Induced Triglyceride Accumulation Research Articles

FABP4 inhibition suppresses PPARγ activity and VLDL-induced foam cell formation in IL-4-polarized human macrophages

ObjectiveMacrophages, converted to lipid-loaded foam cells, accumulate in atherosclerotic lesions. Macrophage lipid metabolism is transcriptionally regulated by peroxisome proliferator-activated receptor gamma (PPARγ), and its target gene fatty acid binding protein 4 (FABP4) accelerates the progression of atherosclerosis in mouse models. Since expression of PPARγ and FABP4 is increased upon interleukin-4 (IL-4)-induced macrophage polarization, we aimed to investigate the role of FABP4 in human IL-4-polarized macrophages. Methods and resultsWe investigated the impact of FABP4 on PPARγ-dependent gene expression in primary human monocytes differentiated to macrophages in the presence of IL-4. IL-4 increased PPARγ and its target genes lipoprotein lipase (LPL) and FABP4 compared to non-polarized or LPS/interferon γ-stimulated macrophages. LPL expression correlated with increased very low density lipoprotein (VLDL)-induced triglyceride accumulation in IL-4-polarized macrophages, which was sensitive to inhibition of lipolysis or PPARγ antagonism. Inhibition of FABP4 during differentiation using chemical inhibitors BMS309403 and HTS01037 or FABP4 siRNA decreased the expression of FABP4 and LPL, and reduced lipid accumulation in macrophages treated with VLDL. FABP4 or LPL inhibition also reduced the expression of inflammatory mediators chemokine (C-C motif) ligand 2 (CCL2) and IL-1β in response to VLDL in IL-4-polarized macrophages. PPARγ luciferase reporter assays confirmed that FABP4 supports fatty acid-induced PPARγ activation. ConclusionOur findings suggest that IL-4 induces a lipid-accumulating macrophage phenotype by activating PPARγ and subsequent LPL expression. Inhibition of FABP4 decreases VLDL-induced foam cell formation, indicating that anti-atherosclerotic effects achieved by FABP4 inhibition in mouse models may be feasible in the human system as well.

Uric acid induces fat accumulation via generation of endoplasmic reticulum stress and SREBP-1c activation in hepatocytes

Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common types of chronic liver injury. Elevated serum uric acid is a strong predictor of the development of fatty liver as well as metabolic syndrome. Here we demonstrate that uric acid induces triglyceride accumulation by SREBP-1c activation via induction of endoplasmic reticulum (ER) stress in hepatocytes. Uric acid-induced ER stress resulted in an increase of glucose-regulated protein (GRP78/94), splicing of the X-box-binding protein-1 (XBP-1), the phosphorylation of protein kinase RNA-like ER kinase (PERK), and eukaryotic translation initiation factor-2α (eIF-2α) in cultured hepatocytes. Uric acid promoted hepatic lipogenesis through overexpression of the lipogenic enzyme, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) via activation of SREBP-1c, which was blocked by probenecid, an organic anion transport blocker in HepG2 cells and primary hepatocytes. A blocker of ER stress, tauroursodeoxycholic acid (TUDCA), and an inhibitor of SREBP-1c, metformin, blocked hepatic fat accumulation, suggesting that uric acid promoted fat synthesis in hepatocytes via ER stress-induced activation of SREBP-1c. Uric acid-induced activation of NADPH oxidase preceded ER stress, which further induced mitochondrial ROS production in hepatocytes. These studies provide new insights into the mechanisms by which uric acid stimulates fat accumulation in the liver.

