Taichi Sugizaki

Lowering Bile Acid Pool Size with a Synthetic Farnesoid X Receptor (FXR) Agonist Induces Obesity and Diabetes through Reduced Energy Expenditure

We evaluated the metabolic impact of farnesoid X receptor (FXR) activation by administering a synthetic FXR agonist (GW4064) to mice in which obesity was induced by a high fat diet. Administration of GW4064 accentuated body weight gain and glucose intolerance induced by the high fat diet and led to a pronounced worsening of the changes in liver and adipose tissue. Mechanistically, treatment with GW4064 decreased bile acid (BA) biosynthesis, BA pool size, and energy expenditure, whereas reconstitution of the BA pool in these GW4064-treated animals by BA administration dose-dependently reverted the metabolic abnormalities. Our data therefore suggest that activation of FXR with synthetic agonists is not useful for long term management of the metabolic syndrome, as it reduces the BA pool size and subsequently decreases energy expenditure, translating as weight gain and insulin resistance. In contrast, expansion of the BA pool size, which can be achieved by BA administration, could be an interesting strategy to manage the metabolic syndrome.

Bile Acid Binding Resin Improves Metabolic Control through the Induction of Energy Expenditure

Background Besides well-established roles of bile acids (BA) in dietary lipid absorption and cholesterol homeostasis, it has recently become clear that BA is also a biological signaling molecule. We have shown that strategies aimed at activating TGR5 by increasing the BA pool size with BA administration may constitute a significant therapeutic advance to combat the metabolic syndrome and suggest that such strategies are worth testing in a clinical setting. Bile acid binding resin (BABR) is known not only to reduce serum cholesterol levels but also to improve glucose tolerance and insulin resistance in animal models and humans. However, the mechanisms by which BABR affects glucose homeostasis have not been established. We investigated how BABR affects glycemic control in diet-induced obesity models. Methods and Findings We evaluated the metabolic effect of BABR by administrating colestimide to animal models for the metabolic syndrome. Administration of BABR increased energy expenditure, translating into significant weight reduction and insulin sensitization. The metabolic effects of BABR coincide with activation of cholesterol and BA synthesis in liver and thermogenesis in brown adipose tissue. Interestingly, these effects of BABR occur despite normal food intake and triglyceride absorption. Administration of BABR and BA had similar effects on BA composition and thermogenesis, suggesting that they both are mediated via TGR5 activation. Conclusion Our data hence suggest that BABR could be useful for the management of the impaired glucose tolerance of the metabolic syndrome, since they not only lower cholesterol levels, but also reduce obesity and improve insulin resistance.

Angiopoietin-like protein 2 increases renal fibrosis by accelerating transforming growth factor-β signaling in chronic kidney disease

Renal fibrosis is a common pathological consequence of chronic kidney disease (CKD) with tissue fibrosis closely associated with chronic inflammation in numerous pathologies. However, molecular mechanisms underlying that association, particularly in the kidney, remain unclear. Here, we determine whether there is a molecular link between chronic inflammation and tissue fibrosis in CKD progression. Histological analysis of human kidneys indicated abundant expression of angiopoietin-like protein 2 (ANGPTL2) in renal tubule epithelial cells during progression of renal fibrosis. Numerous ANGPTL2-positive renal tubule epithelial cells colocalized with cells positive for transforming growth factor (TGF)-β1, a critical mediator of tissue fibrosis. Analysis of M1 collecting duct cells in culture showed that TGF-β1 increases ANGPTL2 expression by attenuating its repression through microRNA-221. Conversely, ANGPTL2 increased TGF-β1 expression through α5β1 integrin-mediated activation of extracellular signal-regulated kinase. Furthermore, ANGPTL2 deficiency in a mouse unilateral ureteral obstruction model significantly reduced renal fibrosis by decreasing TGF-β1 signal amplification in kidney. Thus, ANGPTL2 and TGF-β1 positively regulate each other as renal fibrosis progresses. Our study provides insight into molecular mechanisms underlying chronic inflammation and tissue fibrosis and identifies potential therapeutic targets for CKD treatment.

The Niemann-Pick C1 Like 1 (NPC1L1) Inhibitor Ezetimibe Improves Metabolic Disease Via Decreased Liver X Receptor (LXR) Activity in Liver of Obese Male Mice

