Intracellular Triglyceride Levels Research Articles

Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein

Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

The effect of arginine on the Wnt/β-catenin signaling pathway during porcine intramuscular preadipocyte differentiation.

Dietary l-arginine supplementation decreases backfat thickness and increases intramuscular fat content in growing-finishing pigs, but the underlying mechanisms are unknown. In this study, the effect of arginine on differentiation of porcine intramuscular preadipocytes was investigated in vitro. We showed that the mRNA and protein expressions of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α were upregulated by arginine supplementation. Furthermore, the intracellular triglyceride level was increased by arginine supplementation. We also showed that activation of the Wnt/β-catenin signal pathway by using lithium chloride (LiCl) significantly attenuated arginine-induced upregulation of PPARγ and increased the phospho-β-catenin level. These findings suggested that arginine promotes porcine intramuscular preadipocyte differentiation, which might be via repressing the Wnt/β-catenin signaling pathway.

Artemisia iwayomogi plus Curcuma longa Synergistically Ameliorates Nonalcoholic Steatohepatitis in HepG2 Cells.

The combination of Artemisia iwayomogi and Curcuma longa radix is frequently prescribed for liver diseases in TKM. However, the synergic effects of the two herbs on nonalcoholic steatohepatitis (NASH) have not yet been studied. Therefore, we investigated the anti-NASH effects of the water extract of A. iwayomogi (AI), C. longa radix (CL), and combination of the two herbs (ACE). Hepatic steatosis and NASH were induced in HepG2 cells by treatment with palmitic acid (PA, for 6 h) with/without pretreatment of ACE (25 or 50 μg/mL), AI (50 or 100 μg/mL), CL (50 or 100 μg/mL), curcumin (5 μg/mL), or scopoletin (5 μg/mL). The PA treatment (200 μM) drastically altered intracellular triglyceride levels, total cholesterol, and expression levels of genes related to lipid metabolism (CD36, SREBP1c, PPAR-γ, and PPAR-α), whereas pretreatment with ACE significantly attenuated these alterations. ACE also protected HepG2 cells from PA- (300 μM-) induced endoplasmic reticulum (ER) stress and apoptosis and attenuated the related key molecules including GRP78, eIF2, and CHOP, respectively. In conclusion, we found synergic effects of A. iwayomogi and C. longa on NASH, supporting the clinical potential for fatty liver disorders. In addition, modulation of ER stress-relative molecules would be involved in its underlying mechanism.

Exendin-4 Inhibits Hepatic Lipogenesis by Increasing β-Catenin Signaling.

The aim of this study is to investigate whether the beneficial effect of exendin-4 on hepatic steatosis is mediated by β-catenin signaling. After the HepG2 human hepatoma cells were treated with PA for 24 hours, total triglycerides levels were increased in a dose-dependent manner, and the expression levels of perilipin family members were upregulated in cells treated with 400 μM PA. For our in vitro model of hepatic steatosis, HepG2 cells were treated with 400 μM palmitic acid (PA) in the presence or absence of 100 nM exendin-4 for 24 hours. PA increased the expression of lipogenic genes, such as sterol regulatory element-binding protein 1c (SREBP-1c), peroxisome proliferator-activated receptor gamma (PPARγ), stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) and triglyceride synthesis-involved genes, such as diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) in HepG2 cells, whereas exendin-4 treatment significantly prevented the upregulation of SREBP-1c, PPARγ, SCD1, FAS, ACC, DGAT1 and DGAT2. Moreover, exendin-4 treatment increased the expression of phosphorylated glycogen synthase kinase-3 beta (GSK-3β) in the cytosolic fraction and the expression of β-catenin and transcription factor 4 (TCF4) in the nuclear fraction. In addition, siRNA-mediated inhibition of β-catenin upregulated the expression of lipogenic transcription factors. The protective effects of exendin-4 on intracellular triglyceride content and total triglyceride levels were not observed in cells treated with the β-catenin inhibitor IWR-1. These data suggest that exendin-4 treatment improves hepatic steatosis by inhibiting lipogenesis via activation of Wnt/β-catenin signaling.

