SIRT-1 Activity Sustains Cholesterol Synthesis in the Brain.

SIRT-1 is a potent energy regulator that has been implicated in the aging of different tissues, and cholesterol synthesis demands high amounts of cellular adenosine triphosphate. An efficient synaptic transmission depends on processes that are highly influenced by cholesterol levels, like endocytosis, exocytosis and membrane lateral diffusion of neurotransmitter receptors. We set out to investigate whether SIRT-1 activity affects brain cholesterol metabolism. We found that pharmacological inhibition of SIRT-1 with EX-527 reduces the mRNA amounts of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), Cytochrome P450 46A1 (CYP46A1) and Apolipoprotein E (APO-E) in rat primary cortical cultures. The decreased expression of these genes was paralleled by a significant reduction of the cholesterol levels in this type of neuronal culture. Interestingly, a cholesterol decrease of similar extent was observed in mouse astroglial cultures after EX-527 treatment. In agreement, mice administered with EX-527 for 5 days showed a down-regulation of cholesterol synthesis in the cortex, with significant reductions in the mRNA amounts of the transcription factor Sterol Regulatory Element Binding Protein 2 (SREBP-2) and the enzyme HMGCR, two key regulators of the cholesterol synthesis. These transcriptional changes were paralleled by reduced cholesterol levels at cortical synapses. SIRT-1 inhibition also reduced the amount of cholesterol in the hippocampus but without affecting the HMGCR expression levels. Altogether, these results uncover a role for SIRT-1 in the regulation of cholesterol metabolism, and demonstrate that SIRT-1 is required to sustain adequate levels of cholesterol synthesis in the adult brain.

Impact of high fat diet on the sterol regulatory element-binding protein 2 cholesterol pathway in the testicle.

Male fertility has been shown to be dependent on cholesterol homeostasis. This lipid is essential for testosterone synthesis and spermatogenesis, but its levels must be maintained in an optimal range for proper testicular function. In particular, sperm cells' development is very sensitive to high cholesterol levels, noticeably during acrosomal formation. The aim of this work was to study whether the molecular pathway that regulates intracellular cholesterol, the sterol regulatory element-binding protein (SREBP) pathway, is affected in the testicles of animals under a fat diet. To investigate this, we took advantage of the non-obese hypercholesterolemia (HC) model in New Zealand rabbits that displays poor sperm and seminal quality. The testicular expression of SREBP isoform 2 (SREBP2) and its target molecules 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and low-density lipoprotein receptor (LDLR) were studied under acute (6 months) and chronic (more than 12 months) fat intake by RT-PCR, western blot and immunofluorescence. Our findings showed that fat consumption promoted down-regulation of the SREBP2 pathway in the testicle at 6 months, but upregulation after a chronic period. This was consistent with load of testicular cholesterol, assessed by filipin staining. In conclusion, the intracellular pathway that regulates cholesterol levels in the testicle is sensitive to dietary fats, and behaves differently depending on the duration of consumption: it has a short-term protective effect, but became deregulated in the long term, ultimately leading to a detrimental situation. These results will contribute to the understanding of the basic mechanisms of the effect of fat consumption in humans with idiopathic infertility.

Mitochondrial aquaporin-8 is involved in SREBP-controlled hepatocyte cholesterol biosynthesis.

Cholesterol, via sterol regulatory element-binding protein (SREBP) transcription factors, activates or represses genes involved in its hepatic biosynthetic pathway, and also modulates the expression of hepatocyte mitochondrial aquaporin-8 (mtAQP8), a channel that can function as peroxiporin by facilitating the transmembrane diffusion of H2O2. Here we tested the hypothesis that mtAQP8 is involved in the SREBP-mediated regulation of hepatocyte cholesterol biosynthesis. Using human hepatocyte-derived Huh-7 cells and primary rat hepatocytes, we found that mtAQP8 knockdown significantly downregulated de novo cholesterol synthesis as well as protein expressions of SREBP-2 and its target gene, a rate-limiting enzyme in cholesterol synthesis 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR). In contrast, adenovirus-mediated human AQP8 mitochondrial expression significantly increased de novo cholesterol synthesis and protein expressions of SREBP-2 and HMGCR. In mtAQP8-overexpressed hepatocytes, mitochondrial H2O2 release was found to be increased; and a mitochondria-targeted antioxidant prevented the upregulation of mitochondrial H2O2 release and that of cholesterol synthesis. Our results suggest that peroxiporin mtAQP8 plays a role in the SREBP-controlled hepatocyte cholesterogenesis, a finding that might be relevant to cholesterol-related metabolic disorders.