Niemann–Pick disease type C2 protein induces triglyceride accumulation in silkworm and mammalian cell lines

Silkworm haemolymph induced both the cessation of growth and an increase in triglyceride (triacylglycerol) storage in BmN4 cells. We purified the growth inhibitory factor from the silkworm haemolymph and identified this protein as the Bombyx mori PP (promoting protein), an orthologue of NPC2 (Niemann-Pick disease type C2) protein. Recombinant silkworm NPC2 inhibited cellular proliferation and increased triglyceride accumulation in BmN4 cells. Injection of either the recombinant protein or antiserum of NPC2 into living silkworms increased or decreased respectively triglyceride levels in the fat body. A mutation that depletes the cholesterol-binding capacity did not abolish the activity of NPC2. We further revealed that NPC2 induced the phosphorylation of AMPK (AMP-activated protein kinase) and that an AMPK inhibitor suppressed NPC2-dependent triglyceride accumulation. These findings suggest that NPC2 induces triglyceride accumulation via the activation of AMPK independently of its cholesterol-binding capacity in the silkworm.

Inhibition of ATP citrate lyase induces triglyceride accumulation with altered fatty acid composition in cancer cells

De novo lipogenesis is activated in most cancers and several lipogenic enzymes have been implicated as therapeutic targets. Here, we demonstrate a novel function of the lipogenic enzyme, ATP citrate lyase (ACLY), in lipid metabolism in cancer cells. ACLY depletion by small interfering RNAs caused growth suppression and/or apoptosis in a subset of cancer cell lines. To investigate the effect of ACLY inhibition on lipid metabolism, metabolome and transcriptome analysis was performed. ACLY depletion blocks the fatty acid chain elongation from C16 to C18 in triglyceride (TG), but not in other lipid classes. Meanwhile, wild-type ACLY overexpression enhanced fatty acid elongation of TG, whereas an inactive mutant ACLY did not change it. ACLY depletion-mediated blockade of fatty acid elongation was coincident with downregulation of long-chain fatty acid elongase ELOVL6, which resides in endoplasmic reticulum (ER). Paradoxically, ACLY depletion-mediated growth suppression was associated with TG accumulation. ACLY depletion downregulated the expression of carnitine palmitoyltransferase 1A, which is a mitochondrial fatty acid transporter. Consistent with this finding, metabolome analysis revealed that ACLY positively regulates the carnitine system, which plays as an essential cofactor for fatty acid transport across mitochondrial membrane. AICAR, an activator of mitochondrial fatty acid oxidation (FAO), significantly reduced ACLY depletion-mediated TG accumulation. These data indicate that inhibition of ACLY might affect both fatty acid elongation in ER and FAO in mitochondria, thereby explaining the TG accumulation with altered fatty acid composition. This phenotype may be a hallmark of growth suppression mediated by ACLY inhibition.

ω‐3 Fatty Acids Prevent Hepatic Steatosis, Independent of PPAR‐α Activity, in a Murine Model of Parenteral Nutrition–Associated Liver Disease

ω-3 Fatty acids (FAs), natural ligands for the peroxisome proliferator-activated receptor-α (PPAR-α), attenuate parenteral nutrition-associated liver disease (PNALD). However, the mechanisms underlying the protective role of ω-3 FAs are still unknown. The aim of this study was to determine the effects of ω-3 FAs on hepatic triglyceride (TG) accumulation in a murine model of PNALD and to investigate the role of PPAR-α and microsomal triglyceride transfer protein (MTP) in this experimental setting. 129S1/SvImJ wild-type or 129S4/SvJaePparatm/Gonz/J PPAR-α knockout mice were fed chow and water (controls); oral, fat-free PN solution only (PN-O); PN-O plus intraperitoneal (IP) ω-6 FA-predominant supplements (PN-ω-6); or PN-O plus IP ω-3 FA (PN-ω-3). Control and PN-O groups received sham IP injections of 0.9% NaCl. Hepatic histology, TG and cholesterol, MTP activity, and PPAR-α messenger RNA were assessed after 19 days. In all experimental groups, PN feeding increased hepatic TG and MTP activity compared with controls. Both PN-O and PN-ω-6 groups accumulated significantly greater amounts of TG when compared with PN-ω-3 mice. Studies in PPAR-α null animals showed that PN feeding increases hepatic TG as in wild-type mice. PPAR-α null mice in the PN-O and PN-ω-6 groups demonstrated variable degrees of hepatic steatosis, whereas no evidence of hepatic fat accumulation was found after 19 days of oral PN plus IP ω-3 FAs. PN induces TG accumulation (steatosis) in wild-type and PPAR-α null mice. In PN-fed wild-type and PPAR-α null mice given IP ω-3 FAs, reduced hepatic TG accumulation and absent steatosis are found. Prevention of steatosis by ω-3 FAs results from PPAR-α-independent pathways.

