E4orf1: a novel ligand that improves glucose disposal in cell culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance (IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 'requires' E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as 'sufficient' to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras--the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large (Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif (PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs.     

Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.     

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.     

Celecoxib attenuates hepatosteatosis by impairing de novo lipogenesis via Akt‐dependent lipogenic pathway

Mounting evidence indicates that hepatic de novo lipogenesis is a common abnormality in non‐alcoholic fatty liver disease (NAFLD) patients. We investigated whether a selective COX‐2 inhibitor, celecoxib, alleviates hepatic steatosis by targeting an Akt‐driven lipogenic pathway. We estimated the efficacy of celecoxib in a novel Akt‐driven NAFLD mouse model established via hydrodynamic transfection of activated forms of AKT and in fructose‐fed NAFLD mice that exhibited increased insulin‐independent hepatic lipogenesis. AKT‐transfected and insulin‐stimulated human hepatoma cells were used for the in vitro experiments. Haematoxylin and eosin staining, immunohistochemistry and immunoblotting were performed for mechanistic studies. The results revealed that celecoxib ameliorated hepatic steatosis in the AKT‐triggered NAFLD mice. Mechanistically, celecoxib effectively suppressed AKT/mTORC1 signalling and its downstream lipogenic cascade in the Akt‐driven NAFLD mice and in vitro. Furthermore, celecoxib had limited efficacy in alleviating hepatic lipid accumulation and showed no influence on lipogenic proteins associated with hepatic lipogenesis in fructose‐administered mice. This study suggests that celecoxib may be favourable for the treatment of NAFLD, especially in the subset with Akt‐triggered hepatic lipogenesis.     

Lipocalin 13 protein protects against hepatic steatosis by both inhibiting lipogenesis and stimulating fatty acid β-oxidation

Obesity is associated with hepatic steatosis, partially due to increased lipogenesis and decreased fatty acid β-oxidation in the liver; however, the underlying mechanism of abnormal lipid metabolism is not fully understood. We reported previously that obesity is associated with LCN13 (lipocalin 13) deficiency. LCN13 is a lipocalin family member involved in glucose metabolism, and LCN13 deficiency appears to contribute to hyperglycemia in obese mice. Here, we show that LCN13 is also an important regulator of lipogenesis and β-oxidation in the liver. In primary hepatocytes, recombinant LCN13 directly suppressed lipogenesis and increased fatty acid β-oxidation, whereas neutralization of endogenous LCN13 had an opposite effect. Transgenic overexpression of LCN13 protected against hepatic steatosis in mice with either dietary or genetic (ob/ob) obesity. LCN13 transgenic overexpression also improved hyperglycemia, glucose intolerance, and insulin resistance in ob/ob mice. Short-term LCN13 overexpression via an adenovirus-mediated gene transfer similarly attenuated hepatic steatosis in db/db mice. LCN13 inhibited the expression of important lipogenic genes and stimulated the genes that promote β-oxidation. These results suggest that LCN13 decreases liver lipid levels by both inhibiting hepatic lipogenesis and stimulating β-oxidation. LCN13 deficiency is likely to contribute to fatty liver disease in obese mice.     

Astaxanthin reduces hepatic lipid accumulations in high-fat-fed C57BL/6J mice via activation of peroxisome proliferator-activated receptor (PPAR) alpha and inhibition of PPAR gamma and Akt

