Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steatosis

Objective

Hepatokine fibroblast growth factor (FGF) 21 takes part in the regulation of lipid metabolism in the liver and adipose tissue. We investigated whether exendin-4 regulates the expression of FGF21 in the liver, and whether the effects of exendin-4 on the regulation of FGF21 expression are mediated via silent mating type information regulation 2 homolog (SIRT) 1 or SIRT6.

Materials/methods

The C57BL/6J mice were fed a low fat diet, high fat diet, or high fat diet with 1 nmol/kg/day exendin-4 intraperitoneal injection for 10 weeks. HepG2 used in vitro study was treated with palmitic aicd (0.4 mM) with or without exendin-4 (100 nM) and FGF21 (50 nM) for 24 hours. The change of FGF21 and its receptors expression by exendin-4 were measured using quantitative real-time RT-PCR and Western blot. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Inhibition of FGF21, SIRT1 and SIRT6, by 10 nM siRNA was performed to establish the signaling pathway of exendin-4 action in hepatic lipid metabolism.

Results

Exendin-4 increased the expression of FGF21 and its receptors in high fat diet-induced obese mice. In addition, recombinant FGF21 treatment reduced lipid content in palmitic acid-treated HepG2 cells. We also observed significantly decreased expression of peroxisomal proliferator-activated receptor (PPAR) α and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in hepatocytes transfected with FGF21 siRNA. In cells treated with exendin-4, inhibition of SIRT1, but not SIRT6, by siRNA significantly repressed the expression of FGF21 mRNA, whereas decreased SIRT1 expression by inhibition of FGF21 was not observed.

Conclusions

These data suggest that exendin-4 could improve fatty liver by increasing SIRT1-mediated FGF21.     

Artemisia scoparia extract attenuates non-alcoholic fatty liver disease in diet-induced obesity mice by enhancing hepatic insulin and AMPK signaling independently of FGF21 pathway

Objective

Nonalcoholic fatty liver disease (NAFLD) is a common liver disease which has no standard treatment. In this regard, we sought to evaluate the effects of extracts of Artemisia santolinaefolia (SANT) and Artemisia scoparia (SCO) on hepatic lipid deposition and cellular signaling in a diet-induced obesity (DIO) animal model.

Materials/Methods

DIO C57/B6J mice were randomly divided into three groups, i.e. HFD, SANT and SCO. Both extracts were incorporated into HFD at a concentration of 0.5% (w/w). Fasting plasma glucose, insulin, adiponectin, and FGF21 concentrations were measured.

Results

At the end of the 4-week intervention, liver tissues were collected for analysis of insulin, AMPK, and FGF21 signaling. SANT and SCO supplementation significantly increased plasma adiponectin levels when compared with the HFD mice (P < 0.001). Fasting insulin levels were significantly lower in the SCO than HFD mice, but not in SANT group. Hepatic H&E staining showed fewer lipid droplets in the SCO group than in the other two groups. Cellular signaling data demonstrated that SCO significantly increased liver IRS-2 content, phosphorylation of IRS-1, IR β, Akt1 and Akt2, AMPK α1 and AMPK activity and significantly reduced PTP 1B abundance when compared with the HFD group. SCO also significantly decreased fatty acid synthase (FAS), HMG-CoA Reductase (HMGR), and Sterol regulatory element-binding protein 1c (SREBP1c), but not Carnitine palmitoyltransferase I (CPT-1) when compared with HFD group. Neither SANT nor SCO significantly altered plasma FGF21 concentrations and liver FGF21 signaling.

Conclusion

This study suggests that SCO may attenuate liver lipid accumulation in DIO mice. Contributing mechanisms were postulated to include promotion of adiponectin expression, inhibition of hepatic lipogenesis, and/or enhanced insulin and AMPK signaling independent of FGF21 pathway.     

De novo lipogenesis in health and disease

Background

De novo lipogenesis (DNL) is a complex and highly regulated metabolic pathway. In normal conditions DNL converts excess carbohydrate into fatty acids that are then esterified to storage triacylglycerols (TGs). These TGs could later provide energy via β-oxidation. In human body this pathway is primarily active in liver and adipose tissue. However, it is considered to be a minor contributor to the serum lipid homeostasis. Deregulations in the lipogenic pathway are associated with diverse pathological conditions.

