Kyoichiro Tsuchiya

FoxOs integrate pleiotropic actions of insulin in vascular endothelium to protect mice from atherosclerosis.

Atherosclerotic cardiovascular disease is the leading cause of death in insulin-resistant (type 2) diabetes. Vascular endothelial dysfunction paves the way for atherosclerosis through impaired nitric oxide availability, inflammation, and generation of superoxide. Surprisingly, we show that ablation of the three genes encoding isoforms of transcription factor FoxO in endothelial cells prevents atherosclerosis in low-density lipoprotein receptor knockout mice by reversing these subphenotypes. Paradoxically, the atheroprotective effect of FoxO deletion is associated with a marked decrease of insulin-dependent Akt phosphorylation in endothelial cells, owing to reduced FoxO-dependent expression of the insulin receptor adaptor proteins Irs1 and Irs2. These findings support a model in which FoxO is the shared effector of multiple atherogenic pathways in endothelial cells. FoxO ablation lowers the threshold of Akt activity required for protection from atherosclerosis. The data demonstrate that FoxO inhibition in endothelial cells has the potential to mediate wide-ranging therapeutic benefits for diabetes-associated cardiovascular disease.

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.

FOXO1 inhibition yields functional insulin-producing cells in human gut organoid cultures

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors, we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive, insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells, we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded shRNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells, release C-peptide in response to secretagogues, and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes.

Expanded Granulocyte/Monocyte Compartment in Myeloid-Specific Triple Foxo Knockout Increases Oxidative Stress and Accelerates Atherosclerosis in Mice

Rationale: Increased neutrophil and monocyte counts are often associated with an increased risk of atherosclerosis, but their relationship to insulin sensitivity is unknown. Objective: To investigate the contribution of forkhead transcription factors (FoxO) in myeloid cells to neutrophil and monocyte counts, atherosclerosis, and systemic insulin sensitivity. Methods and Results: Genetic ablation of the 3 genes encoding FoxO isoforms 1, 3a, and 4, in myeloid cells resulted in an expansion of the granulocyte/monocyte progenitor compartment and was associated with increased atherosclerotic lesion formation in low-density lipoprotein receptor knockout mice. In vivo and ex vivo studies indicate that FoxO ablation in myeloid cells increased generation of reactive oxygen species. Accordingly, treatment with the antioxidant N-acetyl-L-cysteine reversed the phenotype, normalizing atherosclerosis. Conclusions: Our data indicate that myeloid cell proliferation and oxidative stress can be modulated via the FoxO branch of insulin receptor signaling, highlighting a heretofore-unknown link between insulin sensitivity and leukocytosis that can affect the predisposition to atherosclerosis.

Increased atherosclerosis and endothelial dysfunction in mice bearing constitutively deacetylated alleles of Foxo1 gene.

Background: Transcription factor FoxO1 is deacetylated in response to oxidative stress and hyperglycemia in diabetes. Results: Mice bearing constitutively deacetylated alleles of Foxo1 develop larger atherosclerotic lesions despite improved plasma lipid levels in a bone marrow transplantation-independent manner. Conclusion: FoxO1 deacetylation predisposes to atherosclerosis and vascular endothelial dysfunction. Significance: The data identify a mechanism whereby oxidative stress, acting through FoxO1 deacetylation, promotes atherosclerosis in diabetic patients. Complications of atherosclerosis are the leading cause of death of patients with type 2 (insulin-resistant) diabetes. Understanding the mechanisms by which insulin resistance and hyperglycemia contribute to atherogenesis in key target tissues (liver, vessel wall, hematopoietic cells) can assist in the design of therapeutic approaches. We have shown that hyperglycemia induces FoxO1 deacetylation and that targeted knock-in of alleles encoding constitutively deacetylated FoxO1 in mice (Foxo1KR/KR) improves hepatic lipid metabolism and decreases macrophage inflammation, setting the stage for a potential anti-atherogenic effect of this mutation. Surprisingly, we report here that when Foxo1KR/KR mice are intercrossed with low density lipoprotein receptor knock-out mice (Ldlr−/−), they develop larger aortic root atherosclerotic lesions than Ldlr−/− controls despite lower plasma cholesterol and triglyceride levels. The phenotype is unaffected by transplanting bone marrow from Ldlr−/− mice into Foxo1KR/KR mice, indicating that it is independent of hematopoietic cells and suggesting that the primary lesion in Foxo1KR/KR mice occurs in the vessel wall. Experiments in isolated endothelial cells from Foxo1KR/KR mice indicate that deacetylation favors FoxO1 nuclear accumulation and exerts target gene-specific effects, resulting in higher Icam1 and Tnfα expression and increased monocyte adhesion. The data indicate that FoxO1 deacetylation can promote vascular endothelial changes conducive to atherosclerotic plaque formation.

Improvement of endothelial function in patients with hypertension and type 2 diabetes after treatment with telmisartan

Telmisartan, a selective antagonist for angiotensin type1 receptor and a partial agonist for peroxisome proliferator-activated receptor-γ, decreases blood pressure and has been shown to improve glucose and lipid metabolism, suggesting potential cardiovascular protective effects. In this study, we investigated whether long-term treatment with telmisartan improved endothelial function in 35 hypertensive patients with type 2 diabetes mellitus (T2DM). Office and home early morning blood pressure levels and flow-mediated vasodilation (FMD) were evaluated before and after 12 months of treatment with telmisartan. Blood samples were also obtained for measurement of several biochemical parameters and of adiponectin (AN) and highly sensitive C-reactive protein (hs-CRP) before and after treatment. After 12 months of treatment, office and morning blood pressure levels had significantly decreased, and levels of plasma glucose, glycosylated hemoglobin, total cholesterol, triglyceride and low-density lipoprotein cholesterol had also significantly decreased. Plasma AN and high-density lipoprotein cholesterol levels increased, but hs-CRP levels decreased. Furthermore, FMD significantly increased; changes in percent FMD showed a significant negative correlation with changes in systolic and diastolic blood pressure and a significant positive correlation with changes in AN. Stepwise multivariate regression analysis revealed that changes in plasma AN and office systolic blood pressure were both independent determinants for endothelial function after telmisartan treatment. In conclusion, this study shows that long-term treatment with telmisartan improves not only blood pressure and glucose and lipid metabolism but also endothelial function in hypertensive patients with T2DM, possibly by increased circulating AN and decreased blood pressure.

