Forkhead Box O6 (FoxO6) Depletion Attenuates Hepatic Gluconeogenesis and Protects against Fat-induced Glucose Disorder in Mice

Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.     

FoxO1 对肝脏脂代谢的影响

Fox O1是叉头转录家族O亚族的一员,因其对胰岛素作用起重要的调控作用而被人所熟知,越来越多的研究表明,Fox O1对于肝脏脂质代谢也起重要的调控作用,其作用机制可能是通过上调微粒体甘油三酯转运蛋白(MTP)、载脂蛋白B(Apo B)的表达,从而促进极低密度脂蛋白(VLDL)在肝脏中的合成,增加循环中VLDL含量;Fox O1还可通过促进载脂蛋白CⅢ(Apo CⅢ)的表达,进而抑制脂蛋白酯酶(LPL)活性,减少循环中甘油三酯(TG)分解,导致高甘油三脂血症的发生;同时,Fox O1还能抑制固醇调节元件结合蛋白SREBP-1c表达,抑制脂肪合成。本文主要通过以上几个方面概述了Fox O1与肝脏脂代谢的影响。     

Hepatic FoxO1 Integrates Glucose Utilization and Lipid Synthesis through Regulation of Chrebp O-Glycosylation

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis.     

Oleanolic Acid Reduces Hyperglycemia beyond Treatment Period with Akt/FoxO1-Induced Suppression of Hepatic Gluconeogenesis in Type-2 Diabetic Mice

The present study investigated the chronic efficacy of oleanolic acid (OA), a triterpenoid selected from our recent screening, on hyperglycemia in type-2 diabetic mice. C57BL/6J mice were fed a high-fat diet followed by low doses of streptozotocin to generate a type-2 diabetic model. OA (100 mg/kg/day) was administered orally for 2 weeks with its effects monitored for 6 weeks. High-fat feeding and streptozotocin generated a steady hyperglycemia (21.2±1.1 mM) but OA administration reversed the hyperglycemia by ～60%. Interestingly, after the cessation of OA administration, the reversed hyperglycemia was sustained for the entire post-treatment period of the study (4 weeks) despite the reoccurrence of dyslipidemia. Examination of insulin secretion and pancreas morphology did not indicate improved β-cell function as a likely mechanism. Urine glucose loss was decreased with substantial improvement of diabetic nephropathy after the OA treatment. Pair-feeding the OA-treated mice to an untreated group ruled out food intake as a main factor attributable for this sustained reduction in hyperglycemia. Studies with the use of glucose tracers revealed no increase in glucose influx into muscle, adipose tissue or liver in the OA-treated mice. Finally, we analyzed key regulators of gluconeogenesis in the liver and found significant increases in the phosphorylation of both Akt and FoxO1 after treatment with OA. Importantly, these increases were significantly correlated with a down-regulation of glucose-6-phosphatase expression. Our findings suggest triterpenoids are a potential source of new efficacious drugs for sustained control of hyperglycemia. The liver appears to be a major site of action, possibly by the suppression of hepatic glucose production via the Akt/FoxO1 axis.     

Reduced Histone H3K9 Acetylation of Clock Genes and Abnormal Glucose Metabolism in ob/ob Mice

Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice. (Author correspondence: akiofuji@jichi.ac.jp)     

Reduced Histone H3K9 Acetylation of Clock Genes and Abnormal Glucose Metabolism in ob/ob Mice

Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice. (Author correspondence: akiofuji@jichi.ac.jp ).     

Genetic Variation in Activity of the Enzymes of Glycolysis and Gluconeogenesis between Inbred Strains of Mice

Variation in the activity of 21 liver and 15 erythrocyte enzymes between seven inbred strains of mice has been studied in a single area of metabolism, glycolysis and gluconeogenesis. Most of the variation between the strains is genetic. From the variation within and between inbred strains heritabilities (H²) were determined. Out of 35, 26 showed significant values above 0.4. A comparison with previously published work suggests that enzyme activities have mainly dominance and interaction components of variance, and this is discussed in relation to the variation in quantitative characters such as growth. In nine of the pairwise comparisons of the strains, the activity of the enzyme varied more than two-fold. In these cases the genetics and biochemistry of the enzyme was studied; F₂ progeny were produced and assessed for segregation, and the heat stability of the enzyme was determined. No unequivocal segregation was observed, although in one case we found a considerable difference in heat stability. The variations found were not considered to be great enough to be useful as models of human inborn errors of metabolism or to study metabolic control. If such variants are to be found, sources of variation other than inbred strains must be used.     

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3^tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.     

Hepatic Gluconeogenesis and the Activity of PDH in Individual Tissues of GTG-Obese Mice Following Adrenalectomy

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 ± 0.7g; GTG: 44.1 ± 0.4g; p<0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 ± 1.6 mmol/L; GTG: 29.4 ± 1.9 mmol/L; p<0.05) and serum insulin levels (GTG ADX: 76 ± 10μ.U/mL; GTG: 470 ± 63μU/mL; p<0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 ± 10 nmol glucose/10⁶ cells; GTG: 403 ± 65 nmol glucose/10⁶ cells; p<0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 ± 27 μmol/liver; GTG: 614 ± 60 pmol/Iiver; p<0.05) and fatty acid content (GTG ADX: 101 ± 9 mg fatty acid/g liver; GTG: 404 ± 40 mg fatty acid/g liver; p<0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.     