Scope of review

The present review focuses on our current understanding of the lipogenic pathway with special reference to the causes and consequences of aberrant DNL.

Major conclusions

The deregulation of DNL in the major lipogenic tissues of the human body is often observed in various metabolic anomalies — including obesity, non-alcoholic fatty liver disease and metabolic syndrome. In addition to that de novo lipogenesis is reported to be exacerbated in cancer tissues, virus infected cells etc. These observations suggest that inhibitors of the DNL pathway might serve as therapeutically significant compounds. The effectiveness of these inhibitors in treatment of cancer and obesity has been suggested by previous works.

General significance

De novo lipogenesis – which is an intricate and highly regulated pathway – can lead to adverse metabolic consequences when deregulated. Therapeutic targeting of this pathway may open a new window of opportunity for combating various lipogenesis-driven pathological conditions — including obesity, cancer and certain viral infections.     

Long-term hypercaloric diet exacerbates metabolic liver disease in PNPLA3 I148M animals

Background & Aims

Nonalcoholic fatty liver disease (NAFLD) is a major health burden associated with the metabolic syndrome leading to liver fibrosis, cirrhosis and ultimately liver cancer. In humans, the PNPLA3 I148M polymorphism of the phospholipase patatin-like phospholipid domain containing protein 3 (PNPLA3) has a well-documented impact on metabolic liver disease. In this study, we used a mouse model mimicking the human PNPLA3 I148M polymorphism in a long-term high fat diet (HFD) experiment to better define its role for NAFLD progression.

Methods

Male mice bearing wild-type Pnpla3 (Pnpla3^WT), or the human polymorphism PNPLA3 I148M (Pnpla3^148M/M) were subjected to HFD feeding for 24 and 52 weeks. Further analysis concerning basic phenotype, inflammation, proliferation and cell death, fibrosis and microbiota were performed in each time point.

Results

After 52 weeks HFD Pnpla3^148M/M animals had more liver fibrosis, enhanced numbers of inflammatory cells as well as increased Kupffer cell activity. Increased hepatocyte cell turnover and ductular proliferation were evident in HFD Pnpla3^148M/M livers. Microbiome diversity was decreased after HFD feeding, changes were influenced by HFD feeding (36%) and the PNPLA3 I148M genotype (12%). Pnpla3^148M/M mice had more faecal bile acids. RNA-sequencing of liver tissue defined an HFD-associated signature, and a Pnpla3^148M/M specific pattern, which suggests Kupffer cell and monocytes-derived macrophages as significant drivers of liver disease progression in Pnpla3^148M/M animals.

Conclusion

With long-term HFD feeding, mice with the PNPLA3 I148M genotype show exacerbated NAFLD. This finding is linked to PNPLA3 I148M-specific changes in microbiota composition and liver gene expression showing a stronger inflammatory response leading to enhanced liver fibrosis progression.     

Glucagon-like peptide-1 production in the GLUTag cell line is impaired by free fatty acids via endoplasmic reticulum stress

Objects

Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-stimulated insulin secretion, and protects pancreas beta cells. However, few studies have examined hypernutrition stress in L cells and its effects on their function. Here, we demonstrated that a high-fat diet reduced glucose-stimulated secretion of GLP-1 and induced expression of an endoplasmic reticulum (ER) stress markers in the intestine of a diet-induced obesity mouse model.

Methods

To clarify whether ER stress in L cells caused the attenuation of GLP-1 secretion, we treated the mouse intestinal L cell line, GLUTag cells with palmitate or oleate.

Results

Palmitate, but not oleate caused ER stress and decreased the protein levels of prohormone convertase 1/3 (PC1/3), an essential enzyme in GLP-1 production. The same phenomena were observed in GLUTag cells treated with in ER stress inducer, thapsigargin. Moreover, oleate improved palmitate-induced ER stress, reduced protein and activity levels of PC1/3, and attenuated GLP-1 secretion from GLUTag cells.

