Yajuan Qi

Hepatic Suppression of Foxo1 and Foxo3 Causes Hypoglycemia and Hyperlipidemia in Mice

Dysregulation of blood glucose and triglycerides are the major characteristics of type 2 diabetes mellitus. We sought to identify the mechanisms regulating blood glucose and lipid homeostasis. Cell-based studies established that the Foxo forkhead transcription factors Forkhead box O (Foxo)-1, Foxo3, and Foxo4 are inactivated by insulin via a phosphatidylinositol 3-kinase/Akt-dependent pathway, but the role of Foxo transcription factors in the liver in regulating nutrient metabolism is incompletely understood. In this study, we used the Cre/LoxP genetic approach to delete the Foxo1, Foxo3, and Foxo4 genes individually or a combination of two or all in the liver of lean or db/db mice and assessed the role of Foxo inactivation in regulating glucose and lipid homeostasis in vivo. In the lean mice or db/db mice, hepatic deletion of Foxo1, rather than Foxo3 or Foxo4, caused a modest reduction in blood glucose concentrations and barely affected lipid homeostasis. Combined deletion of Foxo1 and Foxo3 decreased blood glucose levels, elevated serum triglyceride and cholesterol concentrations, and increased hepatic lipid secretion and caused hepatosteatosis. Analysis of the liver transcripts established a prominent role of Foxo1 in regulating gene expression of gluconeogenic enzymes and Foxo3 in the expression of lipogenic enzymes. Our findings indicate that Foxo1 and Foxo3 inactivation serves as a potential mechanism by which insulin reduces hepatic glucose production and increases hepatic lipid synthesis and secretion in healthy and diabetic states.

Myocardial Loss of IRS1 and IRS2 Causes Heart Failure and Is Controlled by p38α MAPK During Insulin Resistance

Cardiac failure is a major cause of death in patients with type 2 diabetes, but the molecular mechanism that links diabetes to heart failure remains unclear. Insulin resistance is a hallmark of type 2 diabetes, and insulin receptor substrates 1 and 2 (IRS1 and IRS2) are the major insulin-signaling components regulating cellular metabolism and survival. To determine the role of IRS1 and IRS2 in the heart and examine whether hyperinsulinemia causes myocardial insulin resistance and cellular dysfunction via IRS1 and IRS2, we generated heart-specific IRS1 and IRS2 gene double-knockout (H-DKO) mice and liver-specific IRS1 and IRS2 double-knockout (L-DKO) mice. H-DKO mice had reduced ventricular mass; developed cardiac apoptosis, fibrosis, and failure; and showed diminished Akt→forkhead box class O-1 signaling that was accompanied by impaired cardiac metabolic gene expression and reduced ATP content. L-DKO mice had decreased cardiac IRS1 and IRS2 proteins and exhibited features of heart failure, with impaired cardiac energy metabolism gene expression and activation of p38α mitogen-activated protein kinase (p38). Using neonatal rat ventricular cardiomyocytes, we further found that chronic insulin exposure reduced IRS1 and IRS2 proteins and prevented insulin action through activation of p38, revealing a fundamental mechanism of cardiac dysfunction during insulin resistance and type 2 diabetes.

Activation of Foxo1 by Insulin Resistance Promotes Cardiac Dysfunction and β-Myosin Heavy Chain Gene Expression

Background—Heart failure is a leading cause of morbidity and mortality in the USA and is closely associated with diabetes mellitus. The molecular link between diabetes mellitus and heart failure is incompletely understood. We recently demonstrated that insulin receptor substrates 1, 2 (IRS1, 2) are key components of insulin signaling and loss of IRS1 and IRS2 mediates insulin resistance, resulting in metabolic dysregulation and heart failure, which is associated with downstream Akt inactivation and in turn activation of the forkhead transcription factor Foxo1. Methods and Results—To determine the role of Foxo1 in control of heart failure in insulin resistance and diabetes mellitus, we generated mice lacking Foxo1 gene specifically in the heart. Mice lacking both IRS1 and IRS2 in adult hearts exhibited severe heart failure and a remarkable increase in the &bgr;-isoform of myosin heavy chain (&bgr;-MHC) gene expression, whereas deletion of cardiac Foxo1 gene largely prevented the heart failure and resulted in a decrease in &bgr;-MHC expression. The effect of Foxo1 deficiency on rescuing cardiac dysfunction was also observed in db/db mice and high-fat diet mice. Using cultures of primary ventricular cardiomyocytes, we found that Foxo1 interacts with the promoter region of &bgr;-MHC and stimulates gene expression, mediating an effect of insulin that suppresses &bgr;-MHC expression. Conclusions—Our study suggests that Foxo1 has important roles in promoting diabetic cardiomyopathy and controls &bgr;-MHC expression in the development of cardiac dysfunction. Targeting Foxo1 and its regulation will provide novel strategies in preventing metabolic and myocardial dysfunction and influencing MHC plasticity in diabetes mellitus.