NS5ATP6 modulates intracellular triglyceride content through FGF21 and independently of SIRT1 and SREBP1

The prevalence of nonalcoholic fatty liver disease (NAFLD) is rising strikingly in Western countries and China. The molecular biological mechanism of NAFLD remains unclear, with no effective therapies developed so far. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone, with safe lipid lowering effects. FGF21 analogs are being developed for clinical application. Here we demonstrated that a novel gene, NS5ATP6, modulated intracellular triglyceride (TG) content independently of sirtuin1 (SIRT1) and sterol regulatory element binding protein 1 (SREBP1) in HepG2 cells. Interestingly, NS5ATP6 regulated FGF21 expression both at the mRNA and protein levels. The modulatory effects of NS5ATP6 on intracellular TG content depended upon FGF21. Further studies revealed that NS5ATP6 decreased the promoter activity of FGF21. In addition, NS5ATP6 regulated the expression of miR-577, which directly targeted and regulated FGF21. Therefore, miR-577 might be involved in NS5ATP6 regulation of FGF21 at the post-transcriptional level. In conclusion, NS5ATP6 regulates the intracellular TG level via FGF21, and independently of SIRT1 and SREBP1.

Reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1.

Previous studies indicated that miR-200s participated in IL-6-induced hepatic insulin resistance. However, the role of miR-200s in hepatic lipid accumulation has not been elucidated. Here we found that miR-200b and miR-200c were reduced in the steatotic livers of mice fed a high-fat diet (HFD) and patients with nonalcoholic fatty liver disease. This down-regulation was accompanied by an increase in the expression of lipogenic proteins such as sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS). The suppression of miR-200b and miR-200c in Hep1-6 and NCTC1469 hepatocytes enhanced intracellular triglyceride levels, which were associated with increased SREBP-1 and FAS protein levels. In contrast, the over-expression of miR-200b and miR-200c suppressed lipid accumulation and reduced the expression of SREBP1 and FAS in Hep1-6 and NCTC1469 cells transfected with miR-200b or miR-200c mimics. Importantly, the up-regulation of miR-200b and miR-200c could reverse oleic acid/palmitic acid-induced lipid accumulation in hepatocytes. A luciferase reporter assay identified that miR-200b and miR-200c could directly bind the 3′UTR of jun. JUN activated the transcription of srebp1 to increase lipid accumulation. The data also demonstrated that increased miR-200b and miR-200c expression might be associated with sitagliptin-reduced hepatic lipid accumulation in mice fed a HFD. These findings suggest, for the first time, that reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1.

RNA-binding protein HuD reduces triglyceride production in pancreatic β cells by enhancing the expression of insulin-induced gene 1

Although triglyceride (TG) accumulation in the pancreas leads to β-cell dysfunction and raises the chance to develop metabolic disorders such as type 2 diabetes (T2DM), the molecular mechanisms whereby intracellular TG levels are regulated in pancreatic β cells have not been fully elucidated. Here, we present evidence that the RNA-binding protein HuD regulates TG production in pancreatic β cells. Mouse insulinoma βTC6 cells stably expressing a small hairpin RNA targeting HuD (shHuD) (βTC6-shHuD) contained higher TG levels compared to control cells. Moreover, downregulation of HuD resulted in a decrease in insulin-induced gene 1 (INSIG1) levels but not in the levels of sterol regulatory element-binding protein 1c (SREBP1c), a key transcription factor for lipid production. We identified Insig1 mRNA as a direct target of HuD by using ribonucleoprotein immunoprecipitation (RIP) and biotin pulldown analyses. By associating with the 3′-untranslated region (3ʹUTR) of Insig1 mRNA, HuD promoted INSIG1 translation; accordingly, HuD downregulation reduced while ectopic HuD expression increased INSIG1 levels. We further observed that HuD downregulation facilitated the nuclear localization of SREBP1c, thereby increasing the transcriptional activity of SREBP1c and the expression of target genes involved in lipogenesis; likewise, we observed lower INSIG1 levels in the pancreatic islets of HuD-null mice. Taken together, our results indicate that HuD functions as a novel repressor of lipid synthesis in pancreatic β cells.

