Transcription Factor Forkhead Box O1 Mediates Transforming Growth Factor-ß1-Induced Apoptosis in Hepatocytes.

Dysregulation of hepatocyte apoptosis is associated with several types of chronic liver diseases. Transforming growth factor-ß1 (TGF-ß1) is a well-known pro-apoptotic factor in the liver, which constitutes a receptor complex composed of TGF-ß receptor I and II, along with transcription factor Smad proteins. As a member of the forkhead box O (Foxo) class of transcription factors, Foxo1 is a predominant regulator of hepatic glucose production and apoptosis. This study investigated the potential relationship between TGF-ß1 signaling and Foxo1 in control of apoptosis in hepatocytes. TGF-ß1 induced hepatocyte apoptosis in a Foxo1-dependent manner in hepatocytes isolated from both wild-type and liver-specific Foxo1 knockout mice. TGF-ß1 activated protein kinase A through TGF-ß receptor I-Smad3, followed by phosphorylation of Foxo1 at Ser273 in promotion of apoptosis in hepatocytes. Moreover, Smad3 overexpression in the liver of mice promoted the levels of phosphorylated Foxo1-S273, total Foxo1, and a Foxo1-target pro-apoptotic gene Bim, which eventually resulted in hepatocyte apoptosis. The study further demonstrated a crucial role of Foxo1-S273 phosphorylation in the pro-apoptotic effect of TGF-ß1 by using hepatocytes isolated from Foxo1-S273A/A knock-in mice, in which the phosphorylation of Foxo1-S273 was disrupted. Taken together, this study established a novel role of TGF-ß1→protein kinase A→Foxo1 signaling cascades in control of hepatocyte survival.

TGF-ß1 Signaling Impairs Metformin Action on Glycemic Control.

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-ß1) levels are elevated both in the liver and serum of T2D humans and mice. Here, we found that TGF-ß1 treatment impairs metformin action on suppressing HGP via inhibiting AMPK phosphorylation at Threonine 172 (T172). Hepatic TGF-ß1 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-ß1 (TKObeta1) or hepatic Foxo1 (TKOfoxo1). Moreover, blockade of TGF-ß1 signaling by chemical inhibitor of TGF-ß1 type I receptor LY2157299 improves to metformin sensitivity in mice. Taken together, our current study suggests that hepatic TGF-ß1 signaling impairs metformin action on glycemic control, and suppression of TGF-ß1 signaling could serve as part of combination therapy with metformin for T2D treatment.

Hepatic p38α MAPK controls gluconeogenesis via FOXO1 phosphorylation at S273 during glucagon signalling in mice.

AIMS/HYPOTHESIS: Hyperglucagonaemia-stimulated hepatic glucose production (HGP) contributes to hyperglycaemia during type 2 diabetes. A better understanding of glucagon action is important to enable efficient therapies to be developed for the treatment of diabetes. Here, we aimed to investigate the role of p38 MAPK family members in glucagon-induced HGP and determine the underlying mechanisms by which p38 MAPK regulates glucagon action. METHODS: p38α, ß, γ and δ MAPK siRNAs were transfected into primary hepatocytes, followed by measurement of glucagon-induced HGP. Adeno-associated virus serotype 8 carrying p38α MAPK short hairpin RNA (shRNA) was injected into liver-specific Foxo1 knockout, liver-specific Irs1/Irs2 double knockout and Foxo1S273D knockin mice. Foxo1S273A knockin mice were fed a high-fat diet for 10 weeks. Pyruvate tolerance tests, glucose tolerance tests, glucagon tolerance tests and insulin tolerance tests were carried out in mice, liver gene expression profiles were analysed and serum triglyceride, insulin and cholesterol levels were measured. Phosphorylation of forkhead box protein O1 (FOXO1) by p38α MAPK in vitro was analysed by LC-MS. RESULTS: We found that p38α MAPK, but not the other p38 isoforms, stimulates FOXO1-S273 phosphorylation and increases FOXO1 protein stability, promoting HGP in response to glucagon stimulation. In hepatocytes and mouse models, inhibition of p38α MAPK blocked FOXO1-S273 phosphorylation, decreased FOXO1 levels and significantly impaired glucagon- and fasting-induced HGP. However, the effect of p38α MAPK inhibition on HGP was abolished by FOXO1 deficiency or a Foxo1 point mutation at position 273 from serine to aspartic acid (Foxo1S273D) in both hepatocytes and mice. Moreover, an alanine mutation at position 273 (Foxo1S273A) decreased glucose production, improved glucose tolerance and increased insulin sensitivity in diet-induced obese mice. Finally, we found that glucagon activates p38α through exchange protein activated by cAMP 2 (EPAC2) signalling in hepatocytes. CONCLUSIONS/INTERPRETATION: This study found that p38α MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis in both health and disease. The glucagon-induced EPAC2-p38α MAPK-pFOXO1-S273 signalling pathway is a potential therapeutic target for the treatment of type 2 diabetes.