Novel Mechanism of Blood Pressure Regulation By Forkhead Box Class O1–Mediated Transcriptional Control of Hepatic Angiotensinogen

The renin–angiotensin system is a major determinant of blood pressure regulation. It consists of a cascade of enzymatic reactions involving 3 components: angiotensinogen, renin, and angiotensin-converting enzyme, which generate angiotensin II as a biologically active product. Angiotensinogen is largely produced in the liver, acting as a major determinant of the circulating renin–angiotensin system, which exerts acute hemodynamic effects on blood pressure regulation. How the expression of angiotensinogen is regulated is not completely understood. Here, we hypothesize that angiotensinogen is regulated by forkhead transcription factor forkhead box class O1 (Foxo1), an insulin-suppressed transcription factor, and thereby controls blood pressure in mice. We generated liver-specific Foxo1 knockout mice, which exhibited a reduction in plasma angiotensinogen and angiotensin II levels and a significant decrease in blood pressure. Using hepatocyte cultures, we demonstrated that overexpression of Foxo1 increased angiotensinogen expression, whereas hepatocytes lacking Foxo1 demonstrated a reduction of angiotensinogen gene expression and partially impaired insulin inhibition on angiotensinogen gene expression. Furthermore, mouse angiotensinogen prompter analysis demonstrated that the angiotensinogen promoter region contains a functional Foxo1-binding site, which is responsible for both Foxo1 stimulation and insulin suppression on the promoter activity. Together, these data demonstrate that Foxo1 regulates hepatic angiotensinogen gene expression and controls plasma angiotensinogen and angiotensin II levels, modulating blood pressure control in mice.

Activation of AMPK restricts coxsackievirus B3 replication by inhibiting lipid accumulation

Coxsackievirus B3 (CVB3) is the major pathogen of human viral myocarditis. CVB3 has been found to manipulate and modify the cellular lipid metabolism for viral replication. The cellular AMP-activated protein kinase (AMPK) is a key regulator of multiple metabolic pathways, including lipid metabolism. Here we explore the potential roles AMPK plays in CVB3 infection. We found that AMPK is activated by the viral replication during CVB3 infection in Hela cells and primary myocardial cells. RNA interference mediated inhibition of AMPK could increase the CVB3 replication in cells, indicating that AMPK contributed to restricting the viral replication. Next, we showed that CVB3 replication could be inhibited by several different pharmacological AMPK activators including metformin, A769662 and AICAR. And the constitutively active AMPK mutant (CA-AMPK) could also inhibit the CVB3 replication. Furthermore, we found that CVB3 infection increased the cellular lipid levels and showed that the AMPK agonist AICAR both restricted CVB3 replication and reduced lipid accumulation through inhibiting the lipid synthesis associated gene expression. We further found that CVB3 infection would also induce AMPK activated in vivo. The AMPK agonist metformin, which has been widely used in diabetes therapy, could decrease the viral replication and further protect the mice from myocardial histological and functional changes in CVB3 induced myocarditis, and improve the survival rate of infected mice. Lastly, it was demonstrated that the AICAR-mediated restriction of viral replication could be rescued partially by exogenous palmitate, the first product of fatty acid biosynthesis, demonstrating that AMPK activation restricted CVB3 infection through its inhibition of lipid synthesis. Taken together, these data in the present study suggest a model in which AMPK is activated by CVB3 infection and restricts viral replication by inhibiting the cellular lipid accumulation, and inform a potential novel therapeutic strategy for CVB3-associated diseases.

