Activation of Human Stearoyl-Coenzyme A Desaturase 1 Contributes to the Lipogenic Effect of PXR in HepG2 Cells

The pregnane X receptor (PXR) was previously known as a xenobiotic receptor. Several recent studies suggested that PXR also played an important role in lipid homeostasis but the underlying mechanism remains to be clearly defined. In this study, we found that rifampicin, an agonist of human PXR, induced lipid accumulation in HepG2 cells. Lipid analysis showed the total cholesterol level increased. However, the free cholesterol and triglyceride levels were not changed. Treatment of HepG2 cells with rifampicin induced the expression of the free fatty acid transporter CD36 and ABCG1, as well as several lipogenic enzymes, including stearoyl-CoA desaturase-1 (SCD1), long chain free fatty acid elongase (FAE), and lecithin-cholesterol acyltransferase (LCAT), while the expression of acyl:cholesterol acetyltransferase(ACAT1) was not affected. Moreover, in PXR over-expressing HepG2 cells (HepG2-PXR), the SCD1 expression was significantly higher than in HepG2-Vector cells, even in the absence of rifampicin. Down-regulation of PXR by shRNA abolished the rifampicin-induced SCD1 gene expression in HepG2 cells. Promoter analysis showed that the human SCD1 gene promoter is activated by PXR and a novel DR-7 type PXR response element (PXRE) response element was located at -338 bp of the SCD1 gene promoter. Taken together, these results indicated that PXR activation promoted lipid synthesis in HepG2 cells and SCD1 is a novel PXR target gene.     

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.     

Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue

Aims

The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting).

Methods and Results

Male C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters — CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle.

Conclusion

Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.     

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gamma1 (PPARgamma1) overexpression

Peroxisome proliferator activated-receptor (PPAR) isoforms, alpha and gamma, function as important coregulators of energy (lipid) homeostasis. PPARalpha regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPARgamma serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPARgamma isoforms, PPARgamma1 and PPARgamma2 generated by alternative splicing, PPARgamma1 isoform is expressed in liver and other tissues, whereas PPARgamma2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPARgamma1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPARgamma1 in mouse liver. Adenovirus-PPARgamma1 injected into the tail vein induced hepatic steatosis in PPARalpha(-/-) mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPARalpha(-/-) livers with PPARgamma1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, Delta(9) desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosis per se, induced either by feeding a diet deficient in choline or developing in fasted PPARalpha(-/-) mice, failed to induce the expression of these PPARgamma-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPARgamma in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPARgamma activity can lead to the development of a novel type of adipogenic hepatic steatosis.     

Adipocytic differentiation and liver x receptor pathways regulate the accumulation of triacylglycerols in human vascular smooth muscle cells

Lipid accumulation by vascular smooth muscle cells (VSMC) is a feature of atherosclerotic plaques. In this study we describe two mechanisms whereby human VSMC foam cell formation is driven by de novo synthesis of fatty acids leading to triacylglycerol accumulation in intracellular vacuoles, a process distinct from serum lipoprotein uptake. VSMC cultured in adipogenic differentiation medium accumulated lipids and were induced to express the adipocyte marker genes adipsin, adipocyte fatty acid-binding protein, C/EBPalpha, PPARgamma, and leptin. However, complete adipocyte differentiation was not observed as numerous genes present in mature adipocytes were not detected, and the phenotype was reversible. The rate of lipid accumulation was not affected by PPARgamma agonists, but screening for the effects of other nuclear receptor agonists showed that activation of the liver X receptors (LXR) dramatically promoted lipid accumulation in VSMC. Both LXRalpha and LXRbeta were present in VSMC, and their activation with TO901317 resulted in induction of the lipogenic genes fatty acid synthetase, sterol regulatory element binding protein (SREBP1c), and stearoyl-CoA desaturase. 27-Hydroxycholesterol, an abundant oxysterol synthesized by VSMC acted as an LXR antagonist and, therefore, may have a protective role in preventing foam cell formation. Immunohistochemistry showed that VSMC within atherosclerotic plaques express adipogenic and lipogenic markers, suggesting these pathways are present in vivo. Moreover, the development of an adipogenic phenotype in VSMC is consistent with their known phenotypic plasticity and may contribute to their dysfunction in atherosclerotic plaques and, thus, impinge on plaque growth and stability.     