Conclusions/Interpretation

These results suggest that the intake of abundant saturated fatty acids induces ER stress in the intestine and decreases GLP-1 production.     

PNPLA3 I148M (rs738409) genetic variant is associated with hepatocellular carcinoma in obese individuals

Background

Obesity is a risk factor for cancer, including hepatocellular carcinoma. Patatin-like phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) genetic variant has been associated with hepatocellular carcinoma (HCC) in individuals with chronic alcohol abuse or hepatic viral infection. In the present study we examined the association between the PNPLA3 I148M genetic variant and hepatocellular carcinoma in obese individuals from the Swedish Obese Subjects cohort (n = 4047).

Methods

We performed a matched, prospective, controlled, interventional trial, investigating the effect of bariatric surgery (surgery group) compared to conventional treatment (control group) for obesity.

Results

A total of 9 events were observed in the 15-year median follow up (5 in the control group and 4 in the surgery group). A significantly higher incidence of hepatocellular carcinoma in PNPLA3 148M allele carriers was found in obese individuals in the control group (log-rank P-value = 0.001), but not in the surgery group (log-rank P-value = 0.783). Consistently, an increased risk (for each PNPLA3 148M allele, hazard ratio: 5.9; 95% confidence interval 1.5–23.8; P-value = 0.013) of developing hepatocellular carcinoma was observed only in the control group.

Conclusion

The current study is the first prospective report showing the association of the PNPLA3 I148M genetic variant and hepatocellular carcinoma in severely obese individuals.     

Effects of individual branched-chain amino acids deprivation on insulin sensitivity and glucose metabolism in mice

Objective

We recently discovered that leucine deprivation increases hepatic insulin sensitivity via general control nondepressible (GCN) 2/mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways. The goal of the present study was to investigate whether the above effects were leucine specific or were also induced by deficiency of other branched chain amino acids including valine and isoleucine.

Methods

Following depletion of BCAAs, changes in metabolic parameters and the expression of genes and proteins involved in regulation of insulin sensitivity and glucose metabolism were analyzed in mice and cell lines including human HepG2 cells, primary mouse hepatocytes and a mouse myoblast cell line C2C12.

Results

Valine or isoleucine deprivation for 7 days has similar effect on improving insulin sensitivity as leucine, in wild type and insulin-resistant mice models. These effects are possibly mediated by decreased mTOR/S6K1 and increased AMPK signaling pathways, in a GCN2-dependent manner. Similar observations were obtained in in vitro studies. In contrast to leucine withdrawal, valine or isoleucine deprivation for 7 days significantly decreased fed blood glucose levels, possibly due to reduced expression of a key gluconeogenesis gene, glucose-6-phosphatase. Finally, insulin sensitivity was rapidly improved in mice 1 day following maintenance on a diet deficient for any individual BCAAs.

Conclusions

Our results show that while improvement on insulin sensitivity is a general feature of BCAAs depletion, individual BCAAs have specific effects on metabolic pathways, including those that regulate glucose level. These observations provide a conceptual framework for delineating the molecular mechanisms that underlie amino acid regulation of insulin sensitivity.     

Two α-dicarbonyls downregulate migration,invasion, and adhesion of liver cancer cells in a p53-dependent manner

Background

Hepatocellular carcinoma accounts for more than 600,000 deaths per year due to it being a highly invasive tumor. The α-dicarbonyl, methylglyoxal demonstrates efficacy at reducing tumor burden, however the anti-cancerous activities of 3-deoxyglucosone, have never been studied.

Aims

To determine the anti-cancerous potential of methylglyoxal and 3-deoxyglucosone on liver tumor cells.

Methods

The in vitro effects of methylglyoxal and 3-deoxyglucosone were studied by investigating migration, invasion, and adhesion of Huh-7, HepG2, and Hep3B cells.