Deficiency of ATP-Binding Cassette Transporters A1 and G1 in Endothelial Cells Accelerates Atherosclerosis in Mice

Objective— Plasma high-density lipoproteins have several putative antiatherogenic effects, including preservation of endothelial functions. This is thought to be mediated, in part, by the ability of high-density lipoproteins to promote cholesterol efflux from endothelial cells (ECs). The ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) interact with high-density lipoproteins to promote cholesterol efflux from ECs. To determine the impact of endothelial cholesterol efflux pathways on atherogenesis, we prepared mice with endothelium-specific knockout of Abca1 and Abcg1. Approach and Results— Generation of mice with EC-ABCA1 and ABCG1 deficiency required crossbreeding Abca1 fl/fl Abcg1 fl/fl Ldlr −/− mice with the Tie2Cre strain, followed by irradiation and transplantation of Abca1 fl/fl Abcg1 fl/fl bone marrow to abrogate the effects of macrophage ABCA1 and ABCG1 deficiency induced by Tie2Cre. After 20 to 22 weeks of Western-type diet, both single EC-Abca1 and Abcg1 deficiency increased atherosclerosis in the aortic root and whole aorta. Combined EC-Abca1/g1 deficiency caused a significant further increase in lesion area at both sites. EC-Abca1/g1 deficiency dramatically enhanced macrophage lipid accumulation in the branches of the aorta that are exposed to disturbed blood flow, decreased aortic endothelial NO synthase activity, and increased monocyte infiltration into the atherosclerotic plaque. Abca1/g1 deficiency enhanced lipopolysaccharide-induced inflammatory gene expression in mouse aortic ECs, which was recapitulated by ABCG1 deficiency in human aortic ECs. Conclusions— These studies provide direct evidence that endothelial cholesterol efflux pathways mediated by ABCA1 and ABCG1 are nonredundant and atheroprotective, reflecting preservation of endothelial NO synthase activity and suppression of endothelial inflammation, especially in regions of disturbed arterial blood flow.

Liver Sinusoidal Endothelial Cells Link Hyperinsulinemia to Hepatic Insulin Resistance

Insulin signaling in vascular endothelial cells (ECs) is critical to maintain endothelial function but also to mediate insulin action on peripheral glucose disposal. However, gene knockout studies have reached disparate conclusions. Thus, insulin receptor inactivation in ECs does not impair insulin action, whereas inactivation of Irs2 does. Previously, we have shown that endothelial ablation of the three Foxo genes protects mice from atherosclerosis. Interestingly, here we show that mice lacking FoxO isoforms in ECs develop hepatic insulin resistance through excessive generation of nitric oxide (NO) that impairs insulin action in hepatocytes via tyrosine nitration of insulin receptors. Coculture experiments demonstrate that NO produced in liver sinusoidal ECs impairs insulin’s ability to suppress glucose production in hepatocytes. The effects of liver sinusoidal ECs can be mimicked by NO donors and can be reversed by NO inhibitors in vivo and ex vivo. The findings are consistent with a model in which excessive, rather than reduced, insulin signaling in ECs predisposes to systemic insulin resistance, prompting a reevaluation of current approaches to insulin sensitization.

A Mutant Allele Encoding DNA Binding–Deficient FoxO1 Differentially Regulates Hepatic Glucose and Lipid Metabolism

Insulin signaling in the liver blunts glucose production and stimulates triglyceride biosynthesis. FoxO1 is required for cAMP induction of hepatic glucose production and is permissive for the effect of insulin to suppress this process. Moreover, FoxO1 ablation increases lipogenesis. In this study, we investigated the pleiotropic actions of FoxO1 on glucose and lipid metabolism. To this end, we reconstituted FoxO1 function in mice with a liver-specific deletion of Foxo1 using targeted knock-in of an allele encoding a DNA binding–deficient FoxO1 mutant (L-DBD). Chow-reared L-DBD mice showed defects in hepatic glucose production but normal liver triglyceride content despite increased rates of de novo lipogenesis and impaired fatty acid oxidation in isolated hepatocytes. Gene expression studies indicated that FoxO1 regulates the expression of glucokinase via a cell-nonautonomous coregulatory mechanism, while its regulation of glucose-6-phosphatase proceeds via a cell-autonomous action as a direct transcriptional activator. These conclusions support a differential regulation of hepatic glucose and lipid metabolism by FoxO1 based on the mechanism by which it alters the expression of key target genes involved in each process.

Endothelial Acyl-CoA Synthetase 1 Is Not Required for Inflammatory and Apoptotic Effects of a Saturated Fatty Acid-Rich Environment

Objective—Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall, in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSLs). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating endothelial cell responses to a saturated fatty acid-rich environment in vitro and in vivo. Methods and Results—Saturated fatty acids caused increased inflammatory activation, endoplasmic reticulum stress, and apoptosis in mouse microvascular endothelial cells. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and endoplasmic reticulum stress. However, endothelial ACSL1 deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin-resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression. Conclusion—Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.