Role of Pregnane X Receptor in Obesity and Glucose Homeostasis in Male Mice

Clinical obesity is a complex metabolic disorder affecting one in three adults. Recent reports suggest that pregnane X receptor (PXR), a xenobiotic nuclear receptor important for defense against toxic agents and for eliminating drugs and other xenobiotics, may be involved in obesity. Noting differences in ligand specificities between human and mouse PXRs, the role of PXR in high fat diet (HFD)-induced obesity was examined using male PXR-humanized (hPXR) transgenic and PXR-knock-out (PXR-KO) mice in comparison to wild-type (WT) mice. After 16 weeks on either a control diet or HFD, WT mice showed greater weight gain, whereas PXR-KO mice gained less weight due to their resistance to HFD-induced decreases in adipose tissue peroxisome proliferator-activated receptor α and induction of hepatic carnitine palmitoyltransferase 1, suggesting increased energy metabolism. Interestingly, control-fed PXR-KO mice exhibited hepatomegaly, hyperinsulinemia, and hyperleptinemia but hypoadiponectinemia and lower adiponectin receptor R2 mRNA levels relative to WT mice. Evaluation of these biologic indicators in hPXR mice fed a control diet or HFD revealed further differences between the mouse and human receptors. Importantly, although HFD-fed hPXR mice were resistant to HFD-induced obesity, both PXR-KO and hPXR mice exhibited impaired induction of glucokinase involved in glucose utilization and displayed elevated fasting glucose levels and severely impaired glucose tolerance. Moreover, the basal hepatic levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 were increased in hPXR mice compared with WT mice. Altogether, although the mouse PXR promotes HFD-induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and thus provides a model for exploring the role of human PXR in the metabolic syndrome.     

CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine

CD36, a membrane protein that facilitates fatty acid uptake, is highly expressed in the intestine on the luminal surface of enterocytes. Cd36 null (Cd36(-/-)) mice exhibit impaired chylomicron secretion but no overall lipid absorption defect. Because chylomicron production is most efficient proximally we examined whether CD36 function is important for proximal lipid absorption. CD36 levels followed a steep decreasing gradient along three equal-length, proximal to distal intestinal segments (S1-S3). Enterocytes isolated from the small intestines of Cd36(-/-) mice, when compared with wild type counterparts, exhibited reduced uptake of fatty acid (50%) and cholesterol (60%) in S1. The high affinity fatty acid uptake component was missing in Cd36(-/-) cells. Fatty acid incorporation into triglyceride and triglyceride secretion were also reduced in Cd36(-/-) S1 enterocytes. In vivo, proximal absorption was monitored using mass spectrometry from oleic acid enrichment of S1 lipids, 90 min (active absorption) and 5 h (steady state) after intragastric olive oil (70% triolein). Oleate enrichment was 50% reduced at 90 min in Cd36(-/-) tissue consistent with defective uptake whereas no differences were measured at 5 h. In Cd36(-/-) S1, mRNA for L-fabp, Dgat1, and apoA-IV was reduced. Protein levels for FATP4, SR-BI, and NPC1L1 were similar, whereas those for apoB48 and apoA-IV were significantly lower. A large increase in NPC1L1 was observed in Cd36(-/-) S2 and S3. The findings support the role of CD36 in proximal absorption of dietary fatty acid and cholesterol for optimal chylomicron formation, whereas CD36-independent mechanisms predominate in distal segments.     

