Glen Tetzloff

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.

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.

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 β3 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 ∼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.

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.

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.

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.

The effects of atriopeptin II on calcium fluxes in rabbit aorta

Effects of atriopeptin II (AP II) at 10(-7) M on the 45Ca flux and contractile responses to vasoconstrictors including norepinephrine (NE), angiotensin II (angio II) and high K were studied in isolated rabbit aortic strips. Augmentation of 45Ca efflux from aorta in normal physiological saline (PSS) due to NE and angio II each at 3 X 10(-7) M was greatly inhibited by AP II, suggesting that stimulated increase in cytosolic Ca2+ was suppressed. In contrast, the 45Ca efflux responses to KCl (20 and 40 mM) were not affected by AP II. Furthermore, the marked inhibition of contractile responses to NE (-58%) and angio II (-57%) by AP II was accompanied by significant decreases in 45Ca influx, whereas AP II exhibited only a modest inhibition on KCl (40 mM)-induced contraction (-28%) without affecting the accompanying increase in 45Ca influx. The 45Ca efflux from aortae in Ca2+-free PSS due to NE was markedly diminished by AP II, suggesting impairment of intracellular Ca2+ release. With tissues in either a basal or post-stimulation state (tissue Ca2+ previously increased with KCl stimulation), AP II did not stimulate 45Ca efflux in Ca2+-free PSS, suggesting its lack of effect on Ca extrusion. It is concluded that AP II is preferentially antagonistic against vascular responses to NE and angio II vs. high K and that inhibition of Ca entry and release forms the primary basis of its potent vasorelaxant action against vasoconstrictors.

Phorbol 12,13-dibutyrate, an activator of protein kinase C, stimulates both contraction and Ca2+ fluxes in dog saphenous vein

SummaryThe vascular effects of phorbol 12,13-dibutyrate (PDBu) were studied in the dog saphenous vein. PDBu (1 μM) caused contraction (0.58 ± 0.22 g/mg wet wt.) and Ca uptake (74.2 ± 41.2 μmol/kg wet wt.) which were unaffected by 10 μM phentolamine (N = 6). The PDBu-induced contraction was greatly (60–80%) inhibited in Ca2+-free solution. 15 Ca efflux measurements performed in Ca2+-free solution showed that PDBu did not cause Ca release from intracellular storage sites. The contractile response to PDBu (1 nM-1 μM) was significantly correlated with the magnitude of Ca uptake; contraction and the rise in tissue Ca2+ also had a similar time course. Correlation between the two measures persisted when the responses to PDBu were augmented by co-administration with 20 mM KCl. However, no synergism occurred between the two agonists. Both the contraction and Ca uptake responses to PDBu were reduced by nifedipine and verapamil, each at 1 μM. In the Triton X-100 skinned saphenous vein, where the voltage-dependent Ca channel is not functional, 10 μM PDBu did not cause contractions in the presence of 0.1 μM Ca2+. Thus, contraction of the intact saphenous vein by PDBu characteristically exhibits great Ca dependence and PDBu seems to activate the voltage-dependent Ca channel, presumably through stimulation of protein kinase C; the ensuing Ca entry is primarily responsible for contraction. However, the mechanism responsible for the PDBu-induced contractions that are resistant to Ca2+-free PSS or Ca entry blockers remains to be defined. It appears that the dog saphenous vein differs from dog femoral artery, rabbit aorta and pig carotid artery where PDBu contractions do not display dependence on external Ca2+.

Effects of BRL 34915, a putative K channel opener, on transmembrane 45Ca movements in rabbit aortic smooth muscle.

The effects of BRL 34915, a putative K channel opener, on transmembrane Ca2+ movements were studied in isolated rabbit aortic strips, using the 45Ca flux techniques. BRL 34915 (1 and 10 microM) inhibited the contraction and Ca entry in response to norepinephrine (NE) and angiotensin II, each at 0.3 microM. BRL 34915 differed from Na nitroprusside in that it demonstrated little or no inhibition on the contraction and 45Ca efflux provoked by NE in Ca2+-free buffer, suggesting that the effect of BRL 34915 on intracellular Ca release plays a minor role in inhibiting the vasoconstrictor responses to receptor agonists. Moreover, BRL 34915 (100 microM) did not stimulate cyclic GMP formation in rabbit aortic slices. BRL 34915 (10 microM) failed to inhibit the vascular responses to KCl (20 and 40 mM). Tetraethylammonium (20 mM), a K channel blocker, abolished the vasorelaxant action of BRL 34915 against NE. However, Na nitroprusside inhibition of NE contraction was similarly affected. It is concluded that reduction of Ca entry appears to be the predominant mechanism mediating the inhibitory effect of BRL 34915 on the contractile responses to vasoconstrictor stimuli.