Cholesterol can modulate mitochondrial aquaporin-8 expression in human hepatic cells.

Hepatocyte mitochondrial aquaporin-8 (mtAQP8) works as a multifunctional membrane channel protein that facilitates the uptake of ammonia for its detoxification to urea as well as the mitochondrial release of hydrogen peroxide. Since early oligonucleotide microarray studies in liver of cholesterol-fed mice showed an AQP8 downregulation, we tested whether alterations of cholesterol content per se modulate mtAQP8 expression in human hepatocyte-derived Huh-7 cells. Cholesterol loading with methyl-ß-cyclodextrin (mßCD):cholesterol complexes downregulated the proteolytic activation of cholesterol-responsive sterol regulatory element-binding protein (SREBP) transcriptions factors 1 and 2, and the expression of the target gene 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Under such conditions, mtAQP8 mRNA and protein expressions were significantly reduced. In contrast, cholesterol depletion using mßCD alone increased SREBP-1 and 2 activation and upregulated HMGCR and mtAQP8 mRNA and protein expressions. The results suggest that cholesterol can regulate transcriptionally human hepatocyte mtAQP8 expression likely via SREBPs. The functional implications of our findings are discussed. © 2017 IUBMB Life, 69(5):341-346, 2017.

Identification and characterization of SREBF2 expression and its association with chicken carcass traits.

The sterol regulatory element-binding transcription factor 2 gene (SREBF2) plays an important role in regulating lipid homeostasis. To reveal the genetic factors that underlie carcass fat deposition in chickens, we cloned the coding DNA sequence of chicken SREBF2, investigated SREBF2 mRNA expression levels in various tissues, detected single nucleotide polymorphisms (SNPs) in the exon regions of the gene, and conducted association analyses between single markers/haplotypes and carcass traits. The entire 2859-bp cDNA sequence of chicken SREBF2 that encoded 952 amino acids was obtained and characterized. SREBF2 mRNA was highly expressed in the uropygial gland, followed by the liver, breast muscle, and leg muscle. Ten SNPs were detected, and four (g.49363077T>A, g.49357503C>T, g.49355533G>A, and g.49354641G>A) were novel. When analyzing the associations between the single mutations and carcass traits, significant differences were found in three SNPs and g.49357915G>A was highly significantly associated with most carcass traits, except for abdominal fat weight and sebum thickness. In addition, haplotype combinations that were constructed using the SREBF2 SNPs were associated with breast muscle weight. Chickens with the combined genotype H21H21 had the highest live weight, carcass weight, eviscerated weight, and semi-eviscerated weight values. To the best of our knowledge, this is the first study conducted on chicken SREBF2 polymorphisms, which are predictive of the genetics that underlie the economic performance of chickens.

AMFR gene silencing inhibits the differentiation of porcine preadipocytes.

Our study clarifies the role of the autocrine motility factor receptor (AMFR) gene in porcine preadipocyte differentiation. AMFR-siRNA was transfected into porcine preadipocytes and the preadipocytes were induced to differentiation. Subsequently, qRT-PCR was conducted to examine changes in mRNA expression of a series of genes in porcine preadipocytes, including AMFR, sterol-regulatory element-binding protein-1a (SREBP1a), SREBP2, insulin-induced gene 1 (Insig1), and Insig2. Expression changes in the mRNA of genes regulating adipocyte differentiation were also analyzed using qRT-PCR, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and Kruppel-like factor 2 (KLF2). Western blot analysis was conducted to examine the changes in AMFR protein expression in porcine preadipocytes. Additionally, morphological changes in differentiated porcine preadipocytes were examined by oil red O staining, and changes in optical density (OD) values were measured using an ultraviolet spectrophotometer. At 24 h after transfection with AMFR-siRNA, AMFR mRNA expression significantly reduced (P < 0.01), and AMFR protein expression markedly decreased (P < 0.05). The mRNA expression of SREBP1a, SREBP2, Insig1, and C/EBPα was significantly reduced (P < 0.01), whereas the expression of KLF2 mRNA was significantly elevated (P < 0.01). After induction of preadipocyte differentiation, the number of lipid droplets decreased in the AMFR-silenced group, and the OD value markedly reduced (P < 0.05). In addition, the expression of C/EBPα mRNA significantly decreased (P < 0.05), whereas the expression of KLF2 mRNA considerably increased (P < 0.05). Taken together, silencing of the AMFR gene inhibits the differentiation of porcine preadipocytes.

Modulation by geraniol of gene expression involved in lipid metabolism leading to a reduction of serum-cholesterol and triglyceride levels.