Kupffer cells suppress perfluorononanoic acid-induced hepatic peroxisome proliferator-activated receptor α expression by releasing cytokines

Kupffer cells (KCs) have been demonstrated to play a role in the regulation of intra-hepatic lipid metabolism through the synthesis and secretion of biologically active products. The involvement of KCs in the disturbance of lipid metabolism that induced by perfluorononanoic acid (PFNA), a known agonist of the peroxisome proliferator-activated receptor alpha (PPARα), was investigated in this study. Rats were exposed to PFNA or PFNA combined with gadolinium chloride, an inhibitor of KCs, for 14 days. PFNA exposure dose-dependently increased absolute and relative liver weights, induced triglyceride accumulation, up-regulated the expression of both SERBP-1c and PPARα, and stimulated the release of TNFα and IL-1β. Inactivation of KCs markedly lowered TNFα and IL-1β level, enhanced PFNA-induced expression of PPARα and its target genes, and reduced liver triglyceride levels. In vitro, PFNA-induced expression of PPARα in primary cultured hepatocytes was suppressed by recombinant rat TNFα and IL-1β. However, inhibition of the NF-κB pathway prevented this. Transient transfection and promoter analysis further revealed that these two cytokines and NF-κB were coordinately involved in the suppression of PPARα promoter activity. Our data demonstrate that TNFα and IL-1β released from KCs following PFNA exposure can suppress the expression of PPARα via NF-κB pathway, which partially contribute to the evident accumulation of triglycerides in rat liver.

Human Adipose Tissue Extract Induces Angiogenesis and AdipogenesisIn Vitro

The induction of adequate vascularization, a major challenge in tissue engineering, has been tried with numerous methods but with unsatisfactory results. Adipose tissue, an active endocrine organ with dense vasculature, secretes a wide number of angiogenic and adipogenic factors and seems an attractive source for these bioactive factors. We produced a novel cell-free extract from mature human adipose tissue (adipose tissue extract [ATE]) and analyzed the ability of this extract to induce angiogenesis and adipogenesis in vitro and studied the cytokine and growth factor composition of ATE with ELISA and cytokine array. We demonstrate that ATE, when added as cell culture supplement, effectively induced triglyceride accumulation in human adipose stem cells at concentrations from 200 μg/mL upward in less than a week and caused elevated levels of adipocyte differentiation markers (proliferator-activated receptor gamma and acyl-CoA-binding protein) when treated with at least 350 μg/mL of ATE. ATE induced angiogenesis from 450 μg/mL upward after a week in vitro. ATE contained numerous angiogenic and adipogenic factors, for example, vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, angiogenin, leptin, and insulin-like growth factor-I, as well as lower levels of a wide variety of other cytokines. We here present a novel cell-free angiogenesis- and adipogenesis-inducing agent that is cell-free and easy to produce, and its effect is dose dependent and its composition can be easily modified. Therefore, ATE is a promising novel agent to be used for angiogenesis induction to overcome the challenge of vascularization and for adipogenesis induction in a wide variety of tissue engineering applications in vitro and in vivo. ATE is also efficient for reproduction and modeling of natural adipogenesis in vitro for, for example, obesity and diabetes studies.