We have previously reported that astaxanthin (AX), a dietary carotenoid, directly interacts with peroxisome proliferator-activated receptors PPARα and PPARγ, activating PPARα while inhibiting PPARγ, and thus reduces lipid accumulation in hepatocytes in vitro. To investigate the effects of AX in vivo, high-fat diet (HFD)-fed C57BL/6J mice were orally administered AX (6 or 30 mg/kg body weight) or vehicle for 8 weeks. AX significantly reduced the levels of triglyceride both in plasma and in liver compared with the control HFD mice. AX significantly improved liver histology and thus reduced both steatosis and inflammation scores of livers with hematoxylin and eosin staining. The number of inflammatory macrophages and Kupffer cells were reduced in livers by AX administration assessed with F4/80 staining. Hepatic PPARα-responsive genes involved in fatty acid uptake and β-oxidation were upregulated, whereas inflammatory genes were downregulated by AX administration. In vitro radiolabeled assays revealed that hepatic fatty acid oxidation was induced by AX administration, whereas fatty acid synthesis was not changed in hepatocytes. In mechanism studies, AX inhibited Akt activity and thus decreased SREBP1 phosphorylation and induced Insig-2a expression, both of which delayed nuclear translocation of SREBP1 and subsequent hepatic lipogenesis. Additionally, inhibition of the Akt-mTORC1 signaling axis by AX stimulated hepatic autophagy that could promote degradation of lipid droplets. These suggest that AX lowers hepatic lipid accumulation in HFD-fed mice via multiple mechanisms. In addition to the previously reported differential regulation of PPARα and PPARγ, inhibition of Akt activity and activation of hepatic autophagy reduced hepatic steatosis in mouse livers.     

Reduced hepatic fatty acid oxidation in fasting PPARalpha null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression

Glucose and fatty acid metabolism (oxidation versus esterification) has been measured in hepatocytes isolated from 24 h starved peroxisome proliferator-activated receptor-alpha (PPARalpha) null and wild-type mice. In PPARalpha null mice, the development of hypoglycemia during starvation was due to a reduced capacity for hepatic gluconeogenesis secondary to a 70% lower rate of fatty acid oxidation. This was not due to inappropriate expression of the hepatic CPT I gene, which was similar in both genotypes, but to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression in the PPARalpha null mouse liver. We also demonstrate that hepatic steatosis of fasting PPARalpha null mice was not due to enhanced triglyceride synthesis.     

SIRT4 Represses Peroxisome Proliferator-Activated Receptor α Activity To Suppress Hepatic Fat Oxidation

Sirtuins are a family of protein deacetylases, deacylases, and ADP-ribosyltransferases that regulate life span, control the onset of numerous age-associated diseases, and mediate metabolic homeostasis. We have uncovered a novel role for the mitochondrial sirtuin SIRT4 in the regulation of hepatic lipid metabolism during changes in nutrient availability. We show that SIRT4 levels decrease in the liver during fasting and that SIRT4 null mice display increased expression of hepatic peroxisome proliferator-activated receptor α (PPARα) target genes associated with fatty acid catabolism. Accordingly, primary hepatocytes from SIRT4 knockout (KO) mice exhibit higher rates of fatty acid oxidation than wild-type hepatocytes, and SIRT4 overexpression decreases fatty acid oxidation rates. The enhanced fatty acid oxidation observed in SIRT4 KO hepatocytes requires functional SIRT1, demonstrating a clear cross talk between mitochondrial and nuclear sirtuins. Thus, SIRT4 is a new component of mitochondrial signaling in the liver and functions as an important regulator of lipid metabolism.     

Liver Patt1 deficiency protects male mice from age-associated but not high-fat diet-induced hepatic steatosis

Patt1 is a newly identified protein acetyltransferase that is highly expressed in liver. However, the role of Patt1 in liver is still unclear. We generated Patt1 liver-specific knockout (LKO) mice and mainly measured the effect of hepatic Patt1 deficiency on lipid metabolism. Hepatic Patt1 deficiency in male mice markedly decreases fat mass and dramatically alleviates age-associated accumulation of lipid droplets in liver. Moreover, hepatic Patt1 abrogation in male mice significantly reduces the liver triglyceride and free fatty acid levels, but it has no effect on liver cholesterol level, liver weight, and liver function. Consistently, primary cultured Patt1-deficient hepatocytes are resistant to palmitic acid-induced lipid accumulation, but hepatic Patt1 deficiency fails to protect male mice from high-fat diet-induced hepatic steatosis. Further studies show that hepatic Patt1 deficiency decreases fatty acid uptake, reduces lipid synthesis, and enhances fatty acid oxidation, which may contribute to the attenuated hepatic steatosis in Patt1 LKO mice. These results demonstrate that Patt1 plays an important role in hepatic lipid metabolism and have implications toward resolving age-associated hepatic steatosis.     

