Chronic treatment with epoxyeicosatrienoic acids modulates insulin signaling and prevents insulin resistance in hepatocytes.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites produced by cytochrome P450 epoxygenases which are highly expressed in hepatocytes. The functions of EETs in hepatocytes are not well understood. In this study, we investigated the effects of 14,15-EETs treatment on the insulin signal transduction pathway in hepatocytes. We report that chronic treatment, not acute treatment, with 30 µM 14,15-EETs prevents palmitate induced insulin resistance and potentiates insulin action in cultured HepG2 hepatocytes. 14,15-EETs increase Akt phosphorylation at S473, activating Akt, in an insulin dependent manner in HepG2 cells. Under insulin resistant conditions induced by palmitate, 14,15-EETs restore the insulin response by increasing S473-phosphorylated Akt. 8,9-EETs and 11,12-EETs demonstrated similar effects to 14,15-EETs. Furthermore, 14,15-EETs potentiate insulin-suppression of gluconeogenesis in cultured H4IIE hepatocytes. To elucidate the mechanism of EETs function, we analyzed the insulin signaling factors upstream of Akt. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 attenuated the 14,15-EETs-induced activating phosphorylation of Akt. 14,15-EETs reduced palmitate-stimulated phosphorylation of IRS-1 on S312 and phosphorylation of c-Jun N-terminal kinase (JNK) at threonine 183 and tyrosine 185 residues. The regulation of insulin sensitivity in cultured hepatocytes by chronic 14,15-EETs treatment appears to involve the JNK-IRS-PI3K pathway. The requirement of chronic treatment with EETs suggests that the effects of EETs on insulin response may be indirect.

Identification and characterization of dual inhibitors for phospholipid transfer protein and microsomal triglyceride transfer protein.

Phospholipid transfer protein (PLTP) plays an important role in atherogenesis and lipoprotein metabolism. PLTP exerts its functions intracellularly and extracellularly. Both PLTP and microsomal triglyceride transfer protein (MTP) have been shown to regulate the secretion of apolipoprotein B (apoB) in hepatocytes. We have previously reported the characterization of inhibitors that selectively inhibit PLTP activity and reduce apoB secretion in hepatocytes. In the present study, we identified more compounds that inhibit both PLTP and MTP activity to various extents. These dual inhibitors are structurally different from the PLTP-selective inhibitors. In human hepatoma cell lines, dual inhibitors seem to be more effective in reducing apoB secretion than selective PLTP or MTP inhibitors. Furthermore, the dual inhibitors markedly reduced triglyceride secretion from hepatocytes. In the absence of PLTP, the dual inhibitors can further reduce apoB secretion, whereas selective PLTP inhibitors had no effect. We conclude that MTP and PLTP may work coordinately in the process of hepatic apoB assembly and secretion. To avoid liver toxicity mediated by MTP inhibition, selective PLTP inhibitors should be pursued.

Pharmacologic inhibition of phospholipid transfer protein activity reduces apolipoprotein-B secretion from hepatocytes.

Phospholipid transfer protein (PLTP) plays an important role in atherogenesis, and its function goes well beyond that of transferring phospholipids between lipoprotein particles. Previous studies showed that genetic deficiency of PLTP in mice causes a substantially impaired hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To understand whether the impaired apoB secretion is a direct result from lack of PLTP activity, in this study, we further investigated the function of PLTP in apoB secretion by using PLTP inhibitors. We identified a series of compounds containing a 3-benzazepine core structure that inhibit PLTP activity. Compound A, the most potent inhibitor, was characterized further and had little cross-reactivity with microsomal triglyceride transfer protein. Compound A reduced apoB secretion in human hepatoma cell lines and mouse primary hepatocytes. Furthermore, we confirmed that the reduction of apoB secretion mediated by compound A is PLTP-dependent, because the PLTP inhibitor had no effect on apoB secretion from PLTP-deficient hepatocytes. These studies provided evidence that PLTP activity regulates apoB secretion and pharmacologic inhibition of PLTP may be a new therapy for dyslipidemia by reducing apoB secretion.

Pharmacologic inhibition of site 1 protease activity inhibits sterol regulatory element-binding protein processing and reduces lipogenic enzyme gene expression and lipid synthesis in cultured cells and experimental animals.

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 microM PF-429242 (Bioorg Med Chem Lett 17:4411-4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC(50) of 0.5 microM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice.

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.

Aminopyrrolidineamide inhibitors of site-1 protease.

The discovery and efficacy of a series of potent aminopyrrolidineamide-based inhibitors of sterol regulatory element binding protein site-1 protease is described.

Isozyme-nonselective N-substituted bipiperidylcarboxamide acetyl-CoA carboxylase inhibitors reduce tissue malonyl-CoA concentrations, inhibit fatty acid synthesis, and increase fatty acid oxidation in cultured cells and in experimental animals.

Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect the multitude of cardiovascular risk factors associated with the metabolic syndrome. To achieve maximal effectiveness, an ACC inhibitor should inhibit both the lipogenic tissue isozyme (ACC1) and the oxidative tissue isozyme (ACC2). Herein, we describe the biochemical and acute physiological properties of CP-610431, an isozyme-nonselective ACC inhibitor identified through high throughput inhibition screening, and CP-640186, an analog with improved metabolic stability. CP-610431 inhibited ACC1 and ACC2 with IC50s of approximately 50 nm. Inhibition was reversible, uncompetitive with respect to ATP, and non-competitive with respect to bicarbonate, acetyl-CoA, and citrate, indicating interaction with the enzymatic carboxyl transfer reaction. CP-610431 also inhibited fatty acid synthesis, triglyceride (TG) synthesis, TG secretion, and apolipoprotein B secretion in HepG2 cells (ACC1) with EC50s of 1.6, 1.8, 3.0, and 5.7 microm, without affecting either cholesterol synthesis or apolipoprotein CIII secretion. CP-640186, also inhibited both isozymes with IC50sof approximately 55 nm but was 2-3 times more potent than CP-610431 in inhibiting HepG2 cell fatty acid and TG synthesis. CP-640186 also stimulated fatty acid oxidation in C2C12 cells (ACC2) and in rat epitrochlearis muscle strips with EC50s of 57 nm and 1.3 microm. In rats, CP-640186 lowered hepatic, soleus muscle, quadriceps muscle, and cardiac muscle malonyl-CoA with ED50s of 55, 6, 15, and 8 mg/kg. Consequently, CP-640186 inhibited fatty acid synthesis in rats, CD1 mice, and ob/ob mice with ED50s of 13, 11, and 4 mg/kg, and stimulated rat whole body fatty acid oxidation with an ED50 of approximately 30 mg/kg. Taken together, These observations indicate that isozyme-nonselective ACC inhibition has the potential to favorably affect risk factors associated with the metabolic syndrome.