Positive Cross-Talk Between Hypoxia Inducible Factor-1α and Liver X Receptor α Induces Formation of Triglyceride-Loaded Foam Cells

Atherosclerosis is a chronic and progressive inflammatory disease of the arteries that is characterized by subendothelial accumulation of lipid-rich macrophages, called foam cells. We sought to identify the molecular details of cross-talk between liver X receptor α (LXRα) and hypoxia-inducible factor 1α (HIF-1α) for the formation of triglyceride-rich foam cells under hypoxic conditions. We first observed that expression of LXRα and its target lipogenic genes was time-dependently induced in human primary macrophages and RAW 264.7 cells under hypoxia. Similarly, TO901317, an activator of LXRα, enhanced the expression level and the transcriptional activity of HIF-1α. Second, we demonstrated that LXRα increased HIF-1α protein stability through a physical interaction between the ligand binding domain of LXRα and the oxygen-dependent degradation domain of HIF-1α. Third, we found that the activation of HIF-1α or LXRα synergistically induced triglyceride accumulation in macrophages. Finally, we showed that LXRα and HIF-1α were codistributed in the macrophages of atherosclerotic lesions of patients. These results suggest that the positive feed-forward regulation of transcriptional activity and protein stability of LXRα and HIF-1α has an important impact in foam cell formation.

Tamm-Horsfall protein 1 macrophage lipid accumulation unaffected by fatty acid double-bond geometric or positional configuration

Dietary fatty acid type alters atherosclerotic lesion progression and macrophage lipid accumulation. Incompletely elucidated are the mechanisms by which fatty acids differing in double-bond geometric or positional configuration alter arterial lipid accumulation. The objective of this study was to evaluate the suitability of using Tamm-Horsfall protein 1 (THP-1) macrophages as a model for human monocytes/macrophages to address this issue. Our hypothesis was that THP-1 macrophages pretreated with ω-3 polyunsaturated fatty acids (PUFA) or fatty acid containing a cis double bond would accumulate less lipid, particularly cholesteryl ester, compared with ω-6 polyunsaturated fatty acids or a fatty acid containing a trans double bond, respectively. THP-1 monocytes were differentiated into macrophages and then incubated with fatty acids for 48 hours. There was an increase in intracellular lipid in response to all the fatty acids assessed, and by response was similar among the fatty acids. The increase in lipid accumulation was contributed to triglyceride and to a lesser extent cholesterol, primarily free cholesterol. These data suggest that free fatty acids bound to bovine serum albumin, regardless of double-bond geometric or positional configuration, induce triglyceride accumulation but had only a modest effect on cholesterol accumulation in THP-1 macrophages. The cells appeared to respond similarly to the assessed fatty acids in terms of amount and type of lipid accumulated. Hence, the THP-1 cell line was not appropriate to test the hypotheses of interest.

Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1–iNOS–CD36 pathway

Obesity due to nutrient excess leads to chronic pathologies including type 2 diabetes and cardiovascular disease. Related to nutrient excess, FoxO1 has a role in regulating fatty acid uptake and oxidation and triglyceride (TG) storage by mechanisms that are largely unresolved. We examined the mechanism behind palmitate (PA)-induced TG accumulation in cardiomyocytes. To mimic lipid excess, rat ventricular myocytes were incubated with albumin-bound PA (1mM) or rats were administered Intralipid (20%). PA-treated cardiomyocytes showed a substantial increase in TG accumulation, accompanied by amplification of nuclear migration of phospho-p38 and FoxO1, iNOS induction, and translocation of CD36 to the plasma membrane. PA also increased Cdc42 protein and its tyrosine nitration, thereby rearranging the cytoskeleton and facilitating CD36 translocation. These effects were duplicated by TNF-α and reversed by the iNOS inhibitor 1400W. PA increased the nuclear interaction between FoxO1 and NF-κB, reduced the nuclear presence of PGC-1α, and downregulated expression of oxidative phosphorylation proteins. In vivo a robust increase in cardiac TGs after Intralipid administration was also associated with augmentation of nuclear FoxO1 and iNOS expression. Impeding this FoxO1–iNOS–CD36 pathway could decrease cardiac lipid accumulation and oxidative/nitrosative stress and help ameliorate the cardiovascular complications associated with obesity and diabetes.

