High-Efficacy α,β-Dehydromonacolin S Improves Hepatic Steatosis and Suppresses Gluconeogenesis Pathway in High-Fat Diet-Induced Obese Rats

Isolated α,β-dehydromonacolin S (C5) from soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 was recently shown to exhibit an inhibitory effect against 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity in vitro. In this study, we investigated the effects of C5 on lipid-lowering, hepatic steatosis, and hepatic gluconeogenesis in vivo. The control rats received a daily dose of either vehicle or C5 at 10 mg/kg, while the high-fat diet-induced obese (HFD) rats were administered vehicle; 1, 3, or 10 mg/kg C5; or 10 mg/kg lovastatin (LO) for 6 weeks. C5 significantly improved dyslipidemia and diminished liver enzymes, HMGR activity, insulin resistance, and hepatic steatosis, comparable to LO without any hepatotoxicity and nephrotoxicity in HFD rats. A higher efficacy of C5 in lipid-lowering activity and anti-hepatic steatosis was associated with a significant decrease in genes involved in lipid metabolism including sterol regulatory element binding protein (SREBP) 1c, SREBP2, liver X receptor alpha (LXRα), and peroxisome proliferator-activated receptor (PPAR) gamma (PPARγ) together with an increase in the PPAR alpha (PPARα). Correspondingly, C5 was able to down-regulate the lipid transporters cluster of differentiation 36 (CD36) and Niemann-Pick C1 Like 1 (NPC1L1), increase the antioxidant superoxide dismutase gene expression, and decrease the proinflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β). Impairment of hepatic gluconeogenesis and insulin resistance in HFD rats was restored by C5 through down-regulation of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), and the activation of AMP-dependent kinase serine (AMPK) and serine/threonine protein kinase B (Akt). Collectively, this novel C5 may be a therapeutic option for treating dyslipidemia, hepatic steatosis, and reducing potential risk for diabetes mellitus.

Effectors of Rapid Homeostatic Responses of Endoplasmic Reticulum Cholesterol and 3-Hydroxy-3-methylglutaryl-CoA Reductase

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.

Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7α-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7α-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7α-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.

083 Is HMGR the Key Regulator of α-Farnesene Biosynthesis of Apple?

We tested the hypothesis that conversion of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) to mevalonate (MVA) catalyzed by HMG CoA reductase (HMGR) is the rate limiting step for α-farnesene biosynthesis of apples. In higher plants, isopentenyl pyrophosphate (IPP) is derived via two pathways: 1) the classical mevalonate pathway, and 2) the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR action. When apple skin discs were incubated with MVA, or GAP and pyruvate, MVA increased α-farnesene levels in the skin but not GAP and pyruvate. Treating apple fruits with Lovastatin (1000 ppm), a competitive inhibitor of HMGR, inhibited α-farnesene accumulation in the skin by 20% to 50% during storage. Content of α-farnesene in the skin increased during the first 2 to 4 months in storage, and then decreased. In contrast, HMGR activity, as determined by the conversion of [4-3H]HMG CoA to MVA in the total membrane and soluble fraction, was the highest at the time of harvest and gradually decreased during 5 months of storage in air at 0 °C. The potent ethylene action inhibitor 1-MCP inhibited ethylene production and α-farnesene evolution by 99% and 97%, respectively. The effect of 1-MCP on in vitro activity of HMGR was marginal (≈30% inhibition). 1-MCP inhibited respiratory CO2 evolution by 50%, which suggests also that inhibition by 1-MCP of α-farnesene synthesis in apple could be regulated by the acetyl CoA pool. In plants, HMGR is encoded by a small gene family and differentially expressed. As the first step of studying the molecular mechanism of HMGR regulation, we have isolated a 444-bp fragment of apple hmgr gene using apple skin mRNA and degenerate oligonucleotides designed against conserved regions of plant hmgr genes.