Background: Type 2 diabetes (T2D) is a high-risk factor for incident of cardiovascular diseases. Women at young ages show a reduced incidence of both T2D and cardiovascular diseases compared with men, but these disparities disappear in postmenopausal women versus age-matched men. Thus, ovaries and ovarian hormones, such as estrogen, are expected to protect from T2D and cardiovascular diseases. In this study, we aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism using the cardiac IRS (insulin receptor substrate) 1 and IRS2 double genes knockout mice that mimic cardiac insulin resistance. Methods: Control and heart-specific IRS1/2 double genes knockout mice were treated with placebo or 17β-estradiol (E ₂ ) pellets, respectively, through subcutaneous implantation. Female mice were subjected to a bilateral ovariectomy surgery to remove endogenous E ₂ . The cardiac function and energy metabolism were determined using echocardiography and indirect calorimeter, respectively. Results: All male heart-specific IRS1/2 double genes knockout mice died of heart failure at 6 to 8 weeks as we previously described (Qi et al), but all female heart-specific IRS1/2 double genes knockout mice survived >1 year. Removal of ovaries in heart-specific IRS1/2 double genes knockout female mice resulted in cardiac dysfunction, and ultimately animal death. However, E ₂ supplementation prevented the dilated cardiomyopathy, improved cardiac function and energy metabolism, and enhanced lifespan in both male and ovariectomy female mice deficient for cardiac IRS1 and IRS2 genes, largely owing to the activation of Akt (protein kinase B)-Foxo1 (O1 class of forkhead/winged helix transcription factor) signaling cascades. Conclusions: These results show that estrogen protects mice from cardiac insulin resistance-induced diabetic cardiomyopathy. This may provide a fundamental mechanism for the gender difference for the incidence of both T2D and cardiovascular diseases. This study highlights that estrogen signaling could be a potential target for improving cardiac function and energy metabolism in humans with T2D.

Metformin is the first-line pharmacotherapy for type 2 diabetes mellitus (T2D). Metformin exerts its glucose-lowering effect primarily through decreasing hepatic glucose production (HGP). However, the precise molecular mechanisms of metformin remain unclear due to supra-pharmacological concentration of metformin used in the study. Here, we investigated the role of Foxo1 in metformin action in control of glucose homeostasis and its mechanism via the transcription factor Foxo1 in mice, as well as the clinical relevance with co-treatment of aspirin. We showed that metformin inhibits HGP and blood glucose in a Foxo1-dependent manner. Furthermore, we identified that metformin suppresses glucagon-induced HGP through inhibiting the PKA→Foxo1 signaling pathway. In both cells and mice, Foxo1-S273D or A mutation abolished the suppressive effect of metformin on glucagon or fasting-induced HGP. We further showed that metformin attenuates PKA activity, decreases Foxo1-S273 phosphorylation, and improves glucose homeostasis in diet-induced obese mice. We also provided evidence that salicylate suppresses HGP and blood glucose through the PKA→Foxo1 signaling pathway, but it has no further additive improvement with metformin in control of glucose homeostasis. Our study demonstrates that metformin inhibits HGP through PKA-regulated transcription factor Foxo1 and its S273 phosphorylation.

Hyperglycemia is a hallmark of type 2 diabetes (T2D). Metformin, the first-line drug used to treat T2D, maintains blood glucose within a normal range by suppressing hepatic glucose production (HGP). However, resistance to metformin treatment is developed in most T2D patients over time. Transforming growth factor beta 1 (TGF-β1) levels are elevated both in the liver and serum of T2D humans and mice. Here, we found that TGF-β1 treatment impairs metformin action on suppressing HGP via inhibiting AMPK phosphorylation at Threonine 172 (T172). Hepatic TGF-β1 deficiency improves metformin action on glycemic control in high fat diet (HFD)-induced obese mice. In our hepatic insulin resistant mouse model (hepatic insulin receptor substrate 1 (IRS1) and IRS2 double knockout (DKO)), metformin action on glycemic control was impaired, which is largely improved by further deletion of hepatic TGF-β1 (TKObeta1) or hepatic Foxo1 (TKOfoxo1). Moreover, blockade of TGF-β1 signaling by chemical inhibitor of TGF-β1 type I receptor LY2157299 improves to metformin sensitivity in mice. Taken together, our current study suggests that hepatic TGF-β1 signaling impairs metformin action on glycemic control, and suppression of TGF-β1 signaling could serve as part of combination therapy with metformin for T2D treatment.

Aims: Excessive hepatic glucose production (HGP) driven by glucagon contributes to hyperglycaemia in obesity and type 2 diabetes (T2D), yet the molecular mechanisms underlying this dysregulation remain incompletely defined. This study investigates the role of Smad3 signaling and its regulation by CHIP (Carboxy‐terminus of Hsc70‐interacting protein) in modulating glucagon action on HGP. Materials and Methods: We examined glucagon signaling and HGP in primary hepatocytes and in high‐fat diet (HFD)‐induced obese (DIO) mice. Mechanistic studies included hepatic knockdown of CHIP and Smad3, assessment of Smad3 protein stability, ubiquitination assays, and quantification of gluconeogenic gene expression. Results: We identified Smad3 as a key mediator of glucagon‐induced HGP, synergizing with TGF‐β1 signaling to enhance gluconeogenic gene G6pc expression in a Foxo1‐dependent manner. Glucagon elevated Smad3 protein levels by inhibiting CHIP‐mediated ubiquitination, thereby increasing Smad3 stability. CHIP expression was downregulated in the livers of DIO mice. Hepatic CHIP knockdown augmented glucagon‐stimulated HGP and increased Smad3 levels, whereas simultaneous knockdown of Smad3 reversed these effects. Conclusions: Our findings reveal a novel CHIP–Smad3 regulatory axis that enhances glucagon action on HGP in obesity. Targeting this pathway may offer a new therapeutic strategy for improving glycaemic control in T2D.