Oleate upregulates lectin galactoside-binding soluble 2 (LGALS2) in in vitro model of cellular steatosis

IntroductionGalectin-2 (LGALS2) has been shown to co-localise with and bind to lymphotoxin-α (LTA); a cytokine that have been associated with insulin resistance. Thus, LGALS2 has been implicated in metabolic syndrome...

Pachymic acid stimulates glucose uptake through enhanced GLUT4 expression and translocation

In an effort to investigate the effect and mechanism of Poria cocos on glucose uptake, six lanostane-type triterpenoids were isolated and analyzed. Among them, pachymic acid displayed the most significant stimulating activity on glucose uptake in 3T3-L1 adipocytes. The effect of pachymic acid on the expression profile of glucose transporters in differentiated 3T3-L1 adipocytes was also analyzed. Our results demonstrated that pachymic acid induced an increase in GLUT4, but not GLUT1, expression at both the mRNA and protein levels. The role of GLUT4 was further confirmed using the lentiviral vector-derived GLUT4 short hairpin RNA (shRNA). The stimulating activity of pachymic acid on glucose uptake was abolished when the endogenous GLUT4 expression was suppressed in 3T3-L1 adipocytes. In addition to increased GLUT4 expression, pachymic acid stimulated GLUT4 redistribution from intracellular vesicles to the plasma membrane in adipocytes. Exposure of the differentiated adipocytes to pachymic acid increased the phosphorylation of insulin receptor substrate (IRS)-1, AKT and AMP-activated kinase (AMPK). The involvement of PI3K and AMPK in the action of pachymic acid was further confirmed as PI3K and AMPK inhibitors completely blocked the pachymic acid-mediated activities in adipocytes. In addition, pachymic acid was shown to induce triglyceride accumulation and inhibit lipolysis in differentiated adipocytes. Taken together, we demonstrated the insulin-like activities of this compound in stimulating glucose uptake, GLUT4 gene expression and translocation, and promoting triglyceride accumulation in adipocytes. Our study provides important insights into the underlying mechanism of hypoglycemic activity of P. cocos.

Sterol-mediated Regulation of Human Lipin 1 Gene Expression in Hepatoblastoma Cells

Lipin 1 plays a crucial role in lipid metabolism in adipose tissue, skeletal muscle, and liver. Its physiological role involves two cellular functions: regulation of phosphatidate phosphatase activity and regulation of fatty acid oxidation. In this study, we have demonstrated that lipin 1 gene (LPIN1) expression is regulated by cellular sterols, which are key regulators of lipid metabolism. We have also characterized the sterol-response element and nuclear factor Y-binding sites in the human LPIN1 promoter. Using a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of LPIN1 gene, mediated by SREBP-1 (sterol regulatory element-binding protein 1) and nuclear factor Y. Furthermore, we investigated whether lipin 1 is involved in lipogenesis by transfection of LPIN1 small interfering RNA. We infer that sterol-mediated regulation of lipin 1 gene transcription modulates triglyceride accumulation. This modulation involves changes in the activity of phosphatidate phosphatase.

The role of insulin and glucose in goose primary hepatocyte triglyceride accumulation