Hepatocyte FoxO1 Deficiency Protects From Liver Fibrosis via Reducing Inflammation and TGF-ß1-mediated HSC Activation.

BACKGROUND & AIMS: The O-class of the forkhead transcription factor FoxO1 is a crucial factor mediating insulin→PI3K→Akt signaling and governs diverse cellular processes. However, the role of hepatocyte FoxO1 in liver fibrosis has not been well-established. In his study, we investigated the role of hepatocyte FoxO1 in liver fibrosis and uncovered the underlying mechanisms. METHODS: Liver fibrosis was established by carbon tetrachloride (CCL4) administration and compared between liver-specific deletion of FoxO1 deletion (F1KO) and control (CNTR) mice. Using genetic and bioinformatic strategies in vitro and in vivo, the role of hepatic FoxO1 in liver fibrosis and associated mechanisms was established. RESULTS: Increased FoxO1 expression and FoxO1 signaling activation were observed in CCL4-induced fibrosis. Hepatic FoxO1 deletion largely attenuated CCL4-induced liver injury and fibrosis compared with CNTR mice. F1KO mice showed ameliorated CCL4-induced hepatic inflammation and decreased TGF-ß1 mRNA and protein levels compared with those of CNTR mice. In primary hepatocytes, FoxO1 deficiency reduced TGF-ß1 expression and secretion. Conditioned medium (CM) collected from wild-type hepatocytes treated with CCL4 activated human HSC cell line (LX-2); such effect was attenuated by FoxO1 deletion in primary hepatocytes or neutralization of TGF-ß1 in the CM using TGF-ß1 antibody. Hepatic FoxO1 overexpression in CNTR mice promoted CCL4-induced HSC activation; such effect was blocked in L-TGF-ß1KO mice. CONCLUSIONS: Hepatic FoxO1 mediates CCL4-inducled liver fibrosis via upregulating hepatocyte TGF-ß1 expression, stimulating hepatic inflammation and TGF-ß1-mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.

An estrogen receptor α-derived peptide improves glucose homeostasis during obesity.

Estrogen receptor α (ERα) plays a crucial role in regulating glucose and energy homeostasis during type 2 diabetes mellitus (T2DM). However, the underlying mechanisms remain incompletely understood. Here we find a ligand-independent effect of ERα on the regulation of glucose homeostasis. Deficiency of ERα in the liver impairs glucose homeostasis in male, female, and ovariectomized (OVX) female mice. Mechanistic studies reveal that ERα promotes hepatic insulin sensitivity by suppressing ubiquitination-induced IRS1 degradation. The ERα 1-280 domain mediates the ligand-independent effect of ERα on insulin sensitivity. Furthermore, we identify a peptide based on ERα 1-280 domain and find that ERα-derived peptide increases IRS1 stability and enhances insulin sensitivity. Importantly, administration of ERα-derived peptide into obese mice significantly improves glucose homeostasis and serum lipid profiles. These findings pave the way for the therapeutic intervention of T2DM by targeting the ligand-independent effect of ERα and indicate that ERα-derived peptide is a potential insulin sensitizer for the treatment of T2DM.

ZnPP-laden nanoparticles improve glucose homeostasis and chronic inflammation during obesity.

