Insulin-induced Gene Research Articles

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS-induced mastitis in dairy cows.

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long-term manipulated glucose and insulin concentrations in combination with a LPS-induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28±6weeks of lactation) were randomly assigned to one of three infusion treatments for 56h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT-qPCR. During the 48h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha-lactalbumin, insulin-induced gene 1, κ-casein and acetyl-CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS-induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.

Amla prevents fructose-induced hepatic steatosis in ovariectomized rats: role of liver FXR and LXRα

Objectives Increased fructose consumption causes dyslipidemia and fatty liver in postmenopausal women, both independent risk factors for cardiovascular disease. This study explored the potential mechanisms by which amla (Emblica officinalis) reduced hypercholesterolemia and hypertriglyceridemia and prevented fatty liver in a fructose-fed, ovariectomized rat model of menopause.Methods Sham-operated and ovariectomized rats were put on a chow or high fructose diet. They were further divided into groups with or without amla. After 18 weeks of treatment, livers were harvested and subjected to Western blot and histological analyses.Results In all groups, amla increased the protein expression of liver farnesoid X receptor (FXR) and liver X receptor (LXR), key proteins involved in lipid metabolism. Fructose-fed rats developed fatty liver and amla prevented this. Here amla produced an exceptional rise in LXR and insulin-induced gene-2 (Insig-2) which prevented the maturation of sterol regulatory element-binding protein-1 and steroyl CoA desaturase-1, responsible for triglyceride synthesis. Amla also increased the protein expression of ATP binding cassette transporter A1 (ABCA1), involved in high density lipoprotein (HDL) synthesis as well as low density lipoprotein receptor (LDLR) responsible for uptake of LDL cholesterol. Besides this, amla increased the protein expression of peroxisome proliferator activated receptor α (PPARα) involved in β oxidation of fatty acids.Conclusions Amla increased the protein expression of liver FXR, LXRα, PPARα and their downstream proteins Insig-2, ABCA1 and LDLR. This property of amla to modulate some of the key proteins involved in lipid metabolism promises its usefulness as a preventive agent for dyslipidemia and hepatic steatosis.

Retinoid acid-related orphan receptor γ, RORγ, participates in diurnal transcriptional regulation of lipid metabolic genes.

The hepatic circadian clock plays a pivotal role in regulating major aspects of energy homeostasis and lipid metabolism. In this study, we show that RORγ robustly regulates the rhythmic expression of several lipid metabolic genes, including the insulin-induced gene 2a, Insig2a, elongation of very long chain fatty acids-like 3, Elovl3 and sterol 12α-hydroxylase, Cyp8b1, by enhancing their expression at ZT20-4. The time-dependent increase in their expression correlates with the rhythmic expression pattern of RORγ. The enhanced recruitment of RORγ to ROREs in their promoter region, increased histone acetylation, and reporter and mutation analysis support the concept that RORγ regulates the transcription of several lipid metabolic genes directly by binding ROREs in their promoter regulatory region. Consistent with the disrupted expression of a number of lipid metabolic genes, loss of RORγ reduced the level of several lipids in liver and blood in a ZT-preferred manner. Particularly the whole-body bile acid pool size was considerably reduced in RORγ−/− mice in part through its regulation of several Cyp genes. Similar observations were made in liver-specific RORγ-deficient mice. Altogether, our study indicates that RORγ functions as an important link between the circadian clock and the transcriptional regulation of several metabolic genes.

Activation of sterol regulatory element-binding proteins in mice exposed to perfluorooctanoic acid for 28days.