ATP-binding cassette transporter G1 and lipid homeostasis

PURPOSE OF REVIEW: This review briefly discusses the ATP-binding cassette transporter G (ABCG) family members and emphasizes recent studies that identify ABCG1 as a key regulator of cellular lipid homeostasis. RECENT FINDINGS: The in-vivo importance of ABCG1 has recently been demonstrated with both loss-of-function and gain-of-function studies in mice. Administration of a diet high in both fat and cholesterol to ABCG1 mice results in massive cholesterol accumulation in both the liver and lungs. In contrast, lipid accumulation is greatly attenuated in transgenic mice that express both the murine and human ABCG1 genes. Despite the observed tissue lipid accumulation, plasma lipid levels and lipoprotein cholesterol distribution are not significantly different between wild-type, ABCG1, and hABCG1 transgenic mice. Other studies show that ABCG1 expression is induced following activation of the nuclear receptor LXR and that over expression of ABCG1 results in increased efflux of cellular cholesterol to HDL or phospholipid vesicles. SUMMARY: The ABCG1 transporter plays a key role in regulating cellular cholesterol and lipid homeostasis. Elucidation of the molecular mechanism by which ABCG1 controls sterol flux should provide critical information that may link ABCG1 to the reverse cholesterol transport pathway or diseases such as atherosclerosis.     

Pharmacogenetic evidence that cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor gamma and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-type Cd36. In congenic and transgenic SHR with wild-type Cd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Microarray analysis of peroxisome proliferator-activated receptor-gamma induced changes in gene expression in macrophages

We used a combination of expression microarray and Northern blot analyses to identify target genes for peroxisome proliferator-activated receptor (PPAR) gamma in RAW264.7 macrophages. PPARgamma natural ligand 15-deoxy-Delta(12,14) prostaglandin and synthetic ligands ciglitazone and rosiglitazone increased the expression of scavenger receptor CD36 and ATP-binding cassette transporter A1, as well as adipophilin (a lipid droplet coating protein involved in intracellular lipid storage and transport), calpain (a protease implicated in ABCA1 protein degradation), and ADAM8 (a disintegrin and metalloprotease protein involved in cell adhesion). These findings are relevant to understanding the effect of PPARgamma activation on gene expression and cognate pathways in macrophages.     

Anterior gradient 2 increases long-chain fatty acid uptake via stabilizing FABP1 and facilitates lipid accumulation

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a well-established oncogene. Here, we found that Agr2^-/- mice had a decreased fat mass and hepatic and serum lipid levels compared with their wild-type littermates after fasting, and exhibited reduced high-fat diet (HFD)-induced fat accumulation. Transgenic mice overexpressing AGR2 (Agr2/Tg) readily gained fat weight on a HFD but not a normal diet. Proteomic analysis of hepatic samples from Agr2^-/- mice revealed that depletion of AGR2 impaired long-chain fatty acid uptake and activation but did not affect de novo hepatic lipogenesis. Further investigations led to the identification of several effector substrates, particularly fatty acid binding protein-1 (FABP1) as essential for the AGR2-mediated effects. AGR2 was coexpressed with FABP1, and knockdown of AGR2 resulted in a reduction in FABP1 stability. Physical interactions of AGR2 and FABP1 depended on the PDI motif in AGR2 and the formation of a disulfide bond between these two proteins. Overexpression of AGR2 but not a mutant AGR2 protein lacking PDI activity suppressed lipid accumulation in cells lacking FABP1. Moreover, AGR2 deficiency significantly reduced fatty acid absorption in the intestine, which might be resulted from decreased fatty acid transporter CD36 in mice. These findings demonstrated a novel role of AGR2 in fatty-acid uptake and activation in both the liver and intestine, which contributed to the AGR2-mediated lipid accumulation, suggesting that AGR2 is an important regulator of whole-body lipid metabolism and down-regulation of AGR2 may antagonize the development of obesity.     

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.