BACKGROUND: Geraniol (G) is a natural isoprenoid present in the essential oils of several aromatic plants, with various biochemical and pharmacologic properties. Nevertheless, the mechanisms of action of G on cellular metabolism are largely unknown. HYPOTHESIS/PURPOSE: We propose that G could be a potential agent for the treatment of hyperlipidemia that could contribute to the prevention of cardiovascular disease. The aim of the present study was to advance our understanding of its mechanism of action on cholesterol and TG metabolism. STUDY DESIGN/METHODS: NIH mice received supplemented diets containing 25, 50, and 75 mmol G/kg chow. After a 3-week treatment, serum total-cholesterol and triglyceride levels were measured by commercial kits and lipid biosynthesis determined by the [(14)C] acetate incorporated into fatty acids plus nonsaponifiable and total hepatic lipids of the mice. The activity of the mRNA encoding HMGCR-the rate-limiting step in cholesterol biosynthesis-along with the enzyme levels and catalysis were assessed by real-time RT-PCR, Western blotting, and HMG-CoA-conversion assays, respectively. In-silico analysis of several genes involved in lipid metabolism and regulated by G in cultured cells was also performed. Finally, the mRNA levels encoded by the genes for the low-density-lipoprotein receptor (LDLR), the sterol-regulatory-element-binding transcription factor (SREBF2), the very-low-density-lipoprotein receptor (VLDLR), and the acetyl-CoA carboxylase (ACACA) were determined by real-time RT-PCR. RESULTS: Plasma total-cholesterol and triglyceride levels plus hepatic fatty-acid, total-lipid, and nonsaponifiable-lipid biosynthesis were significantly reduced by feeding with G. Even though an up-regulation of the mRNA encoding HMGCR occurred in the G treated mouse livers, the protein levels and specific activity of the enzyme were both inhibited. G also enhanced the mRNAs encoding the LDL and VLDL receptors and reduced ACACA mRNA, without altering the transcription of the mRNA encoding the SREBF2. CONCLUSIONS: The following mechanisms may have mediated the decrease in plasma lipids levels in mice: a down-regulation of hepatocyte-cholesterol synthesis occurred as a result of decreased HMGCR protein levels and catalytic activity; the levels of LDLR mRNA became elevated, thus suggesting an increase in the uptake of serum LDL, especially by the liver; and TG synthesis became reduced very likely because of a decrease in fatty-acid synthesis.

Possible molecular mechanisms soy-mediated in preventing and treating nonalcoholic fatty liver disease.

The aim of this review is to describe the molecular mechanisms of nonalcoholic fatty liver disease (NAFLD) and to present evidence regarding the mechanisms of soy-mediated therapeutic activity in preventing and treating NAFLD. NAFLD is induced by multiple metabolic pathways, including an increase in the release of fatty acids from the adipose tissue (lipolysis), insulin resistance (IR), and an increase in "de novo" fatty acid synthesis. Furthermore, NAFLD is correlated with a decrease in liver ß-oxidation, an increase in oxygen free radical production, and an increase in pro-inflammatory cytokine production, which leads to an increase in liver fat and, subsequently, to tissue damage. The bioactive compounds in soy can prevent and treat NAFLD by modulating lipid metabolism and regulating the expression of related transcription factors. Soy intake decreases the expression of sterol regulatory-element binding protein-lc (SREBP-1) and increases the expression of SREBP-2, which are transcription factors associated with the regulation of hepatic lipogenesis and reduction of cholesterol synthesis and absorption in the liver, respectively. Besides, interactions between soy components, such as standard amino acids, polyunsaturated fat, and the isoflavonoid-enriched fraction, are believed to improve fatty acid oxidation in the liver parenchyma by increasing the expression of peroxisome proliferator-activated receptor α (PPARα)-regulated genes, thus decreasing lipid accumulation in the liver. Therefore, including soy-derived foods in the diet as a therapeutic tool for patients with NAFLD might improve their clinical evolution.

The role of dietary protein on lipotoxicity.

The worldwide increase in degenerative diseases is in part due to modifications in the lifestyle including the diet. Epidemiological, clinical, and experimental evidence shows that soy protein may prevent lipotoxicity in non-adipose tissues during obesity. The molecular mechanism by which soy protein prevents lipotoxicity involves a reduction in the insulin/glucagon ratio, resulting in a down-regulation of lipogenic genes mediated by the transcription factor sterol regulatory element-binding protein (SREBP)-1, and up-regulation of SREBP-2 to reduce serum cholesterol. In addition, soy protein maintains the functionality of adipose tissue-liver axis to prevent hepatic steatosis during the development of obesity.