Perfluoroalkyl acids are widely used in numerous industrial and commercial applications due to their unique physical and chemical characteristics. Although perfluorooctanoic acid (PFOA) is associated with hepatomegaly through peroxisome proliferator-activated receptor α (PPARα) activation, liver fat accumulation and changes in gene expression related to fatty acid metabolism could still be found in PPARα-null mice exposed to PFOA. To explore the potential effects of PFOA on sterol regulatory element-binding proteins (SREBPs) activity, male mice were dosed with either Milli-Q water or PFOA at doses of 0.08, 0.31, 1.25, 5, and 20mg/kg/day by gavage for 28days. Liver total cholesterol concentrations and PFOA contents showed a dose-dependent decrease and increase, respectively. Transcriptional activity of PPARα and SREBPs was significantly enhanced in livers. Protein expression analyzed by Western blotting showed that PFOA exposure stimulated SREBP maturation. Furthermore, proteins blocked SREBP precursor transport, insulin-induced gene 1 (INSIG1) and INSIG2 proteins, as well as a protein-mediated nuclear SREBP proteolysis, F-box and WD-40 domain protein 7, decreased in mouse liver exposed to PFOA. The expression levels of the miR-183-96-182 cluster, which is possibly involved in a regulatory loop intermediated by SREBPs maturation, were also increased in the mouse liver after PFOA exposure. We also observed that PFOA induced lipid content and PPARα in Hepa 1-6 cells after exposure to PFOA for 72h but SREBPs were not activated in vitro. These results demonstrated that SREBPs were maturated by activating the miR-183-96-182 cluster-SREBP regulatory loop in PFOA-exposed mouse liver.

Inhibition of microRNA-24 expression in liver prevents hepatic lipid accumulation and hyperlipidemia

The incidence of nonalcoholic fatty liver disease (NAFLD) and hyperlipidemia, with their associated risks of endstage liver and cardiovascular diseases, is increasing rapidly due to the prevalence of obesity. Although the mechanisms of NAFLD have been studied extensively, the underlying pathogenesis and the role of microRNAs in this process remain relatively unclear. MicroRNA (miRNA)-dependent posttranscriptional gene silencing is now recognized as a key element of lipid metabolism. Here we report that the expression of microRNA-24 (miR-24) is significantly increased in the livers of high-fat diet-treated mice and in isolated human hepatocytes incubated with fatty acid. Knockdown of miR-24 in those mice caused impaired hepatic lipid accumulation and reduced plasma triglycerides. Bioinformatic and in vitro and in vivo studies led us to identify insulin-induced gene 1 (Insig1), an inhibitor of lipogenesis, as a novel target of miR-24. Inhibition of endogenous miR-24 expression by way of miR-24 inhibitors led to up-regulation of Insig1, and subsequently decreased hepatic lipid accumulation. It is well established that liver-specific deletion of Insig1 leads to higher hepatic and plasma triglyceride levels by inhibiting the processing of sterol regulatory element-binding proteins (SREBPs), transcription factors that activate lipid synthesis. As expected, miR-24 knockdown prevented SREBP processing, and subsequent expression of lipogenic genes. In contrast, the opposite result was observed with overexpression of miR-24, which enhanced SREBP processing. Thus, our study defines a potentially critical role for deregulated expression of miR-24 in the development of fatty liver by way of targeting of Insig1. Our findings show a novel mechanism by which miR-24 promotes hepatic lipid accumulation and hyperlipidemia by repressing Insig1, and suggest the use of miR-24 inhibitor as a potential therapeutic agent for NAFLD and/or atherosclerosis.

A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

Dietary intake of palmitate and oleate has broad impact on systemic and tissue lipid profiles in humans

Epidemiologic evidence has suggested that diets with a high ratio of palmitic acid (PA) to oleic acid (OA) increase risk of cardiovascular disease (CVD). To gain additional insights into the relative effect of dietary fatty acids and their metabolism on CVD risk, we sought to identify a metabolomic signature that tracks with diet-induced changes in blood lipid concentrations and whole-body fat oxidation. We applied comprehensive metabolomic profiling tools to biological specimens collected from 18 healthy adults enrolled in a crossover trial that compared a 3-wk high-palmitic acid (HPA) with a low-palmitic acid and high-oleic acid (HOA) diet. A principal components analysis of the data set including 329 variables measured in 15 subjects in the fasted state identified one factor, the principal components analysis factor in the fasted state (PCF1-Fasted), which was heavily weighted by the PA:OA ratio of serum and muscle lipids, that was affected by diet (P < 0.0001; HPA greater than HOA). One other factor, the additional principal components analysis factor in the fasted state (PCF2-Fasted), reflected a wide range of acylcarnitines and was affected by diet in women only (P = 0.0198; HPA greater than HOA). HOA lowered the ratio of serum low-density lipoprotein to high-density lipoprotein (LDL:HDL) in men and women, and adjustment for the PCF1-Fasted abolished the effect. In women only, adjustment for the PCF2-Fasted eliminated the HOA-diet effect on serum total- and LDL-cholesterol concentrations. The respiratory exchange ratio in the fasted state was lower with the HPA diet (P = 0.04), and the diet effect was eliminated after adjustment for the PCF1-Fasted. The messenger RNA expression of the cholesterol regulatory gene insulin-induced gene-1 was higher with the HOA diet (P = 0.008). These results suggest that replacing dietary PA with OA reduces the blood LDL concentration and whole-body fat oxidation by modifying the saturation index of circulating and tissue lipids. In women, these effects are also associated with a higher production and accumulation of acylcarnitines, possibly reflecting a shift in fat catabolism.