In order to obtain some information on how fatty liver arises in geese, we investigated the role of insulin and glucose in triglyceride (TG) accumulation in goose primary hepatocytes. Goose primary hepatocytes were isolated and treated with insulin and glucose. Compared with the control group, 100 and 150 nmol l(-1) insulin increased TG accumulation, acetyl-CoA carboxylase-alpha (ACCalpha) and fatty acid synthase (FAS) activity, and the mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1), FAS and ACCalpha genes. Insulin at 200 nmol l(-1) had an inhibiting effect on TG accumulation and the activity of ACC and FAS, but increased the gene expression of SREBP-1, FAS and ACCalpha. We also found that high glucose (30 mmol l(-1)) increased the TG level, ACC and FAS activity, and the mRNA levels of SREBP-1 and FAS. However, there was no effect of high glucose on ACCalpha mRNA level. In addition, the interaction between insulin and glucose was observed to induce TG accumulation, ACC and FAS activity, and gene expression of SREBP-1, FAS and ACCalpha, and increase SREBP-1 nuclear protein level and binding of nuclear SREBP-1 and the SRE response element of the ACC gene. The result also indicated that the glucose-induced TG accumulation decreased after 96 h when the hepatocytes were cultured with 30 mmol l(-1) glucose. In conclusion, insulin and glucose may affect hepatic lipogenesis by regulating lipogenic gene expression and lipogenic enzyme activity in goose hepatocytes, and SREBP-1 might play an important role in the synergetic activation of lipogenic genes. We propose that the utilization of accumulated TG in hepatocytes is the reason for the reversible phenomenon in goose hepatocellular steatosis.

Very-Low-Density Lipoprotein-Induced Triglyceride Accumulation in Human Mesangial Cells Is Mainly Mediated by Lipoprotein Lipase

Background: Very-low-density lipoprotein (VLDL) in vitro can induce foam cell formation in human mesangial cells. Lipoprotein lipase (LPL) expressed in the arterial wall plays a key role in atherogenesis by actions of enzymolysis and ‘molecular bridge’, and, thereby, leads to the formation of lipid-loaded foam cells. It is known that LPL is expressed by glomerular mesangial cells. This study was designed to investigate if LPL plays a role in VLDL-induced lipid accumulation in human mesangial cells and its underlying mechanism. Methods: Human wild-type LPL (hLPLwt), catalytically inactive LPL (hLPL194) or control alkaline phosphatase (hAP) were expressed in human mesangial cell line (HMCLs) via adenoviral vectors. Orlistat (tetrahydrolipstatin), a specific inhibitor of the lipoidolytic activity of endogenous LPL, and heparinase, which degrades cell-surface heparan sulfate proteoglycans, were also used to estimate the role of either the enzymolysis or ‘molecular bridge’ actions of LPL in the uptake of VLDL. Anti-low-density lipoprotein receptor (LDLr) antibody and anti-LDL receptor-related protein antibody were used to evaluate the effect of lipoprotein receptors on VLDL-induced lipid accumulation in HMCLs. Cellular lipid deposition was visualized by Oil Red O staining and analyzed quantitatively by standard enzymatic procedures. LPL protein expression and activity were measured by Western blot and a chemical analysis, respectively. Results: VLDL induced triglyceride accumulation in HMCLs in a time- and dose-dependent manner. Compared with Ad-hAP transfected HMCLs, cellular triglyceride content increased 4.55-fold (p < 0.05) in Ad-hLPLwt-transfected HMCLs and 1.52-fold (p < 0.05) in Ad-hLPL194-transfected HMCLs. Triglyceride accumulation in response to VLDL was mostly blocked by orlistat. Pretreatment of the cells with heparinase slightly reduced cellular triglyceride accumulation in the present of high concentrations of VLDL. The blockade of some lipoprotein receptors, such as LDLr, did not significantly reduce cellular triglyceride accumulation. LPL expression was upregulated by VLDL. Conclusions: VLDL-induced triglyceride accumulation in human mesangial cells is mainly mediated by LPL, and the enzymolysis action of LPL could be a major factor in this process. These results suggest that LPL may be an important factor participating in the initiation and progression of VLDL-mediated lipid renal injury.