Triglyceride Regulation of Lysosomal Cholesterol in THP-1 Macrophages

The central question I investigated in my research this semester was how the cholesterol and triglyceride metabolic pathways interact in macrophages when cholesterol levels are high. What factors prevent or enhance macrophage ability to regulate cholesterol metabolism? We have preliminary evidence that excessive cholesterol accumulation in macrophages alters triglyceride metabolism in these cells. When cellular cholesterol levels are normal, the primary site of triglyceride hydrolysis is on the surface of the macrophage. There, lipases hydrolyze triglycerides within triglyceride-rich lipoproteins to glycerol and fatty acids. The fatty acids are carried across the cell membrane into the cytoplasm. In the cytoplasm, the fatty acids can be utilized to make triglycerides, phospholipids and cholesteryl esters. Cholesterol influences how the cell breaks down triglycerides. If cholesterol levels are high then triglyceride metabolism tends to occur more frequently in the lysosome. In contrast, when the cells have excess cholesterol, particularly within their lysosomes, the surface hydrolysis is suppressed and lipoproteins are taken up by endocytosis and delivered to lysosomes for hydrolysis. The effect of inhibiting the surface lipolysis was evaluated to determine the impact on intracellular cholesterol and triglyceride levels. The data indicates that the presence of triglyceride restores lysosomal metabolism of cholesterol.

Inhibitory effects of Aster spathulifolius extract on adipogenesis and lipid accumulation in 3T3-L1 preadipocytes.

Aster spathulifolius Maxim (AS), known for its anti-viral and anti-allergic activity, is also known to reduce body weight gain in high fat diet-induced obese rats. But its molecular mechanism of the anti-obesity effects is still unclear. So, we investigated the inhibitory effect of AS extract (ASE) on adipogenesis and lipid accumulation to determine the underlying cellular molecular mechanism. To perform this study, the contents of intracellular triglyceride were analysed. Real-time polymerase chain reaction and Western blotting were carried out to investigate the expression of adipogenic transcriptional factors. ASE showed the suppression of adipogenic differentiation and the considerable reduction of the lipid accumulation in 3T3-L1 cells. Especially, ASE inhibited the early stage of differentiation via the downregulation of C/EBP-β and C/EBP-δ, which are early adipogenic factors. Major adipogenic factors, such as PPAR-γ and C/EBP-α, were also subsequently inhibited. These findings were supported by Oil Red O staining and intracellular triglyceride levels. A molecular mechanism liking the effect of ASE was identified through the activation of AMPKα pathway. ASE increased protein levels of phosphorylated AMPKα and phosphorylated ACC. ASE showed anti-adipogenic and anti-lipogenic effects through the regulation of adipogenic factors and AMPKα pathway.

Amelioration of free fatty acid-induced fatty liver by quercetin-3-O-β-D-glucuronide through modulation of peroxisome proliferator-activated receptor-alpha/sterol regulatory element-binding protein-1c signaling.

To investigate the therapeutic effect and potential mechanisms of the natural flavonoid quercetin-3-O-β-D-glucuronide (Q3GA) against lipid metabolism disorder in free fatty acid (FFA)-induced fatty liver in vivo and in vitro. Fat accumulation was documented by oil red O staining, and intracellular triglyceride levels were detected by triglyceride(TG) enzymatic assay. Flow cytometry and enzyme-linked immunoassay assay were performed to observe the effect of Q3GA on lipotoxicity and inflammation response of primary rat hepatocytes with FFA treatment. Administration with Q3GA at doses of 25 and 50 mg/kg from the fifth week during high fat diet (HFD) induced non-alcoholic fatty liver disease (NAFLD) model for 8 weeks. Expression of the genes involved in the lipogenesis and fatty acid β-oxidation were assayed by reverse transcription polymerase chain reaction. Q3GA reduced bodyweight gain, liver weight, liver index, dyslipidemia and hepatic TG level in a dose-dependant manner. In the FFA-overloaded primary rat hepatocytes, Q3GA decreased the fat overload and TG content, inhibited hepatocyte apoptosis and reduced inflammation cytokine expression. Importantly, the histopathological examination of liver showed that Q3GA could decrease hepatic lipid accumulation and liver injury. Besides, Q3GA decreased the expression of sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase and increased the expression of peroxisome proliferator-activated receptor-α (PPAR-α), carnitine palmitoyl-transferase 1 and medium-chain acyl-coenzyme A dehydrogenase in vivo and in vitro. The therapeutic effect of Q3GA on lipid metabolism disorder in FFA-induced fatty liver rats is partly due to downregulating SREBP-1c and upregulating PPAR-α-mediated metabolic pathways.