Novel Mechanism of Foxo1 Phosphorylation in Glucagon Signaling in Control of Glucose Homeostasis

Dysregulation of hepatic glucose production (HGP) serves as a major underlying mechanism for the pathogenesis of type 2 diabetes. The pancreatic hormone glucagon increases and insulin suppresses HGP, controlling blood glucose homeostasis. The forkhead transcription factor Foxo1 promotes HGP through increasing expression of genes encoding the rate-limiting enzymes responsible for gluconeogenesis. We previously established that insulin suppresses Foxo1 by Akt-mediated phosphorylation of Foxo1 at Ser256 in human hepatocytes. In this study, we found a novel Foxo1 regulatory mechanism by glucagon, which promotes Foxo1 nuclear translocation and stability via cAMP- and protein kinase A–dependent phosphorylation of Foxo1 at Ser276. Replacing Foxo1-S276 with alanine (A) or aspartate (D) to block or mimic phosphorylation, respectively, markedly regulates Foxo1 stability and nuclear localization in human hepatocytes. To establish in vivo function of Foxo1-Ser276 phosphorylation in glucose metabolism, we generated Foxo1-S273A and Foxo1-S273D knock-in (KI) mice. The KI mice displayed impaired blood glucose homeostasis, as well as the basal and glucagon-mediated HGP in hepatocytes. Thus, Foxo1-Ser276 is a new target site identified in the control of Foxo1 bioactivity and associated metabolic diseases.

Novel Mechanism of Blood Pressure Regulation By Foxo1-Mediated Transcriptional Control of Hepatic Angiotensinogen

The renin–angiotensin system is a major determinant of blood pressure regulation. It consists of a cascade of enzymatic reactions involving 3 components: angiotensinogen, renin, and angiotensin-converting enzyme, which generate angiotensin II as a biologically active product. Angiotensinogen is largely produced in the liver, acting as a major determinant of the circulating renin–angiotensin system, which exerts acute hemodynamic effects on blood pressure regulation. How the expression of angiotensinogen is regulated is not completely understood. Here, we hypothesize that angiotensinogen is regulated by forkhead transcription factor forkhead box class O1 (Foxo1), an insulin-suppressed transcription factor, and thereby controls blood pressure in mice. We generated liver-specific Foxo1 knockout mice, which exhibited a reduction in plasma angiotensinogen and angiotensin II levels and a significant decrease in blood pressure. Using hepatocyte cultures, we demonstrated that overexpression of Foxo1 increased angiotensinogen expression, whereas hepatocytes lacking Foxo1 demonstrated a reduction of angiotensinogen gene expression and partially impaired insulin inhibition on angiotensinogen gene expression. Furthermore, mouse angiotensinogen prompter analysis demonstrated that the angiotensinogen promoter region contains a functional Foxo1-binding site, which is responsible for both Foxo1 stimulation and insulin suppression on the promoter activity. Together, these data demonstrate that Foxo1 regulates hepatic angiotensinogen gene expression and controls plasma angiotensinogen and angiotensin II levels, modulating blood pressure control in mice.

Novel Mechanism of Blood Pressure Regulation By Forkhead Box Class O1–Mediated Transcriptional Control of Hepatic AngiotensinogenNovelty and Significance

The renin–angiotensin system is a major determinant of blood pressure regulation. It consists of a cascade of enzymatic reactions involving 3 components: angiotensinogen, renin, and angiotensin-converting enzyme, which generate angiotensin II as a biologically active product. Angiotensinogen is largely produced in the liver, acting as a major determinant of the circulating renin–angiotensin system, which exerts acute hemodynamic effects on blood pressure regulation. How the expression of angiotensinogen is regulated is not completely understood. Here, we hypothesize that angiotensinogen is regulated by forkhead transcription factor forkhead box class O1 (Foxo1), an insulin-suppressed transcription factor, and thereby controls blood pressure in mice. We generated liver-specific Foxo1 knockout mice, which exhibited a reduction in plasma angiotensinogen and angiotensin II levels and a significant decrease in blood pressure. Using hepatocyte cultures, we demonstrated that overexpression of Foxo1 increased angiotensinogen expression, whereas hepatocytes lacking Foxo1 demonstrated a reduction of angiotensinogen gene expression and partially impaired insulin inhibition on angiotensinogen gene expression. Furthermore, mouse angiotensinogen prompter analysis demonstrated that the angiotensinogen promoter region contains a functional Foxo1-binding site, which is responsible for both Foxo1 stimulation and insulin suppression on the promoter activity. Together, these data demonstrate that Foxo1 regulates hepatic angiotensinogen gene expression and controls plasma angiotensinogen and angiotensin II levels, modulating blood pressure control in mice.