The anti-diabetic properties of Guazuma ulmifolia Lam are mediated by the stimulation of glucose uptake in normal and diabetic adipocytes without inducing adipogenesis

Ethnopharmacological importance Guazuma ulmifolia Lam (Sterculiaceae) is a plant extensively used in México for the empirical treatment of type 2 diabetes. Aim of the study To investigate the anti-diabetic mechanisms of Guazuma ulmifolia. Materials and methods Non-toxic concentrations of Guazuma ulmifolia aqueous extracts (GAE) were assayed on adipogenesis and 2-NBDglucose uptake in the murine 3T3-F442A preadipose cell line. Results GAE added to adipogenic medium (AM) did not affect adipogenesis at any of the tested concentrations (1–70 μg/ml), whereas in AM lacking insulin GAE 70 μg/ml induced triglyceride accumulation by 23%. On the other hand, GAE 70 μg/ml stimulated 2-NBDG uptake by 40% in insulin-sensitive 3T3-F442A adipocytes and by 24% in insulin-resistant adipocytes, with respect to the incorporation showed by insulin-sensitive adipocytes stimulated with the hormone. Conclusion Guazuma ulmifolia exerts its anti-diabetic effects by stimulating glucose uptake in both insulin-sensitive and insulin-resistant adipocytes without inducing adipogenesis.

High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes

The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c) and reduced that of microsomal triglyceride transfer protein (MTP). Finally, d4T-induced alteration of SREBP1c and MTP expression was partially prevented by l-carnitine. Thus, short-term incubation with high concentrations of d4T can rapidly induce accumulation of neutral lipids within rat hepatocytes, which can be fully prevented by l-carnitine. Furthermore, our investigations suggested that lipid accumulation could be the consequence of a dual mechanism, namely a mtDNA-independent impairment of mitochondrial FAO and a reduction of lipid export from the hepatocytes.

CCAAT/Enhancer-binding Protein β Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Lepr Mice

CCAAT/enhancer-binding protein beta (C/EBPbeta) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPbeta in hepatic lipogenesis remains undefined. Here we show that C/EBPbeta inactivation in Lepr(db/db) mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPbeta(-/-) x Lepr(db/db) mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPbeta deletion in Lepr(db/db) mice down-regulated peroxisome proliferator-activated receptor gamma2 (PPARgamma2) and stearoyl-CoA desaturase-1 and up-regulated PPARalpha independent of SREBP1c. Conversely, C/EBPbeta overexpression in wild-type mice increased PPARgamma2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPbeta or C/EBPbeta RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARgamma2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPbeta expression in hepatocytes, whereas fatty acids up-regulate C/EBPbeta expression. These data provide novel evidence linking C/EBPbeta expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease.

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein alpha (C/EBPalpha), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells: role of fatty acid β-oxidation and glucose

Mitochondrial cytopathy has been associated with modifications of lipid metabolism in various situations, such as the acquisition of an abnormal adipocyte phenotype observed in multiple symmetrical lipomatosis or triglyceride (TG) accumulation in muscles associated with the myoclonic epilepsy with ragged red fibers syndrome. However, the molecular signaling leading to fat metabolism dysregulation in cells with impaired mitochondrial activity is still poorly understood. Here, we found that preadipocytes incubated with inhibitors of mitochondrial respiration such as antimycin A (AA) accumulate TG vesicles but do not acquire specific markers of adipocytes. Although the uptake of TG precursors is not stimulated in 3T3-L1 cells with impaired mitochondrial activity, we found a strong stimulation of glucose uptake in AA-treated cells mediated by calcium and phosphatidylinositol 3-kinase/Akt1/glycogen synthase kinase 3beta, a pathway known to trigger the translocation of glucose transporter 4 to the plasma membrane in response to insulin. TG accumulation in AA-treated cells is mediated by a reduced peroxisome proliferator-activated receptor gamma activity that downregulates muscle carnitine palmitoyl transferase-1 expression and fatty acid beta-oxidation, and by a direct conversion of glucose into TGs accompanied by the activation of carbohydrate-responsive element binding protein, a lipogenic transcription factor. Taken together, these results could explain how mitochondrial impairment leads to the multivesicular phenotype found in some mitochondria-originating diseases associated with a dysfunction in fat metabolism.