Controversial association results for INSIG2 on body mass index may be explained by interactions with age and with MC4R

Among the single-nucleotide polymorphisms (SNPs) previously reported to be associated with body mass index (BMI) and obesity, we focus on a common risk variant rs7566605 upstream of the insulin-induced gene 2 (INSIG2) gene and a rare protective variant rs2229616 on the melanocortin-4 receptor (MC4R) gene. INSIG2 is involved in adipogenesis and MC4R effects hormonal appetite control in response to the amount of adipose tissue. The influence of rs2229616 (MC4R) on BMI and obesity has been confirmed repeatedly and insight into the underlying mechanism provided. However, a main effect of rs7566605 (INSIG2) is under debate because of inconsistent replications of association. Interaction of rs7566605 with age may offer an explanation. SNP-age and SNP-SNP interaction models were tested on independent individuals from three population-based longitudinal cohorts, restricting the analysis to an observed age of 25-74 years. KORA S3/F3, KORA S4/F4 (Augsburg, Germany, 1994-2005, 1999-2008), and Framingham-Offspring data (Framingham, USA, 1971-2001) were analysed, with a total sample size of N=6926 in the joint analysis. The effect of interaction between rs7566605 and age on BMI and obesity status is significant and consistent across studies. This new evidence for rs7566605 (INSIG2) complements previous research. In addition, the interaction effect of rs7566605 with the MC4R variant rs2229616 on BMI was observed. This effect size was three times larger than that in a previously reported single-locus main effect of rs2229616. This leads to the conclusion that SNP-age or SNP-SNP interactions can mask genetic effects for complex diseases if left unaccounted for.

Autism Associated Gene, ENGRAILED2, and Flanking Gene Levels Are Altered in Post-Mortem Cerebellum

BackgroundPrevious genetic studies demonstrated association between the transcription factor ENGRAILED2 (EN2) and Autism Spectrum Disorder (ASD). Subsequent molecular analysis determined that the EN2 ASD-associated haplotype (rs1861972-rs1861973 A-C) functions as a transcriptional activator to increase gene expression. EN2 is flanked by 5 genes, SEROTONIN RECEPTOR5A (HTR5A), INSULIN INDUCED GENE1 (INSIG1), CANOPY1 HOMOLOG (CNPY1), RNA BINDING MOTIF PROTEIN33 (RBM33), and SONIC HEDGEHOG (SHH). These flanking genes are co-expressed with EN2 during development and coordinate similar developmental processes. To investigate if mRNA levels for these genes are altered in individuals with autism, post-mortem analysis was performed.MethodsqRT-PCR quantified mRNA levels for EN2 and the 5 flanking genes in 78 post-mortem cerebellar samples. mRNA levels were correlated with both affection status and rs1861972-rs1861973 genotype. Molecular analysis investigated whether EN2 regulates flanking gene expression.Results EN2 levels are increased in affected A-C/G-T individuals (p = .0077). Affected individuals also display a significant increase in SHH and a decrease in INSIG1 levels. Rs1861972-rs1861973 genotype is correlated with significant increases for SHH (A-C/G-T) and CNPY1 (G-T/G-T) levels. Human cell line over-expression and knock-down as well as mouse knock-out analysis are consistent with EN2 and SHH being co-regulated, which provides a possible mechanism for increased SHH post-mortem levels.Conclusions EN2 levels are increased in affected individuals with an A-C/G-T genotype, supporting EN2 as an ASD susceptibility gene. SHH, CNPY1, and INSIG1 levels are also significantly altered depending upon affection status or rs1861972-rs1861973 genotype. Increased EN2 levels likely contribute to elevated SHH expression observed in the post-mortem samples

Variation in the MC4R Gene Is Associated with Bone Phenotypes in Elderly Swedish Women