Dyslipidemic patients with diabetes mellitus, including metabolic syndrome, are at increased risk of coronary heart disease. It has been reported that ezetimibe, a cholesterol absorption inhibitor, improves metabolic diseases in mice and humans. However, the underlying mechanism has been unclear. Here we explored the effects of ezetimibe on lipid and glucose homeostasis. Male KK-A(y) mice were fed a high-fat diet, which is the mouse model of metabolic syndrome, with or without ezetimibe for 14 weeks. Ezetimibe improved dyslipidemia, steatosis, and insulin resistance. Ezetimibe decreased hepatic oxysterols, which are endogenous agonists of liver X receptor (LXR), to decrease hepatic lipogenic gene expressions, especially in stearoyl-CoA desaturase-1 (SCD1), leading to a remarkable reduction of hepatic oleate content that would contribute to the improvement of steatosis by reducing triglycerides and cholesterol esters. Simultaneously, hepatic β-oxidation, NADPH oxidase and cytochrome P450 2E1 (CYP2E1) were reduced, and thus reactive oxygen species (ROS) and inflammatory cytokines were also decreased. Consistent with these changes, ezetimibe diminished c-Jun N-terminal kinase (JNK) phosphorylation and improved insulin signaling in the liver. In vitro study using primary hepatocytes obtained from male SD rats, treated with oleate and LXR agonist, showed excess lipid accumulation, increased oxidative stress and impaired insulin signaling. Therefore, in obese subjects, ezetimibe reduces hepatic LXR activity by reducing hepatic oxysterols to lower hepatic oleate content. This improves steatosis and reduces oxidative stress, and this reduction improves insulin signaling in the liver. These results provide insight into pathogenesis and strategies for treatment of the metabolic syndrome.

ANGPTL2 activity in cardiac pathologies accelerates heart failure by perturbing cardiac function and energy metabolism

A cardioprotective response that alters ventricular contractility or promotes cardiomyocyte enlargement occurs with increased workload in conditions such as hypertension. When that response is excessive, pathological cardiac remodelling occurs, which can progress to heart failure, a leading cause of death worldwide. Mechanisms underlying this response are not fully understood. Here, we report that expression of angiopoietin-like protein 2 (ANGPTL2) increases in pathologically-remodeled hearts of mice and humans, while decreased cardiac ANGPTL2 expression occurs in physiological cardiac remodelling induced by endurance training in mice. Mice overexpressing ANGPTL2 in heart show cardiac dysfunction caused by both inactivation of AKT and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a signalling and decreased myocardial energy metabolism. Conversely, Angptl2 knockout mice exhibit increased left ventricular contractility and upregulated AKT-SERCA2a signalling and energy metabolism. Finally, ANGPTL2-knockdown in mice subjected to pressure overload ameliorates cardiac dysfunction. Overall, these studies suggest that therapeutic ANGPTL2 suppression could antagonize development of heart failure.

Mice Deficient in Angiopoietin-like Protein 2 (Angptl2) Gene Show Increased Susceptibility to Bacterial Infection Due to Attenuated Macrophage Activity

Macrophages play crucial roles in combatting infectious disease by promoting inflammation and phagocytosis. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor that induces tissue inflammation by attracting and activating macrophages to produce inflammatory cytokines in chronic inflammation-associated diseases such as obesity-associated metabolic syndrome, atherosclerosis, and rheumatoid arthritis. Here, we asked whether and how ANGPTL2 activates macrophages in the innate immune response. ANGPTL2 was predominantly expressed in proinflammatory mouse bone marrow-derived differentiated macrophages (GM-BMMs) following GM-CSF treatment relative to anti-inflammatory cells (M-BMMs) established by M-CSF treatment. Expression of the proinflammatory markers IL-1β, IL-12p35, and IL-12p40 significantly decreased in GM-BMMs from Angptl2-deficient compared with wild-type (WT) mice, suggestive of attenuated proinflammatory activity. We also report that ANGPTL2 inflammatory signaling is transduced through integrin α5β1 rather than through paired immunoglobulin-like receptor B. Interestingly, Angptl2-deficient mice were more susceptible to infection with Salmonella enterica serovar Typhimurium than were WT mice. Moreover, nitric oxide (NO) production by Angptl2-deficient GM-BMMs was significantly lower than in WT GM-BMMs. Collectively, our findings suggest that macrophage-derived ANGPTL2 promotes an innate immune response in those cells by enhancing proinflammatory activity and NO production required to fight infection.

Bile acid binding resin improves hepatic insulin sensitivity by reducing cholesterol but not triglyceride levels in the liver

AIMS Bile acid binding resin (BAR) improves glycaemic control in patients with type 2 diabetes. Although the mechanism is hypothesised to involve the clearance of excess hepatic triglyceride, this hypothesis has not been examined in appropriately designed studies. Therefore, we investigated whether reduced hepatic triglyceride deposition is involved in BAR-mediated improvements in glycaemic control in spontaneous fatty liver diabetic mice without dietary interventions. METHODS Male 6-week-old fatty liver Shionogi (FLS) mice were fed a standard diet without or with 1.5% BAR (colestilan) for 6 weeks. Glucose tolerance, insulin sensitivity, hepatic lipid content, and gene expression were assessed. A liver X receptor (LXR) agonist was also administered to activate the LXR pathway. We also retrospectively analysed the medical records of 21 outpatients with type 2 diabetes who were treated with colestilan for ≥6 months. RESULTS BAR enhanced glucose tolerance and insulin sensitivity in FLS mice without altering fat mass. BAR improved hepatic insulin sensitivity, increased IRS2 expression, and decreased SREBP expression. BAR reduced hepatic cholesterol levels but not hepatic triglyceride levels. BAR also reduced the expression of LXR target genes, and LXR activation abolished the BAR-mediated improvements in glycaemic control. Colestilan significantly lowered serum cholesterol levels and improved glycaemic control in patients with type 2 diabetes. CONCLUSIONS BAR improved hepatic insulin resistance in FLS mice by reducing hepatic cholesterol without affecting hepatic triglyceride levels or body fat distribution. Our study revealed that BAR improves glycaemic control at least in part by downregulating the hepatic cholesterol-LXR-IRS2 pathway.