BACKGROUND AND PURPOSE: Chronic inflammation plays a pivotal role in the development of Type 2 diabetes mellitus (T2DM). Previous studies have shown that haem oxygenase-1 (HO-1) plays a proinflammatory role during metabolic stress, suggesting that HO-1 inhibition could be an effective strategy to treat T2DM. However, the application of HO-1 inhibitors is restricted due to solubility-limited bioavailability. In this study, we encapsulated the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP), within nanoparticles and investigated their role in regulating glucose homeostasis and chronic inflammation during obesity. EXPERIMENTAL APPROACH: We delivered DMSO-dissolved ZnPP (DMSO-ZnPP) and ZnPP-laden nanoparticles (Nano-ZnPP) to diet-induced obese male mice for 6 weeks. Glucose and insulin tolerance tests were carried out, liver and adipose tissue gene expression profiles analysed, and systemic inflammation analysed using flow cytometry. KEY RESULTS: Nanoparticles significantly increased the delivery efficiency of ZnPP in both cells and mice. In mice with diet-induced obesity, inhibition of HO-1 by Nano-ZnPP significantly decreased adiposity, increased insulin sensitivity, and improved glucose tolerance. Moreover, Nano-ZnPP treatment attenuated both local and systemic inflammatory levels during obesity. Mechanistically, Nano-ZnPP significantly attenuated glucagon, TNF, and fatty acid synthesis signalling pathways in the liver. In white adipose tissue, the oxidative phosphorylation signalling pathway was enhanced and the inflammation signalling pathway diminished by Nano-ZnPP. Our results show that Nano-ZnPP has better effects on the improvement of glucose homeostasis and attenuation of chronic inflammation, than those of DMSO-dissolved ZnPP. CONCLUSIONS AND IMPLICATIONS: These findings indicate that ZnPP-laden nanoparticles are potential therapeutic agents for treating T2DM.

Reciprocal Regulation of Hepatic TGF-ß1 and Foxo1 Controls Gluconeogenesis and Energy Expenditure.

Obesity and insulin resistance are risk factors for the pathogenesis of type 2 diabetes (T2D). Here, we report that hepatic TGF-ß1 expression positively correlates with obesity and insulin resistance in mice and humans. Hepatic TGF-ß1 deficiency decreased blood glucose levels in lean mice and improved glucose and energy dysregulations in diet-induced obese (DIO) mice and diabetic mice. Conversely, overexpression of TGF-ß1 in the liver exacerbated metabolic dysfunctions in DIO mice. Mechanistically, hepatic TGF-ß1 and Foxo1 are reciprocally regulated: fasting or insulin resistance caused Foxo1 activation, increasing TGF-ß1 expression, which, in turn, activated protein kinase A, stimulating Foxo1-S273 phosphorylation to promote Foxo1-mediated gluconeogenesis. Disruption of TGF-ß1→Foxo1→TGF-ß1 looping by deleting TGF-ß1 receptor II in the liver or by blocking Foxo1-S273 phosphorylation ameliorated hyperglycemia and improved energy metabolism in adipose tissues. Taken together, our studies reveal that hepatic TGF-ß1→Foxo1→TGF-ß1 looping could be a potential therapeutic target for prevention and treatment of obesity and T2D. ARTICLE HIGHLIGHTS: Hepatic TGF-ß1 levels are increased in obese humans and mice. Hepatic TGF-ß1 maintains glucose homeostasis in lean mice and causes glucose and energy dysregulations in obese and diabetic mice. Hepatic TGF-ß1 exerts an autocrine effect to promote hepatic gluconeogenesis via cAMP-dependent protein kinase-mediated Foxo1 phosphorylation at serine 273, endocrine effects on brown adipose tissue action, and inguinal white adipose tissue browning (beige fat), causing energy imbalance in obese and insulin-resistant mice. TGF-ß1→Foxo1→TGF-ß1 looping in hepatocytes plays a critical role in controlling glucose and energy metabolism in health and disease.

Suppression of FOXO1 attenuates inflamm-aging and improves liver function during aging.