녹차씨 압착박 열수 추출물의 지방세포 분화 억제 효과

The effect of the hot water extract of defatted green tea seed cake (GTSE) on lipid metabolism and the underlying mechanisms of lipolysis in mature 3T3-L1 adipocytes were investigated. In this study, we found that the naringenin content of GTSE was 5.5 ㎎/g; however, catechins were not detected. The intracellular lipid droplets were stained with Oil Red O dye and quantified. Compared to the control, lipid accumulation was significantly decreased by 52%, and intracellular triglyceride (TG) level was reduced by 33% after treatment with GTSE at a concentration of 40 ㎍/mL. To determine the mechanism of reduction in TG content, we determined the level of fatty acid synthase (FAS), phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), and acetyl-coenzyme A carboxylase (ACC) in the cell model. Incubation of the 3T3-L1 adipocytes with GTSE stimulated AMPK and ACC phosphorylation in a dosedependent manner, and decreased the expression of FAS.

HCBP6 Modulates Triglyceride Homeostasis in Hepatocytes Via the SREBP1c/FASN Pathway

Hypertriglyceridemia leads to liver steatosis, cardiovascular disease, and type 2 diabetes. Although HCBP6 (hepatitis C virus core-binding protein 6) was previously shown to be an HCV (hepatitis C virus) core-binding protein, its biological function remains unclear. Here, we demonstrate that HCBP6 negatively regulates intracellular triglyceride (TG) levels in hepatocytes. We found that bidirectional manipulation of hepatocyte HCBP6 expression by knockdown or overexpression results in increased or decreased TG accumulation, respectively. In addition, HCBP6 mRNA and protein levels exhibited significant time- and dose-dependent increases in a cellular model of lipid-overload hepatic steatosis. Furthermore, TG levels are regulated by HCBP6-sterol regulatory element binding protein 1c (SREBP1c)-mediated fatty acid synthase (FASN) expression. We also demonstrate that HCBP6 mRNA and protein expression is inhibited by microRNA-122 (miR-122), and miR-122 overexpression elicited more robust translational repression of luciferase activity driven by the full 3'-UTR of HCBP6. Taken together, our results provide new evidence that miR-122-regulated HCBP6 functions as a sensor protein to maintain intrahepatocyte TG levels.

Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain.

Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme.

Resveratrol ameliorates lipid accumulation in HepG2 cells, associated with down-regulation of lipin1 expression.

The pathogenesis of alcoholic fatty liver (AFL) disease is associated with the excessive accumulation of lipids in hepatocytes as well as oxidative stress. Resveratrol (RES), a dietary polyphenol found in red wine and grapes, has been shown to protect against AFL disease. However, the precise mechanisms that lead to this protective effect remain elusive. In this study, we used HepG2 cells to investigate the effects of RES on lipid metabolism and the mechanisms underlying these effects. HepG2 cells were cultured with oleic acid and alcohol for 48 h to induce excessive lipid accumulation. Oil red O staining showed that administration of oleic acid and alcohol induced more lipid accumulation than was observed in the control group, and that RES (15, 45, or 135 μmol/L) treatment reduced intracellular lipid droplets. RES treatment also significantly attenuated hepatic steatosis and lowered levels of intracellular triglycerides (TG). Western blot analysis showed that RES enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and down-regulated the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and lipin1. However, compound C, an AMPK inhibitor, reversed these effects of RES. In conclusion, RES reduced lipid accumulation and protected HepG2 cells. This effect may be associated with the down-regulation of SREBP-1c and lipin1 expression, increased levels of phosphorylated AMPK and ACC, and the activation of AMPK-lipin1 signaling.