Results

3-Deoxyglucosone inhibited migration of Huh-7 and HepG2 cells. Methylglyoxal decreased migration of HepG2 cells. Additionally, 3-deoxyglucosone and methylglyoxal impaired invasion, and adhesion of Huh-7 and HepG2 cells. In Hep3B cells, a p53 null cell line, 3-deoxyglucosone and methylglyoxal had no effect on migration, invasion, or adhesion. However, both compounds inhibited invasion of wild-type p53 transfected Hep3B cells. Silencing of p53 in Huh-7 and HepG2 cells abrogated the effects of the α-dicarbonyls on cell invasion. 3DG and MG did not alter p53 total protein but promoted nuclear translocation of p53.

Conclusions

These studies suggest that 3-deoxyglucosone and methylglyoxal impair invasion, migration, and adhesion of hepatocellular carcinoma. The effects of both compounds on cell invasion are dependent on p53 and imply that α-dicarbonyls could be efficacious in the treatment of p53-expressing invasive liver tumors.     

High glucose,insulin and free fatty acid concentrations synergistically enhance perilipin 3 expression and lipid accumulation in macrophages

Objective

Perilipin (PLIN) 3, an intracellular lipid droplet (LD)-associated protein, is implicated in foam cell formation. Since metabolic derangements found in metabolic syndrome, such as high serum levels of glucose, insulin and free fatty acids (FFAs), are major risk factors promoting atherosclerosis, we investigated whether PLIN3 expression is affected by glucose, insulin and oleic acid (OA) using RAW264.7 cells.

Methods

Real-time PCR and Western blotting were performed to detect PLIN3 or PLIN2 expression. Oil-red O staining and Lipid Analysis were employed to measure cellular content of triacylglycerides (TAG) and cholesterol.

Results

PLIN3 mRNA was stimulated by high glucose or insulin concentrations individually, but not by OA. A combination of any two factors did not enhance PLIN3 expression any more than that evoked by glucose alone at 24 h. Interestingly, however, simultaneous addition of all three factors synergistically enhanced the PLIN3 expression. This synergistic effect was not apparent for PLIN2 mRNA expression. Inhibitors of Src family tyrosine kinase and/or phosphatidylinositol 3-kinase, both of which are activated by insulin and FFA signaling, partially suppressed PLIN3 expression induced by the combination of the three factors. While simultaneous addition of glucose, insulin and OA remarkably increased the cellular content of TAG and cholesterol, knocking-down of PLIN3 predominantly reduced TAG content.

Conclusions

These results indicate that PLIN3 expression is synergistically stimulated by high glucose, insulin and FFA concentrations, in parallel with TAG accumulation in macrophages. This finding raises new evidence of PLIN3 involvement in conversion of macrophages into foam cells     

Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism

Objective

The bacteria Staphylococcus aureus is part of the normal bacterial flora and produces a repertoire of enterotoxins which can cause food poisoning and toxic shock and might contribute to the pathogenesis of inflammatory diseases. These enterotoxins directly cross-link the T cell receptor with MHC class II, activating large amounts of T cells and are therefore called superantigens. It was recently discovered that the superantigen SEA binds to the cytokine receptor gp130. As obesity and type 2 diabetes are highly associated with inflammation of the adipose tissue and gp130 has been shown to play an important role in adipocytes, we wanted to investigate the effect of SEA on adipocyte signaling and function.

Materials/methods

Binding of SEA to gp130 was examined using surface plasmon resonance in a cell free system. Effects of SEA on adipocyte signaling, insulin sensitivity and function were studied using western blotting and biological assays for lipolysis, lipogenesis and glucose uptake.

Results

We demonstrate that SEA binds to gp130 with a medium affinity. Furthermore, SEA induces phosphorylation of a key downstream target, STAT3, in adipocytes. SEA also inhibits insulin-induced activation of PKB and PKB downstream signaling which was associated with reduced basal and insulin induced glucose uptake, reduced lipogenesis as well as reduced ability of insulin to inhibit lipolysis.

Conclusions

SEA inhibits insulin signaling as well as insulin biological responses in adipocytes supporting that bacterial infection might contribute to the development of insulin resistance and type 2 diabetes.     