Increased Adiposity,Dysregulated Glucose Metabolism and Systemic Inflammation in Galectin-3 KO Mice

Obesity and type 2 diabetes are associated with increased production of Galectin-3 (Gal-3), a protein that modulates inflammation and clearance of glucose adducts. We used Lean and Diet-induced Obese (DIO) WT and Gal-3 KO mice to investigate the role of Gal-3 in modulation of adiposity, glucose metabolism and inflammation. Deficiency of Gal-3 lead to age-dependent development of excess adiposity and systemic inflammation, as indicated by elevated production of acute-phase proteins, number of circulating pro-inflammatory Ly6C^high monocytes and development of neutrophilia, microcytic anemia and thrombocytosis in 20-week-old Lean and DIO male Gal-3 KO mice. This was associated with impaired fasting glucose, heightened response to a glucose tolerance test and reduced adipose tissue expression of adiponectin, Gal-12, ATGL and PPARγ, in the presence of maintained insulin sensitivity and hepatic expression of gluconeogenic enzymes in 20-week-old Gal-3 KO mice compared to their diet-matched WT controls. Expression of PGC-1α and FGF-21 in the liver of Lean Gal-3 KO mice was comparable to that observed in DIO animals. Impaired fasting glucose and altered responsiveness to a glucose load preceded development of excess adiposity and systemic inflammation, as demonstrated in 12-week-old Gal-3 KO mice. Finally, a role for the microflora in mediating the fasting hyperglycemia, but not the excessive response to a glucose load, of 12-week-old Gal-3 KO mice was demonstrated by administration of antibiotics. In conclusion, Gal-3 is an important modulator of glucose metabolism, adiposity and inflammation.     

Proglucagon Promoter Cre-Mediated AMPK Deletion in Mice Increases Circulating GLP-1 Levels and Oral Glucose Tolerance

Background

Enteroendocrine L-cells synthesise and release the gut hormone glucagon-like peptide-1 (GLP-1) in response to food transit. Deletion of the tumour suppressor kinase LKB1 from proglucagon-expressing cells leads to the generation of intestinal polyps but no change in circulating GLP-1 levels. Here, we explore the role of the downstream kinase AMP-activated protein kinase (AMPK) in these cells.

Method

Loss of AMPK from proglucagon-expressing cells was achieved using a preproglucagon promoter-driven Cre (iGluCre) to catalyse recombination of floxed alleles of AMPKα1 and α2. Oral and intraperitoneal glucose tolerance were measured using standard protocols. L-cell mass was measured by immunocytochemistry. Hormone and peptide levels were measured by electrochemical-based luminescence detection or radioimmunoassay.

Results

Recombination with iGluCre led to efficient deletion of AMPK from intestinal L- and pancreatic alpha-cells. In contrast to mice rendered null for LKB1 using the same strategy, mice deleted for AMPK displayed an increase (WT: 0.05 ± 0.01, KO: 0.09±0.02%, p<0.01) in L-cell mass and elevated plasma fasting (WT: 5.62 ± 0.800 pg/ml, KO: 14.5 ± 1.870, p<0.01) and fed (WT: 15.7 ± 1.48pg/ml, KO: 22.0 ± 6.62, p<0.01) GLP-1 levels. Oral, but not intraperitoneal, glucose tolerance was significantly improved by AMPK deletion, whilst insulin and glucagon levels were unchanged despite an increase in alpha to beta cell ratio (WT: 0.23 ± 0.02, KO: 0.33 ± 0.03, p<0.01).

Conclusion

AMPK restricts L-cell growth and GLP-1 secretion to suppress glucose tolerance. Targeted inhibition of AMPK in L-cells may thus provide a new therapeutic strategy in some forms of type 2 diabetes.     

非编码RNA与肝脏糖脂代谢调控

随着经济的迅速发展、人们饮食习惯的改变和身体活动的减少,糖尿病成为了现代社会的非传染性的流行病,给家庭和社会造成了极大的危害和经济负担。其中以全身性胰岛素抵抗及胰岛功能衰竭为主要发病特征的2型糖尿病(type 2 diabetes,T2D)已在世界范围引起广泛关注。T2D的发生发展涉及许多组织及糖代谢的各个环节,遗传因素和环境因素共同引起的糖脂代谢通路任一环节的失调均可导致T2D的发生。近年来,包括microRNA(miRNA)及长非编码RNA(LncRNA)在内的非编码RNA(ncRNA)的发现及其在人体生理和病理生理过程中的重要调控作用不断被揭示,为进一步了解T2D的发病机制注入了新理念和信息。miRNAs及LncRNAs的表达具有组织特异性,其表达水平的异常通常与疾病相关。本文主要对miRNAs和LncRNAs在肝脏糖脂代谢调控及T2D的发生发展中的作用及机制的最新研究进展作简要综述。     

Hepatic ACAT2 Knock Down Increases ABCA1 and Modifies HDL Metabolism in Mice

Objectives

ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism.

Design

WT and LXRα/β double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet.

Results

ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE.

Conclusions

The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1.     

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury

FXR regulates bile acid metabolism, and FXR null (Fxr^?/?) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr^?/? mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr^?/? compared with 10-week-old Fxr^?/? mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr^?/? mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr^?/?Xbp1^LKO) and Fxr^?/?Xbp1^fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr^?/? mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.     

脂联素对肝脏糖脂代谢调节作用的研究进展

脂联素因其具有抗糖尿病作用而备受关注,它能控制血糖,并且在肝脏、脂肪和胰腺中能影响脂质代谢。脂联素通过刺激脂肪细胞,对抗炎症、控制脂质过氧化和脂肪分解的速率来调控脂质流入非脂肪组织。肝脏是脂联素发挥作用的重要靶器官;在肝脏中,脂联素与脂联素受体1、2或T-钙黏着蛋白结合,激活下游的AMPK、APPL1、神经酰胺酶等发挥其调节作用。我们总结了脂联素改善肝脏胰岛素敏感性和糖脂代谢的相关机制。