Activation of OR1A1 suppresses PPAR-γ expression by inducing HES-1 in cultured hepatocytes

Olfactory receptors (ORs) comprise the largest G protein-coupled receptor gene superfamily. Recent studies indicate that ORs are also expressed in non-olfactory organs, including metabolically active tissues, although their biological functions in these tissues are largely unknown. In this study, OR1A1 expression was detected in HepG2 liver cells. OR1A1 activation by (−)-carvone, a known OR1A1 ligand, increased the cyclic adenosine monophosphate (cAMP), but not intracellular Ca2+ concentration, thereby inducing protein kinase A (PKA) activity with subsequent phosphorylation of cAMP response element-binding protein (CREB) and upregulation of the CREB-responsive gene hairy and enhancer of split (HES)-1, a corepressor of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hepatocytes. In (−)-carvone-stimulated cells, the repression of PPAR-γ reduced the expression of the target gene, mitochondrial glycerol-3-phosphate acyltransferase, which encodes a key enzyme involved in triglyceride synthesis. Intracellular triglyceride level and lipid accumulation were reduced in cells stimulated with (−)-carvone, effects that were diminished following the loss of OR1A1 function. These results indicate that OR1A1 may function as a non-redundant receptor in hepatocytes that regulates the PKA-CREB-HES-1 signaling axis and thereby modulates hepatic triglyceride metabolism.

Effects of individual and multiple fatty acids (palmitate, oleate and docosahaexenoic acid) on cell viability and lipid metabolism in LO2 human liver cells.

This study was designed to investigate the direct effects of fatty acids (FAs) on the cell viability and the expression levels of genes involved in lipid metabolism in LO2 human liver cells. Palmitate (PA), oleate (OA) and docosahaexenoic acid (DHA) were used to represent saturated, mono-unsaturated and polyunsaturated FAs, respectively. At concentrations of ≤3.2 µg/ml, treatment with single FAs increased the viability of the LO2 cells. At FA concentrations of >3.2 µg/ml, cell viability following OA treatment was increased, but PA or DHA treatment at these concentrations reduced cell viability. Administration of mixtures of these FAs in three ratios (PA:OA:DHA = 1:2:1, 1:1:1 and 1:1:2, respectively) increased the cell viability compared with the control group. The intracellular triglyceride (TG) levels following all types of treatment were significantly increased and the accumulation of TGs was markedly increased with high doses of DHA. In addition, peroxisome proliferator-activated receptor-γ was significantly upregulated in all groups, with the exception of the 1:1:1 group at 3.2 µg/ml and the 1:1:2 group at 12.8 µg/ml. The expression levels of sterol regulatory-element binding protein‑1c, liver X receptor α and apolipoprotein C‑I were significantly reduced in all groups with the exception of the DHA‑treated group and the 1:2:1 groups at 3.2 and 12.8 µg/ml. In conclusion, these results indicate that the type, concentration and mixture ratios of FAs are all important in determining the cell viability and lipid metabolism-related gene expression in LO2 hepatocytes.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) levels in the form of large lipid droplets (LDs), reduced adenosine triphosphate (ATP) levels, and elevated reactive oxygen species (ROS) levels, and this contributes to their elevated sensitivity to ischemia/reperfusion injury during transplantation. Reducing macrosteatosis in living donors through dieting has been shown to improve transplant outcomes. Accomplishing the same feat for deceased donor grafts would require ex vivo exposure to potent defatting agents. Here we used a rat hepatocyte culture system exhibiting a macrosteatotic LD morphology, elevated TG levels, and an elevated sensitivity to hypoxia/reoxygenation (H/R) to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48 hours with a defatting cocktail that was previously developed to promote TG catabolism reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but it did not ameliorate sensitivity to H/R. Supplementation of this cocktail with l-carnitine, together with hyperoxic exposure, yielded a similar reduction in the number of macrosteatotic LDs and a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl coenzyme A to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27% (to levels near those of lean controls), and completely abolished H/R sensitivity as indicated by approximately 85% viability after H/R and the reduction of cytosolic lactate dehydrogenase release to levels seen in lean controls. Cultures maintained for 48 hours after H/R were approximately 83% viable and exhibited superior urea secretion and bile canalicular transport in comparison with untreated macrosteatotic cultures. In conclusion, these findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, and they suggest a possible route for the recovery of discarded macrosteatotic grafts.