Irisin in response to acute and chronic whole-body vibration exercise in humans

Objective

Irisin is a recently identified myokine, suggested to mediate the beneficial effects of exercise by inducing browning of white adipocytes and thus increasing energy expenditure. In humans, the regulation of irisin by exercise is not completely understood. We investigated the effect of acute and chronic whole-body vibration exercise, a moderate-intensity exercise that resembles shivering, on circulating irisin levels in young healthy subjects.

Materials/Methods

Healthy untrained females participated in a 6-week program of whole-body vibration exercise training. Blood was drawn before and immediately after an acute bout of exercise at baseline (week 0) and after 6 weeks of training.

Results

The resting irisin levels were not different at baseline (week 0) and after 6 weeks of training. At both 0 and 6 weeks of training, an acute bout of vibration exercise significantly elevated circulating irisin levels by 9.5% and 18.1%, respectively (p = 0.05 for the percent change of irisin levels).

Conclusions

Acute bouts of whole-body vibration exercise are effective in increasing circulating irisin levels but chronic training does not change levels of baseline irisin levels in humans.     

New players in high fat diet-induced obesity: LETM1 and CTMP

Objective

Obesity contributes to insulin resistance and is a risk factor for diabetes. C-terminal modulator protein (CTMP) and leucine zipper/EF-hand-containing transmembrane protein 1 (LETM1) have been reported to influence the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) signaling pathway via the modulation of PKB activity, a key player for insulin signaling. However, it remains unclear whether CTMP and LETM1 are associated with PI3K/PKB signaling in mouse models of obesity.

Materials/Methods

To address this question, we used two different mouse models of obesity, including high-fat diet (HFD)-induced diabetic mice and genetically modified obese mice (ob/ob mice). The levels of insulin-signaling molecules in these mice were determined by immunohistochemical and Western blot analyses. The involvement of CTMP and LETM1 in PI3K/PKB signaling was investigated in HEK293 cells by transient transfection and adenovirus-mediated infection.

Results

We found that the levels of insulin receptor, phosphorylated PKB, and LETM1 were lower and the level of CTMP was higher in the adipose tissue of obese mice on an HFD compared to lean mice on a chow diet. Similar results were obtained in ob/ob mice. In HEK293 cells, the activation of PKB increased the LETM1 level, and inhibition of PKB increased the CTMP level. The overexpression of CTMP suppressed the insulin-induced increase in PKB phosphorylation, which was abrogated by co-overexpression with LETM1.

Conclusion

These results suggest that CTMP and LETM1 may participate in impaired insulin signaling in the adipose tissue of obese mice, raising the possibility that these parameters may serve as new candidate biomarkers or targets in the development of new therapeutic approaches for diabetes.     

Lipid droplet-associated proteins in high-fat fed mice with the effects of voluntary running and diet change

Objective

The relation between lipid accumulation and influence of exercise on insulin sensitivity is not straightforward. A proper balance between lipid droplet synthesis, lipolysis, and oxidative metabolism would ensure low local intramyocellular fatty acid levels, thereby possibly protecting against lipotoxicity-associated insulin resistance. This study investigated whether the accumulation of triglycerides and lipid droplets in response to high availability of fatty acids after high-fat feeding would parallel the abundance of intramyocellular perilipin proteins, especially PLIN5. The effects on these variables after diet change or voluntary running exercise intervention in skeletal muscle were also investigated.

Methods

During a 19-week experiment, C57BL/6J mice were studied in six different groups: low-fat diet sedentary, low-fat diet active, high-fat diet sedentary, high-fat diet active and two groups which were high-fat sedentary for nine weeks, after which divided into low-fat sedentary or low-fat active groups. Myocellular triglyceride concentration and perilipin protein expression levels were assessed.

Results

We show that, concurrently with impaired insulin sensitivity, the expression level of PLIN5 and muscular triglyceride concentration increased dramatically after high-fat diet. These adaptations were reversible after the diet change intervention with no additional effect of exercise.

Conclusions

After high-fat diet, lipid droplets become larger providing more surface area for PLIN5. We suggest that PLIN5 is an important regulator of lipid droplet turnover in altered conditions of fatty acid supply and consumption. Imbalances in lipid droplet metabolism and turnover might lead to lipotoxicity-related insulin resistance.