Effect of Octreotide on Hepatic Steatosis in Diet-Induced Obesity in Rats

Background

Non-alcoholic fatty liver disease (NAFLD) caused by liver lipid dysregulation is linked to obesity. Somatostatin (SST) and its analogs have been used to treat pediatric hypothalamic obesity. However, the application of such drugs for the treatment of NAFLD has not been evaluated.

Objective

This study aimed to investigate the expression levels of important regulators of hepatic lipid metabolism and the possible effect of the SST analog octreotide on these regulators.

Methods

SD rats were assigned to a control group and a high-fat diet group. Obese rats from the high-fat diet group were further divided into the obese and octreotide-treated groups. The body weight, plasma SST, fasting plasma glucose (FPG), insulin, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acid (FFA) levels were measured. Hepatic steatosis was evaluated based on the liver TG content, HE staining and oil red O staining. The SREBP-1c, ACC1, FAS, MTP, apoB and ADRP expression levels in the liver were also determined by RT-PCR, qRT-PCR, western blot or ELISA.

Results

The obese rats induced by high-fat diet expressed more SREBP-1c, FAS and ADRP but less MTP protein in the liver than those of control rats, whereas octreotide intervention reversed these changes and increased the level of apoB protein. Compared to the control group, obese rats showed increased liver ACC1, SREBP-1c and apoB mRNA levels, whereas octreotide-treated rats showed decreased mRNA levels of apoB and SREBP-1c. This was accompanied by increased body weight, liver TG contents, FPG, TG, TC, LDL-C, FFA, insulin and derived homeostatic model assessment (HOMA) values. Octreotide intervention significantly decreased these parameters. Compared to the control group, the obese group showed a decreasing trend on plasma SST levels, which were significantly increased by the octreotide intervention.

Conclusion

Octreotide can ameliorate hepatic steatosis in obese rats, possibly by decreasing hepatic lipogenesis and increasing TG export from hepatocytes.     

IL-1 Signaling in Obesity-Induced Hepatic Lipogenesis and Steatosis

Non-alcoholic fatty liver disease is prevalent in human obesity and type 2 diabetes, and is characterized by increases in both hepatic triglyceride accumulation (denoted as steatosis) and expression of pro-inflammatory cytokines such as IL-1β. We report here that the development of hepatic steatosis requires IL-1 signaling, which upregulates Fatty acid synthase to promote hepatic lipogenesis. Using clodronate liposomes to selectively deplete liver Kupffer cells in ob/ob mice, we observed remarkable amelioration of obesity-induced hepatic steatosis and reductions in liver weight, triglyceride content and lipogenic enzyme expressions. Similar results were obtained with diet-induced obese mice, although visceral adipose tissue macrophage depletion also occurred in response to clodronate liposomes in this model. There were no differences in the food intake, whole body metabolic parameters, serum β-hydroxybutyrate levels or lipid profiles due to clodronate-treatment, but hepatic cytokine gene expressions including IL-1β were decreased. Conversely, treatment of primary mouse hepatocytes with IL-1β significantly increased triglyceride accumulation and Fatty acid synthase expression. Furthermore, the administration of IL-1 receptor antagonist to obese mice markedly reduced obesity-induced steatosis and hepatic lipogenic gene expression. Collectively, our findings suggest that IL-1β signaling upregulates hepatic lipogenesis in obesity, and is essential for the induction of pathogenic hepatic steatosis in obese mice.