Osteoporosis is characterized by reduced bone mineral density (BMD) and increased fracture risk. Fat mass is a determinant of bone strength and both phenotypes have a strong genetic component. In this study, we examined the association between obesity associated polymorphisms (SNPs) with body composition, BMD, Ultrasound (QUS), fracture and biomarkers (Homocysteine (Hcy), folate, Vitamin D and Vitamin B12) for obesity and osteoporosis. Five common variants: rs17782313 and rs1770633 (melanocortin 4 receptor (MC4R); rs7566605 (insulin induced gene 2 (INSIG2); rs9939609 and rs1121980 (fat mass and obesity associated (FTO) were genotyped in 2 cohorts of Swedish women: PEAK-25 (age 25, n = 1061) and OPRA (age 75, n = 1044). Body mass index (BMI), total body fat and lean mass were strongly positively correlated with QUS and BMD in both cohorts (r2 = 0.2–0.6). MC4R rs17782313 was associated with QUS in the OPRA cohort and individuals with the minor C-allele had higher values compared to T-allele homozygotes (TT vs. CT vs. CC: BUA: 100 vs. 103 vs. 103; p = 0.002); (SOS: 1521 vs. 1526 vs. 1524; p = 0.008); (Stiffness index: 69 vs. 73 vs. 74; p = 0.0006) after adjustment for confounders. They also had low folate (18 vs. 17 vs. 16; p = 0.03) and vitamin D (93 vs. 91 vs. 90; p = 0.03) and high Hcy levels (13.7 vs 14.4 vs. 14.5; p = 0.06). Fracture incidence was lower among women with the C-allele, (52% vs. 58%; p = 0.067). Variation in MC4R was not associated with BMD or body composition in either OPRA or PEAK-25. SNPs close to FTO and INSIG2 were not associated with any bone phenotypes in either cohort and FTO SNPs were only associated with body composition in PEAK-25 (p≤0.001). Our results suggest that genetic variation close to MC4R is associated with quantitative ultrasound and risk of fracture.

Association of INSIG2 rs9308762 With ALT Level Independent of BMI

An increasing number of people are at risk for developing nonalcoholic fatty liver disease (NAFLD). Because obesity is a risk factor for NAFLD, the common variants of obesity-susceptible genes may be associated with NAFLD. Our aim was to identify whether the obesity-susceptible gene variants (rs9939609, rs9930506, and rs4783819 in fat mass and obesity-associated gene (FTO); rs12970134 and rs17782313 in melanocortin-4 receptor gene (MC4R); and rs7566605, rs13428113, and rs9308762 in insulin-induced gene 2 [INSIG2]) were associated with NAFLD. The case-control study recruited 1027 Chinese children ages 7 to 18 years, including 162 children with NAFLD and 865 children without NAFLD. Anthropometric measurements, alanine transaminase (ALT) detection, liver ultrasound examination, and genotyping of 8 gene variants were performed. The A-allele of FTO rs9939609 was associated with increased NAFLD risk (P = 0.029, odds ratio 1.43), but was not significant after being adjusted for body mass index (BMI) (P = 0.268). We also found an association between the 2 variants (rs12970134 in MC4R and rs9308762 in INSIG2) and ALT level. For rs12970134, each additional A-allele increased ALT level by 1.87 IU/L (P = 0.032). For rs9308762, the homozygotes of the C-allele had a higher ALT level than the T-allele carriers (β = 3.19, P = 0.007). After adjustment for BMI, the former association did not exist, whereas the latter reminded significant (P = 0.003). The FTO rs9939609 A-allele increased risk of NAFLD and MC4R rs12970134 was associated with ALT level through an effect on BMI. The association between INSIG2 rs9308762 and ALT level was independent of BMI. The results provided evidence for identifying genetic factors of NAFLD and may be useful for risk assessment and personalized medicine of NAFLD.