Effect of intermittent hypoxia on lipid metabolism in liver cells and the underlying mechanism

To determine the effect of intermittent hypoxia on lipid metabolism in liver cells and to explore the possible molecular pathways involved in this process. An intermittent hypoxia cell model system was established by incubating the human hepatic cell lines L02 and HepG2 in an atmosphere of 2% O₂, 5% CO₂ and 93% N₂ for 8 hours per day over a period of 1, 2, 3, 4 or 5 days. Cells cultured in normoxia conditions (21% O₂) served as controls. Changes in intracellular lipid droplets and triglyceride (TG) levels were assessed by biochemical assays and oil red staining. Changes in intracellular reactive oxygen species (ROS) were assessed by inverted fluorescence microscopy and flow cytometry. Changes in expression of hypoxia-inducible factor (HIF)-1a and HIF-2a proteins, and the downstream ADFP, SREBP-1c and FAS proteins, were detected by western blotting. For both L02 and HepG2 cell lines, the cells grown under hypoxic conditions showed significantly higher lipid droplet accumulation and TG content than the cells grown under normoxic conditions (F(L02) =61.83, FHepG2 =104.19, P less than 0.01). Both oxygen concentration and time appeared to be correlated with these lipid-related changes (F(L02) =39.60, FHepG2 =76.39, P less than 0.01). The ROS fluorescence index was significantly increased after 2 days of intermittent hypoxia L02: 0.703 ± 0.129 vs. 3.310 ± 0.198, t =22.0637 and HepG2:0.617 ± 0.156 vs. 2.33 ± 0.42, t =7.2003, P < 0.05); in addition, increasing trends were observed in the ROS content and intensity of green fluorescence in conjunction with increased time of exposure to intermittent hypoxia (F(L02) =1021.84, FHepG2 =49.89, P less than 0.01). Compared with their respective control groups, the L02 and HepG2 cells both showed significantly upregulated expression of HIF-1a ADFP, SREBP-1c and FAS (L02:FHIF-1a =371.19, FsREBP-1c =204.49, FFAS =38.20, FADFP =154.31, P less than 0.05 and HepG2:FHF-1a =150.84, FSRERBP-1c =107.35, FFAs =279.71, FADFP =352.06, P less than 0.01). In contrast, the HIF-2a level was markedly decreased in the cells after 4 and 5 days of exposure to intermittent hypoxia (F(L02) =125.29, FHcpG2 =10.68, P less than 0.05). Under intermittent hypoxic conditions, ROS may regulate the expression of hypoxia-inducible factors and the adipose differentiation-related protein,as well as influence fatty acid metabolism via a HIF-1 a-SREBP-1 c-FAS signal and upregulation of the ADFP protein, in liver cells.

Comparative gene identification-58/α/β hydrolase domain 5

Comparative gene identification-58 (CGI-58) is a lipid droplet-associated protein that controls intracellular triglyceride levels by its ability to activate adipose triglyceride lipase (ATGL). Additionally, CGI-58 was described to exhibit lysophosphatidic acid acyl transferase (LPAAT) activity. This review focuses on the significance of CGI-58 in energy metabolism in adipose and nonadipose tissue. Recent studies with transgenic and CGI-58-deficient mouse strains underscored the importance of CGI-58 as a regulator of intracellular energy homeostasis by modulating ATGL-driven triglyceride hydrolysis. In accordance with this function, mice and humans that lack CGI-58 accumulate triglyceride in multiple tissues. Additionally, CGI-58-deficient mice develop an ATGL-independent severe skin barrier defect and die soon after birth. Although the premature death prevented a phenotypical characterization of adult global CGI-58 knockout mice, the characterization of mice with tissue-specific CGI-58 deficiency revealed new insights into its role in neutral lipid and energy metabolism. Concerning the ATGL-independent function of CGI-58, a recently identified LPAAT activity for CGI-58 was shown to be involved in the generation of signaling molecules regulating inflammatory processes and insulin action. Although the function of CGI-58 in the catabolism of cellular triglyceride depots via ATGL is well established, further studies are required to consolidate the function of CGI-58 as LPAAT and to clarify the involvement of CGI-58 in the metabolism of skin lipids.