Bile acid binding resin prevents fat accumulation through intestinal microbiota in high-fat diet-induced obesity in mice

BACKGROUND Bile acid binding resin (BAR) absorbs intestinal bile acids, and improves obesity and metabolic disorders, but the precise mechanism remains to be clarified. Recent findings reveal that obesity is associated with skewed intestinal microbiota. Thus, we investigated the effect of BAR on intestinal microbiota and the role of microbiota in the prevention of obesity in high-fat diet-induced obesity in mice. PROCEDURES Male Balb/c mice were fed a low-fat diet (LFD), high-fat diet (HFD), or HFD with BAR (HFD+BAR), and then metabolic parameters, caecal microbiota, and metabolites were investigated. The same interventions were conducted in germ-free and antibiotic-treated mice. MAIN FINDINGS The frequency of Clostridium leptum subgroup was higher in both HFD-fed and HFD+BAR-fed mice than in LFD-fed mice. The frequency of Bacteroides-Prevotella group was lower in HFD-fed mice than in LFD-fed mice, but the frequency was higher in HFD+BAR-fed mice than in HFD-fed mice. Caecal propionate was lower in HFD-fed mice than in LFD-fed mice, and higher in HFD+BAR-fed mice than in HFD-fed mice. HFD+BAR-fed mice showed lower adiposity than HFD-fed mice, and the reduction was not observed in germ-free or antibiotic-treated mice. Colonized germ-free mice showed a reduction in adiposity by BAR administration. Energy expenditure was lower in HFD-fed mice and higher in HFD+BAR-fed mice, but the increments induced by administration of BAR were not observed in antibiotic-treated mice. CONCLUSIONS Modulation of intestinal microbiota by BAR could be a novel therapeutic approach for obesity.

Intestinal Bile Acid Composition Modulates Prohormone Convertase 1/3 (PC1/3) Expression and Consequent GLP-1 Production in Male Mice

Besides an established medication for hypercholesterolemia, bile acid binding resins (BABRs) present antidiabetic effects. Although the mechanisms underlying these effects are still enigmatic, glucagon-like peptide-1 (GLP-1) appears to be involved. In addition to a few reported mechanisms, we propose prohormone convertase 1/3 (PC1/3), an essential enzyme of GLP-1 production, as a potent molecule in the GLP-1 release induced by BABRs. In our study, the BABR colestimide leads to a bile acid-specific G protein-coupled receptor TGR5-dependent induction of PC1/3 gene expression. Here, we focused on the alteration of intestinal bile acid composition and consequent increase of total TGR5 agonistic activity to explain the TGR5 activation. Furthermore, we demonstrate that nuclear factor of activated T cells mediates the TGR5-triggered PC1/3 gene expression. Altogether, our data indicate that the TGR5-dependent intestinal PC1/3 gene expression supports the BABR-stimulated GLP-1 release. We also propose a combination of BABR and dipeptidyl peptidase-4 inhibitor in the context of GLP-1-based antidiabetic therapy.

Interstitial pneumonia induced by bleomycin treatment is exacerbated in Angptl2-deficient mice

Angiopoietin-like protein 2 (ANGPTL2) is a chronic inflammatory mediator that, when deregulated, is associated with various pathologies. However, little is known about its activity in lung. To assess a possible lung function, we generated a rabbit monoclonal antibody that specifically recognizes mouse ANGPTL2 and then evaluated protein expression in mouse lung tissue. We observed abundant ANGPTL2 expression in both alveolar epithelial type I and type II cells and in resident alveolar macrophages under normal conditions. To assess ANGPTL2 function, we compared lung phenotypes in Angptl2 knockout (KO) and wild-type mice but observed no overt changes. We then generated a bleomycin-induced interstitial pneumonia model using wild-type and Angptl2 KO mice. Bleomycin-treated wild-type mice showed specifically upregulated ANGPTL2 expression in areas of severe fibrosing interstitial pneumonia, while Angptl2 KO mice developed more severe lung fibrosis than did comparably treated wild-type mice. Lung fibrosis seen following bone marrow transplant was comparable in wild-type or Angptl2 KO mice treated with bleomycin, suggesting that Angptl2 loss in myeloid cells does not underlie fibrotic phenotypes. We conclude that Angptl2 deficiency in lung epithelial cells and resident alveolar macrophages causes severe lung fibrosis seen following bleomycin treatment, suggesting that ANGPTL2 derived from these cell types plays a protective role against fibrosis in lung.