Assisted reproductive technologies impair the expression and methylation of insulin-induced gene 1 and sterol regulatory element-binding factor 1 in the fetus and placenta

To evaluate the cholesterol metabolism linked to assisted reproductive technology (ART) by analyzing the expression levels and DNA methylation patterns of the insulin-induced gene (INSIG), sterol regulatory element-binding protein (SREBP), and SREBP cleavage-activating protein in the fetus and placenta. Experimental research study. An IVF center, university-affiliated teaching hospital. Four patients groups were recruited: pregnancies after IVF/intracytoplasmic sperm injection (ICSI) (n = 55), natural pregnancies (n = 40), multifetal reduction after IVF/ICSI (n = 56), and multifetal reduction after controlled ovarian hyperstimulation (COH) (n = 42). Expression and DNA methylation of INSIG-SREBP- SREBP cleavage-activating protein in the fetus and placenta samples were determined. The expression and DNA methylation patterns were tested by real-time quantitative polymerase chain reaction (PCR) and pyrosequencing. In the ICSI treatment group, significantly higher levels of triglycerides and apolipoprotein-B were observed in cord blood compared with controls. Meanwhile, in ICSI-conceived fetuses, the expression of INSIG1 was significantly higher, and methylation rates were lower, than in the IVF and control groups. Furthermore, in the placenta, the INSIG1 and SREBF1 transcripts were also significantly higher with lower methylation rates in the ICSI group than in the IVF and control groups. Our results indicated that the dysregulation of INSIG1 and SREBF1 caused by ART were observed not only in the fetus but also in the placenta, primarily in the ICSI group. However, the long-term sequelae of this dysregulation should be closely followed.

Dietary macronutrients, genetic variation, and progression of coronary atherosclerosis among women

Previous studies observed the surprising finding that saturated fat was inversely associated with atherosclerosis progression in postmenopausal women, whereas polyunsaturated fat (PUFA) and carbohydrates were positively associated. Whether certain genes modify the association of diet with atherosclerotic progression is unknown. Using Haplotype-tagging single nucleotide polymorphisms, we evaluated gene-diet interactions with 3 preselected genes involved in fatty acid and carbohydrate metabolism: sterol regulatory element binding protein-1 (SREBP1), insulin-induced gene-1 (INSIG1), and SREBP cleavage-activating protein (SCAP). Diet was assessed at baseline. Quantitative coronary angiography was performed at baseline and after a mean of follow-up of 3.09 years in 2,227 coronary segments in 234 postmenopausal women. Global effects of each gene and gene-diet interactions for different fats, total fat, and carbohydrate were evaluated. Global tests revealed no main effects between SCAP, INSIG1, and SREBP1 haplotypes and progression of atherosclerosis (P = .87, P = .58, and P = .44). After correction for 5 nutrients evaluated (Bonferroni-corrected 2-tailed α = .01), no significant gene-nutrient interactions were seen, except for a borderline global interaction between SREBP1 and PUFA intake (P interaction = .013). This interaction was specific to the G-C haplotype (frequency 35%) and was driven by n-6 rather than n-3 PUFA (P for interaction < .0001). The interaction was robust to estimated isocaloric replacement of PUFA with any other nutrient. Per each 5% energy from n-6 PUFA, a 0.21-mm greater decline in mean minimal coronary artery diameter was seen among women per each copy of the second most frequent haplotype of SREBP1. We observed an interaction between SREBP1 and PUFA consumption that might explain the positive association of PUFA with atherosclerosis progression in this cohort.

Genetic associations of the INSIG2 rs7566605 polymorphism with obesity-related metabolic traits in Malaysian Malays.

A genome-wide association study showed that the tagging single nucleotide polymorphism (SNP) rs7566605 in the insulin-induced gene 2 (INSIG2) was associated with obesity. Attempts to replicate this result in different populations have produced inconsistent findings. We aimed to study the association between the rs7566605 SNP with obesity and other metabolic parameters in Malaysian Malays. Anthropometric and obesity-related metabolic parameters and DNA samples were collected. We genotyped the rs7566605 polymorphism in 672 subjects using real-time polymerase chain reaction. No significant associations were found between the rs7566605 tagging SNP of INSIG2 with obesity or other metabolic parameters in the Malaysian Malay population. The INSIG2 rs7566605 SNP may not play a role in the development of obesity-related metabolic traits in Malaysian Malays.

