Quan Pan

Aim: During fasting, the liver undergoes coordinated metabolic adaptations to maintain systemic energy homeostasis. These adaptations are driven in part by glucagon, which promotes hepatic glucose production (HGP) and fatty acid oxidation (FAO) through mechanisms involving the transcription factor Foxo1. Autophagy is also activated during nutrient deprivation and maintains intracellular nutrient availability by recycling cellular components. However, whether autophagy supports glucagon‐Foxo1 signalling and the associated hepatic metabolic responses remains unclear. Materials and Methods: Hydroxychloroquine sulphate (HCQ) and AAV8‐shRNA‐ Atg7 were used to inhibit autophagy in control and liver‐specific Foxo1 knockout (L‐FKO) mice, and chloroquine (CQ) and siRNA‐ Atg7 were used to inhibit autophagy in mouse primary hepatocytes. Pyruvate and glucagon tolerance tests were performed in mice. HGP assays, gene expression analysis, western blotting and HPLC‐based amino acid measurements were conducted to evaluate glucose production, Foxo1 signalling, gluconeogenic and FAO‐related pathways and amino acid availability. Results: We found that inhibition of autophagy reduced pyruvate‐stimulated glucose excursions in mice and glucose production in isolated primary hepatocytes, while showing a trend towards reducing glucagon‐stimulated glucose excursions in vivo. It also attenuated glucagon‐responsive gluconeogenic and FAO‐related gene expression in primary hepatocytes. However, these effects of autophagy inhibition were markedly blunted in Foxo1‐deficient models. Mechanistically, suppression of autophagy significantly decreased Foxo1 protein abundance in association with impaired hepatic amino acid homeostasis, without corresponding changes in Foxo1 gene expression and not fully explained by proteasome‐dependent degradation. Importantly, supplementation with exogenous amino acids restored Foxo1 protein levels by 1.89‐fold under basal conditions and 1.35‐fold under glucagon‐treated conditions ( p < 0.05), and partially rescued HGP under autophagy‐inhibited conditions. Conclusion: These findings identify autophagy‐regulated amino acid homeostasis as a critical mechanism that sustains Foxo1 protein abundance and supports glucagon‐responsive hepatic metabolic adaptation during fasting.

Background: Endotoxemia is a severe inflammatory condition that is characterized by acute immune responses and oxidative stress; endotoxemia can further develop into a cytokine storm and sepsis leading to severe organ damage. Our recent studies revealed that the growth hormone secretagogue receptor (GHSR) regulates macrophage polarization in obesity- and aging-associated chronic inflammation. However, its role in acute inflammation during endotoxemia remains unclear. Methods: We subjected myeloid-specific Ghsr knockout mice (LysM-Cre;Ghsr^f/f) to lipopolysaccharide (LPS)-induced endotoxemia in vivo and treated bone marrow-derived macrophages (BMDMs) with LPS in vitro. Subsequently, mouse survival rate and inflammatory signatures in the blood, peritoneal cavity, liver, and BMDM were assessed. In the ex vivo study, conditioned medium (CM) from BMDMs was applied to primary hepatocytes to assess how BMDM-derived CM influences hepatocyte inflammatory responses. Results: Myeloid-specific Ghsr knockout mice exhibited a significantly improved survival rate following LPS-induced endotoxemia, accompanied by reduced systemic inflammation, evident in the blood, peritoneal macrophages, and liver. In addition, Ghsr deficiency suppressed LPS-induced caspase-1 activation and pro-inflammatory cytokine secretion in macrophages. Consistent with these results, conditioned media from Ghsr-deficient BMDMs attenuated the inflammatory responses of primary hepatocytes. Mechanistically, LPS increased GHSR expression in BMDMs, and Ghsr-deficient BMDMs activated mitochondrial respiration and suppressed production of mitochondrial reactive oxygen species (ROS), resulting in downregulation of inflammatory activation of macrophages following LPS exposure. Conclusions: These data demonstrate that macrophage GHSR promotes systemic and tissue inflammation during endotoxemia by regulating mitochondria-associated macrophage polarization. The findings suggest that macrophage GHSR may represent a promising immunomodulatory target for acute inflammatory states, including endotoxemia and sepsis.

Phospholipids are important components of the bilayer of biological membranes. Alterations of phospholipids are associated with metabolic disorders, including insulin resistance. However, how impaired insulin signaling impacts phospholipids has not been well established. Disruption of hepatic insulin signaling is achieved by insulin receptor substrate 1 (IRS1) and IRS2 double deletion (DKO) in the liver. Further deletion of TGF‐β1 or Foxo1 in the liver of DKO mice was used to examine the role of TGF‐β1 or Foxo1 in contributing to the alterations of phospholipid metabolism in DKO mice. Disruption of hepatic insulin signaling led to the dysregulation of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), cardiolipin (CL), and lysophospholipids in the liver. Mechanistically, disruption of hepatic insulin signaling dysregulated the expression of genes related to phospholipid metabolism. Interestingly, further deletion of Tgfb1 in the liver of DKO mice (TKObeta1) attenuated the alterations of phospholipids and rescued the abnormal expression of genes related to phospholipid metabolism. Moreover, deletion of transcription factor Foxo1 , a key mediator of insulin signaling, achieved similar beneficial effects as Tgfb1 deletion in DKO mice. Our study suggests that insulin signaling plays a crucial role in maintaining phospholipids balance in the liver via TGF‐β1 or Foxo1. Targeting TGF‐β1 or Foxo1 could be promising strategies to combat phospholipids alterations and related metabolic dysfunctions.

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.

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.

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.

The heme oxygenase-1 (HO1) catalyzes the degradation of heme into biliverdin, ferrous iron and carbon monoxide (CO). Although HO1 has long been considered as an anti-inflammatory molecule, recent studies showed that HO1 disrupts the mitochondrial function in hepatocytes, as well as drives inflammation and insulin resistance in mice and humans. Such different findings from the historical view impelled us to further investigate the roles of HO1 in glucose metabolism. In the present study, we found that overexpression of HO1 promoted hepatic glucose production (HGP) in hepatocytes from the wild type (WT) mice, which was comparable to the treatment of glucagon. Since the regulatory roles of the transcription factor forkhead box O1 (Foxo1) in HGP have been well recognized, we then generated the liver-specific Foxo1 knockout (L-FKO) mice to study the contribution of Foxo1 in mediating the effects of HO1. We found that HO1 overexpression did not have a significant effect in promoting HGP in hepatocytes from the L-FKO mice, suggesting HO1 improved HGP dependent on Foxo1. Besides, HO1 overexpression improved the mRNA levels of Foxo1-targeted gluconeogenic genes and increased the phosphorylation of Foxo1 at Ser273, which is a phosphorylation site integrating into the glucagon signaling. We also found that HO1 overexpression activated p38α and p65/NF-κB in hepatocytes. Further study using siRNA or specific inhibitor demonstrated that p65/NF-κB, but not p38α, mediated the HO1-induced pFoxo1-S273 and HGP in hepatocytes. We then explored the role of HO1-induced ferrous iron in activating p65/NF-κB and promoting HGP. We found that HO1 overexpression in hepatocytes substantially increased the intracellular ferrous iron. The pretreatment of iron chelator deferoxamine mesylate diminished the effects of HO1 overexpression on p65/NF-κB, pFoxo1-S273, and HGP. Taken together, results from this study revealed the mechanisms of HO1 in driving HGP, which is through the generation of ferrous iron. Disclosure: W. Liao: None. W. Yang: None. Q. Pan: None. Z. Shen: None. W. Ai: None. S. Guo: None. Funding: National Institutes of Health (R01DK120968)