Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9.

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.

Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.

G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1(-/-) mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1(-/-) mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1(-/-) mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1(-/-) mice on HFD displayed increased hepatic steatosis when compared with Gpbar1(+)(/)(+) males and Gpbar1(-/-) females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.

Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease.

OBJECTIVE: FFAR1/GPR40 is a G-protein-coupled receptor expressed predominantly in pancreatic islets mediating free fatty acid-induced insulin secretion. However, the physiological role of FFAR1 remains controversial. It was previously reported that FFAR1 knockout (Ffar1(-/-)) mice were resistant to high-fat diet-induced hyperinuslinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. A more recent report suggested that although FFAR1 was necessary for fatty acid-induced insulin secretion in vivo, deletion of FFAR1 did not protect pancreatic islets against fatty acid-induced islet dysfunction. This study is designed to investigate FFAR1 function in vivo using a third line of independently generated Ffar1(-/-) mice in the C57BL/6 background. RESEARCH DESIGN AND METHODS: We used CL-316,243, a beta3 adrenergic receptor agonist, to acutely elevate blood free fatty acids and to study its effect on insulin secretion in vivo. Ffar1(+/+) (wild-type) and Ffar1(-/-) (knockout) mice were placed on two distinct high-fat diets to study their response to diet-induced obesity. RESULTS: Insulin secretion was reduced by approximately 50% in Ffar1(-/-) mice, confirming that FFAR1 contributes significantly to fatty acid stimulation of insulin secretion in vivo. However, Ffar1(+/+) and Ffar1(-/-) mice had similar weight, adiposity, and hyperinsulinemia on high-fat diets, and Ffar1(-/-) mice showed no improvement in glucose or insulin tolerance tests. In addition, high-fat diet induced comparable levels of lipid accumulation in livers of Ffar1(+/+) and Ffar1(-/-) mice. CONCLUSIONS: FFAR1 is required for normal insulin secretion in response to fatty acids; however, Ffar1(-/-) mice are not protected from high-fat diet-induced insulin resistance or hepatic steatosis.

Cholesterol homeostasis by the intestine: lessons from Niemann-Pick C1 Like 1 [NPC1L1).

Ezetimibe is a selective cholesterol absorption inhibitor, which potently inhibits the uptake and absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat-soluble vitamins, triglycerides or bile acids. Identification and characterization of Niemann-Pick C1 Like 1 (NPC1L1) has established NPC1L1 as an essential protein in the intestinal cholesterol absorption process. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that ezetimibe specifically binds to NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice were resistant to diet-induced hypercholesterolemia, and when crossed with apoE null mice, were completely resistant to the development of atherosclerosis. In Npc1l1/apoE null mice or apoE null mice treated with ezetimibe plasma cholesterol levels were reduced primarily in the apoB48 containing chylomicron remnant lipoproteins relative to untreated apoE null mice. SR-B1 has been proposed to play a role in intestinal cholesterol uptake, but in Npc1l1/SR-B1 double null mice intestinal cholesterol absorption was not different than Npc1l1 null alone mice. Therefore, NPC1L1 is the critical intestinal sterol transporter which influences whole body cholesterol homeostasis, and is the molecular target of ezetimibe.

Ezetimibe improves high fat and cholesterol diet-induced non-alcoholic fatty liver disease in mice.

Ezetimibe is a novel cholesterol and plant sterol absorption inhibitor that reduces plasma low-density lipoprotein-cholesterol by selectively binding to the intestinal cholesterol transporter, Niemann-Pick C1-Like 1. Mice deficient in Niemann-Pick C1-Like 1 are protected from high fat/cholesterol diet-induced fatty liver as well as hypercholesterolemia. The object of the present study was to determine whether ezetimibe treatment could reduce hepatic steatosis in diet-induced obese mice. C57BL/6J mice were fed a high fat/cholesterol containing semi-purified diet (45% Kcal fat and 0.12% cholesterol) for 7 months after weaning. These mice were not only obese, but also developed hepatomegaly and hepatic steatosis, with varying degrees of liver fibrosis and steatohepatitis. About 87% of the mice on the high fat/cholesterol diet for 7 months had elevated plasma alanine aminotransferase activity, a biomarker for non-alcoholic fatty liver disease. Chronic administration of ezetimibe for 4 weeks significantly reduced hepatomegaly by decreasing hepatic triglyceride, cholesteryl ester and free cholesterol in diet-induced obese mice fed high fat/cholesterol diet for 7 months. Chronic ezetimibe treatment also significantly decreased plasma alanine aminotransferase activity. These results suggest that ezetimibe may be a novel treatment for high fat/cholesterol-induced non-alcoholic fatty liver disease.

Deficiency of Niemann-Pick C1 Like 1 prevents atherosclerosis in ApoE-/- mice.

OBJECTIVE: The objective of this study was to determine whether the deficiency of Niemann-Pick C1 Like 1 (Npc1l1) prevents atherosclerosis in apoE null mice. METHODS AND RESULTS: Npc1l1(-/-)/apoE null-/- mice were generated and found to have a significant reduction in cholesterol absorption (-77%) compared with wild-type or apoE-/- mice. Npc1l1/apoE-/- mice were fed a chow or Western diet for 24 weeks, then lipoprotein, hepatic, and biliary cholesterol, and atherosclerosis development was compared with apoE-/-, Npc1l1-/-, wild-type, and ezetimibe-treated apoE-/- mice. Chylomicron remnant/VLDL cholesterol levels were reduced 80% to 90% in both chow and Western diet-fed Npc1l1/apoE-/- mice relative to apoE-/- mice. Male Npc1l1-/- and Npc1l1/apoE-/- mice were completely resistant to diet induced hypercholesterolemia, and both male and female mice were completely resistant to increases in hepatic and biliary cholesterol levels. Atherosclerosis was reduced 99% in aortic lesion surface area, 94% to 97% in innominate artery intimal lesion area, and >90% in aortic root lesion area in both male and female Npc1l1/apoE-/- mice relative to apoE-/- mice. CONCLUSIONS: Lack of Npc1l1, the molecular target of the cholesterol absorption inhibitor ezetimibe, in apoE-/- mice results in a significant reduction in cholesterol absorption and plasma cholesterol levels, and causes a nearly complete protection from the development of atherosclerosis, under both cholesterol-fed and non-cholesterol-fed conditions.

Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.

The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7alpha-hydroxylase pathway.

Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein.

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.

Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1 Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.

The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption.

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K(d) approximately 18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing 'western' diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.