Scavenger receptor CD36 mediates inhibition of cholesterol synthesisviaactivation of the PPARγ/PGC‐1α pathway and Insig1/2 expression in hepatocytes

The scavenger receptor CD36 plays a central role in lipid metabolism by promoting macrophage cholesterol efflux with the potential to reduce atherosclerotic lesions. However, the effect of CD36 on de novo cholesterol synthesis is not known. Here, we describe the cellular mechanism by which CD36 activation induces cholesterol depletion in HepG2 cells. Using the CD36 ligand hexarelin, we found a rapid phosphorylation of HMG-CoA reductase Ser-872 in treated cells, resulting in inactivation of the rate-limiting enzyme in sterol synthesis. Degradation of HMG-CoA reductase by the ubiquitin-proteasome pathway was also enhanced by hexarelin, through an increased recruitment of the anchor proteins insulin-induced gene (Insig)-1 and Insig-2. Genes encoding key enzymes involved in cholesterol synthesis and under the control of transcription factor sterol regulatory element-binding protein (SREBP)-2 remained unresponsive to sterol depletion, due to retention of the SREBP-2 escort protein Scap by Insig-1/2. Insig1 and Insig2 gene expression was also increased through activation of nuclear receptor peroxisome-proliferator activating receptor γ (PPARγ) by CD36, which lifted the inhibitory effect of PPARγ1 Ser-84 phosphorylation. Recruitment of coactivator peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) to activated AMPKα was also promoted, resulting in PGC-1α transcriptional activation through Sirt1-mediated deacetylation, increased recruitment of PPARγ, and up-regulation of Insig-1/2, revealing a regulatory role of CD36 on PGC-1α signaling. Our data identify CD36 as a novel regulator of HMG-CoA reductase function and Insig-1/2 expression, 2 critical steps regulating cholesterol synthesis in hepatocytes.

The Expression of Neuronal Sorting Nexin 8 (SNX8) Exacerbates Abnormal Cholesterol Levels

SNX8 is a PX-BAR domain sub-family of sorting nexins (SNXs), which is reported as a β-amyloid (Aβ) toxicity enhancer and associated with Alzheimer's disease. We have also described SNX8 as a novel activator of the sterol regulatory element binding protein (SREBP) transcription factor, a major regulator of cholesterol homeostasis. In that study, we have showed that SNX8 reduced an insulin-induced gene (INSIG)-dependent block of SREBP-mediated transcription. Here, for the first time, we investigated the expression and function of SNX8 within the CNS. We found that SNX8 was expressed within neurons, but not astrocytes or microglia, with neuronal localisation primarily in the soma. The protein levels of neuronal SNX8 were unchanged in the presence of moderately high cholesterol but were decreased by mevinolin (a cholesterol-lowering statin) and U18666a (which causes cholesterol to accumulate within the lysosome). To determine if SNX8 overexpression alters the levels of cholesterol, we engineered a GFP-SNX8 lentivirus. The overexpression of GFP-SNX8 had no effect on cholesterol in neurons under control conditions or in already strongly altered cholesterol conditions of mevinolin or U18666a. In contrast, in moderately high cholesterol, the overexpression of GFP-SNX8 caused redistribution of cholesterol within neurons creating a phenotype similar to U18666a treatment. Taken together, these data suggest that extreme changes in cholesterol reduce SNX8 expression and that overexpression of SNX8 exacerbates aberrant handling of neuronal cholesterol. This work further supports the role for SNX8 in regulating cholesterol levels, which could be important in understanding its role as an Aβ toxicity enhancer and its association with Alzheimer's disease.

Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

Response to a Long-term High-Fat Diet in the Signature of Genes Involved in Lipid Metabolism in ApoE−/− Mice

High-fat diet (HFD)-induced lipid metabolism disorders are a critical feature of diet-induced insulin resistance (IR); however, the mechanisms underlying the tissue-specific effects of HFDs on the signature of genes involved in lipid metabolism have not been fully investigated. Glucose homeostasis and insulin sensitivity were monitored in chow-fed and HFD-fed ApoE−/− mice using the hyperinsulinemiceuglycemic clamp technique. The signatures of genes involved in lipid metabolism were measured in the liver and adipose tissues by quantitative real-time polymerase chain reaction and Western blot analysis. Long-term HFD (for 16 weeks) resulted in marked abnormalities in glucose and lipid metabolism and induced IR in ApoE−/− mice. Long-term HFD also markedly upregulated the messenger RNA (mRNA) expression of insulin-induced gene-2, sterol regulatory element-binding protein cleavage-activating protein and sterol regulatory element-binding protein-1 in the liver and/or adipose tissues, in parallel with an elevated insulin-induced gene-2 protein in the liver. However, HFD markedly downregulated the mRNA expression of sterol regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase and low-density lipoprotein receptor in the liver. Interestingly, long-term HFD markedly decreased adipose triglyceride lipase and peroxisome proliferator-activated receptor-γ mRNA, and adipose triglyceride lipase protein contents in the liver and/or adipose tissues. These findings provide a framework to understand the mechanisms by which long-term HFD regulates the gene signatures involved in lipid metabolism in the pathogenesis of diet-induced IR.