The liver is a key metabolic organ that maintains whole-body nutrient homeostasis. Aging-induced liver function alterations contribute to systemic susceptibility to aging-related diseases. However, the molecular mechanisms of liver aging remain insufficiently understood. In this study, we performed bulk RNA-Seq and single-cell RNA-Seq analyses to investigate the underlying mechanisms of the aging-induced liver function changes. We found that liver inflammation, glucose intolerance, and liver fat deposition were aggravated in old mice. Aging significantly increased pro-inflammation in hepatic macrophages. Furthermore, we found that Kupffer cells (KCs) were the major driver to induce pro-inflammation in hepatic macrophages during aging. In KCs, aging significantly increased pro-inflammatory levels; in monocyte-derived macrophages (MDMs), aging had a limited effect on pro-inflammation but led to a functional quiescence in antigen presentation and phagosome process. In addition, we identified an aging-responsive KC-specific (ARKC) gene set that potentially mediates aging-induced pro-inflammation in KCs. Interestingly, FOXO1 activity was significantly increased in the liver of old mice. FOXO1 inhibition by AS1842856 significantly alleviated glucose intolerance, hepatic steatosis, and systemic inflammation in old mice. FOXO1 inhibition significantly attenuated aging-induced pro-inflammation in KCs partially through downregulation of ARKC genes. However, FOXO1 inhibition had a limited effect on aging-induced functional quiescence in MDMs. These results indicate that aging induces pro-inflammation in liver mainly through targeting KCs and FOXO1 is a key player in aging-induced pro-inflammation in KCs. Thus, FOXO1 could be a potential therapeutic target for the treatment of age-associated chronic diseases.

Heme Oxygenase-1 Regulates Ferrous Iron and Foxo1 in Control of Hepatic Gluconeogenesis.

The liver is a key player for maintaining glucose homeostasis. Excessive hepatic glucose production is considered to be a key for the onset of type 2 diabetes. The primary function of heme oxygenase-1 (HO1) is to catalyze the degradation of heme into biliverdin, ferrous iron, and carbon monoxide. Previous studies have demonstrated that the degradation of heme by HO1 in the liver results in mitochondrial dysfunction and drives insulin resistance. In this study, by overexpressing HO1 in hepatocytes and mice, we showed that HO1 promotes gluconeogenesis in a Foxo1-dependent manner. Importantly, HO1 overexpression increased the generation of ferrous iron in the liver, which further activates nuclear factor-κB and phosphorylates Foxo1 at Ser273 to enhance gluconeogenesis. We further assessed the role of HO1 in insulin-resistant liver-specific knockout of IRS1 and IRS2 genes (L-DKO) mice, which exhibit upregulation of HO1 in the liver and hepatic ferrous iron overload. HO1 knockdown by shRNA or treatment of iron chelator rescued the aberrant gluconeogenesis in L-DKO mice. In addition, we found that systemic iron overload promotes gluconeogenesis by activating the hepatic protein kinase A→Foxo1 axis. Thus, our results demonstrate the role of HO1 in regulating hepatic iron status and Foxo1 to control gluconeogenesis and blood glucose.

Metformin Targets Foxo1 to Control Glucose Homeostasis.

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.

Epigallocatechin Gallate Inhibits Hepatic Glucose Production in Primary Hepatocytes via Downregulating PKA Signaling Pathways and Transcriptional Factor FoxO1.

Forkhead/winged helix transcription factor O-class member 1 (FoxO1) is a key mediator of insulin and glucagon signaling in control of glucose homeostasis. Although epigallocatechin gallate (EGCG) has attracted interest owing to its potential to combat hyperglycemic diabetes, molecular mechanisms underlying its antihyperglycemic effect, in particular the effect on FoxO1, is poorly understand. This study aims to assess the impact of EGCG on the glucagon signaling pathway in regulating glucose metabolism. Primary hepatocytes from wild-type (WT), liver-specific FoxO1 knock out (FKO), and FoxO1-S273D knock-in (KI) mice were isolated, cultured, and treated with EGCG and/or glucagon. Our data showed the treatment of 10 µM EGCG for 6 h decreased hepatic glucose production by 20 and 23% in WT and FKO primary hepatocytes, respectively. EGCG repressed both gluconeogenesis and glycogenolysis in primary hepatocytes, coupled with activating AMPK. In addition, EGCG decreased mitochondrial oxygen consumption. We further investigated the effects of EGCG on glucagon-stimulated cAMP/PKA signaling pathway. EGCG reduced p-PKA-T197/T-PKA and p-CREB-S133/T-CREB levels by 39 and 20%, blocked p-FoxO1-S273, and suppressed nuclear FoxO1 translocation, suggesting that FoxO1 and CREB were possible downstream targets. A novel mechanism of EGCG in restraining hepatic glucose production (HGP) is through antagonizing glucagon signaling and suppressing FoxO1 via Ser273. EGCG may serve as a promising compound for regulating glucose homeostasis.