates hepatic glucose metabolism and offers potential strategies for treatment or prevention of obesity-associated glucose intolerance.

Antifibrotic effect of pirfenidone in a mouse model of human nonalcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis with lobular inflammation and hepatocyte injury. Pirfenidone (PFD) is an orally bioavailable pyridone derivative that has been clinically used for the treatment of idiopathic pulmonary fibrosis. However, it remains unknown whether PFD improves liver fibrosis in a mouse model with human NASH-like phenotypes. In this study, we employed melanocortin 4 receptor-deficient (MC4R-KO) mice as a mouse model with human NASH-like phenotypes to elucidate the effect and action mechanisms of PFD on the development of NASH. PFD markedly attenuated liver fibrosis in western diet (WD)-fed MC4R-KO mice without affecting metabolic profiles or steatosis. PFD prevented liver injury and fibrosis associated with decreased apoptosis of liver cells in WD-fed MC4R-KO mice. Pretreatment of PFD inhibited the tumor necrosis factor-α (TNF-α)-induced liver injury and fibrogenic responses associated with decreased apoptosis of liver cells in wild-type mice. PFD also prevented TNF-α-induced hepatocyte apoptosis in vitro with reduced activation of caspase-8 and -3. This study provides evidence for the antifibrotic effect of PFD in a mouse model of human NASH. The data of this study highlight hepatocyte apoptosis as a potential therapeutic target, and suggest that PFD can be repositioned as an antifibrotic drug for human NASH.

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.

Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue fibrosis.

In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.