FTO and INSIG2 Genotyping Combined with Metabolic and Anthropometric Phenotyping of Morbidly Obese Patients

Obesity is a major health problem worldwide. Associations of obesity with common variants of the fat mass- and obesity-associated gene (FTO) and insulin-induced gene 2 (INSIG2) have been reported in various studies. We aimed to further investigate the association of 2 single nucleotide polymorphisms (SNPs), rs9939609 in FTO and rs7566605 in INSIG2, with body mass index (BMI) and other anthropometric and metabolic parameters in subjects with morbid obesity (BMI ≥40). SNPs rs9939609 and rs7566605 were genotyped in 124 unrelated morbidly obese patients (mean BMI = 50, range 40.1-77.1) from Mainz, Germany, and in 253 normal controls without a history of morbid obesity. Metabolic and anthropometric parameters were analyzed in 109 of the 124 patients, and associations with the genotype data were examined. The high-risk AA genotype for FTO rs9939609 was observed in 32.3% of patients versus 15.8% of controls (p = 0.0004) and was associated with an increased obesity risk [odds ratio (OR) = 2.54, 95% confidence interval (CI) = 1.53-4.21]. The intermediate-risk AT genotype was found in patients and controls at similar frequencies (48.4 vs. 48.6%, OR = 0.99). The low-risk TT genotype for rs9939609 was found in 19.4% of patients (35.5% of controls; p = 0.0013) and was associated with a decreased risk for morbid obesity (OR = 0.43, CI = 0.26-0.73). In contrast, INSIG2 rs7566605 showed no association with obesity in our patients. Evaluation of metabolic data indicated associations between the high-risk FTO genotype (rs9939609_AA) and increased levels of serum glutamic oxaloacetic transaminase (GOT) and between the high-risk INSIG2 genotype (rs7566605_CC) and lower waist-to-hip ratio and lower hemoglobin A1c (HbA1c) levels. Our results confirm an association of the FTO SNP with extreme obesity. However, we found no association of the potential obesity risk allele of INSIG2 in our sample and thus cannot confirm an association of the INSIG2 CC genotype with obesity. We identified an association between the high-risk FTO genotype (rs9939609_AA) and higher GOT levels, which could possibly reflect the increased frequency of nonalcoholic steatohepatitis with obesity. We also detected associations of the high-risk INSIG2 genotype (rs7566605_CC) with lower waist-to-hip ratios and lower HbA1c levels, which may indicate amelioration of impaired glucose tolerance and type 2 diabetes for patients with this genotype after bariatric surgery.

Association between intramuscular fat in the arm following arm training and INSIG2

Insulin-induced gene 2 (INSIG2) single-nucleotide polymorphism (SNP rs7566605) is linked to lipid metabolism, and this study assessed its potential influence on fat in the upper arm following arm training. Twenty healthy sedentary volunteers (22.0 ± 1.1 years, body mass index 25.4 ± 4.0 kg/m(2) ; mean ± standard deviation) carried out a 12-week two-arm elbow extensor training (10 maximal extensions with 1 min recovery between bouts) five times per day, five times per week. For 17 volunteers, upper arm muscle and adipose tissue [subcutaneous (SCAT) and intramuscular (IMAT)] volumes were evaluated by magnetic resonance imaging before, immediately after, and 12 months after training and variables were related to the subjects' INSIG2 SNP rs7566605 genotype. Muscle volume and SCAT for the upper arm, as the decrease in IMAT during training were not related to INSIG2 SNP rs7566605: GG: %IMAT 1.0 ± 0.9%; GC/CC: %IMAT 0.6 ± 0.5% (P > 0.05). However, in the year following the training, accumulation of upper arm IMAT was twice as large in participants homozygous for the G allele (GG: Δ%IMAT +2.5 ± 0.8%; GC/CC: Δ%IMAT +1.1 ± 0.7%; P < 0.01). This study suggests that the G allele in the INSIG2 SNP rs7566605 is more relevant for changes in IMAT following training than for the amount of subcutaneous fat.