Harry R. Davis

Diet-induced obese mice develop peripheral, but not central, resistance to leptin.

Leptin administration reduces obesity in leptin-deficient ob/ob mice; its effects in obese humans, who have high circulating leptin levels, remain to be determined. This longitudinal study was designed to determine whether diet-induced obesity in mice produces resistance to peripheral and/or central leptin treatment. Obesity was induced in two strains of mice by exposure to a 45% fat diet. Serum leptin increased in proportion to body weight (P < 0.00001). Whereas C57BL/6 mice initially responded to peripherally administered leptin with a marked decrease in food intake, leptin resistance developed after 16 d on high fat diet; mice on 10% fat diet retained leptin sensitivity. In AKR mice, peripheral leptin significantly decreased food intake in both 10 and 45% fat-fed mice after 16 d of dietary treatment. However, after 56 d, both groups became resistant to peripherally administered leptin. Central administration of leptin to peripherally leptin-resistant AKR mice on 45% fat diet resulted in a robust response to leptin, with a dose-dependent decrease in food intake (P < 0.00001) and body weight (P < 0.0001) after a single intracerebroventricular infusion. These data demonstrate that, in a diet-induced obesity model, mice exhibit resistance to peripherally administered leptin, while retaining sensitivity to centrally administered leptin.

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.

Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men

Inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) inhibitors has been associated with an increase in intestinal cholesterol absorption. This study examined how HMG-CoAR inhibition by atorvastatin modulates expression of key genes involved in intestinal cholesterol metabolism. A crossover study was conducted in which 22 hyperlipidemic men received atorvastatin, 40 mg/day, or placebo, each for 12 weeks. Gene expression was assessed by real-time PCR using duodenal biopsy samples obtained at the end of each phase of treatment. Treatment with atorvastatin was associated with a 76% reduction in lathosterol and significant increases in sitosterol (70%). Atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (59%), LDL receptor (LDLR) (52%), PCSK9 (187%), SREBP-2 (44%), and HNF-4α (13%). Furthermore, atorvastatin significantly increased intestinal mRNA levels of NPC1L1 by 19% and decreased mRNA levels of both ABCG5 and ABCG8 by 14%. Positive correlations were observed between changes in SREBP-2 and HNF-4α expression and concurrent changes in the intestinal mRNA levels of HMG-CoAR, LDLR, and NPC1L1. These results indicate that HMG-CoAR inhibition with atorvastatin stimulates the intestinal expression of NPC1L1, LDLR, and PCSK9; increases cholesterol absorption; and reduces expression of ABCG5/8; these effects are most likely mediated by upregulation of the transcription factors SREBP-2 and HNF-4α.

In Vivo Responsiveness to Ezetimibe Correlates with Niemann-Pick C1 Like-1 (NPC1L1) Binding Affinity: Comparison of Multiple Species NPC1L1 Orthologs

Ezetimibe is the first in class 2-azetidinone that decreases plasma cholesterol by blocking intestinal cholesterol absorption. Ezetimibe effectively reduces plasma cholesterol in several species including human, monkey, dog, hamster, rat, and mouse, but the potency ranges widely. One potential factor responsible for this variation in responsiveness is diversity in ezetimibe metabolism. After oral administration, ezetimibe is glucuronidated. Both ezetimibe and the glucuronide lower plasma cholesterol; however, the glucuronide exhibits greater potency. Recent identification of Niemann-Pick C1 Like-1 (NPC1L1) as the molecular target of ezetimibe enables direct binding studies to be performed. Here, we report the cloning of NPC1L1 derived from multiple species and assess amino acid sequence homology among human, monkey, dog, hamster, rat, and mouse. The rank order of affinity of glucuronidated ezetimibe for NPC1L1 in each species correlates with the rank order of in vivo activity with monkey > dog > hamster and rat ≫ mouse. Ezetimibe analogs that bind to NPC1L1 exhibit in vivo cholesterol-lowering activity, whereas compounds that do not bind NPC1L1 are inactive. Specific structural components of ezetimibe are identified as critical for binding to NPC1L1. The results demonstrate that small variations in ezetimibe structure or in NPC1L1 amino acid sequence can profoundly influence ezetimibe/NPC1L1 interaction and consequently in vivo activity. The results demonstrate that the ability of compounds to bind to NPC1L1 is the major determinant of in vivo responsiveness.

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.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects gene expression pathways beyond cholesterol metabolism in liver cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces degradation of low‐density lipoprotein receptor (LDLR) in the liver. It is being pursued as a therapeutic target for LDL‐cholesterol reduction. Earlier genome‐wide gene expression studies showed that PCSK9 over‐expression in HepG2 cells resulted in up‐regulation of genes in cholesterol biosynthesis and down‐regulation of genes in stress response pathways; however, it was not known whether these changes were directly regulated by PCSK9 or were secondary to PCSK9‐induced changes to the intracellular environment. In order to further understand the biological function of PCSK9 we treated HepG2 cells with purified recombinant wild type (WT) and D374Y gain‐of‐function PCSK9 proteins for 8, 24, and 48 h, and used microarray analysis to identify genome‐wide expression changes and pathways. These results were compared to the changes induced by culturing HepG2 cells in cholesterol‐free medium, mimicking the intracellular environment of cholesterol starvation. We determined that PCSK9‐induced up‐regulation of cholesterol biosynthesis genes resulted from intracellular cholesterol starvation. In addition, we identified novel pathways that are presumably regulated by PCSK9 and are independent of its effects on cholesterol uptake. These pathways included “protein ubiquitination,” “xenobiotic metabolism,” “cell cycle,” and “inflammation and stress response.” Our results indicate that PCSK9 affects metabolic pathways beyond cholesterol metabolism in HepG2 cells. J. Cell. Physiol. 224:273–281, 2010

Glucagon receptor antagonism induces increased cholesterol absorption

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Inverse relationship between LDL cholesterol and PCSK9 plasma levels in dyslipidemic cynomolgus monkeys: Effects of LDL lowering by ezetimibe in the absence of statins

OBJECTIVES To assess the lipid-lowering efficacy of ezetimibe in dyslipidemic cynomolgus monkeys comparing two dosing methods, and to evaluate PCSK9 plasma levels during dyslipidemia induction by feeding a high-fat/high-cholesterol diet (HFD), ezetimibe (Zetia(®), Ezetrol(®)) treatment, ezetimibe washout, and HFD washout. METHODS AND RESULTS Twenty dyslipidemic cynomolgus monkeys on HFD for seven months (LDL cholesterol 100-400 mg/dL) were randomized into two groups and treated with ezetimibe for two weeks, either by oral gavage or by using food treats. The lipid-lowering effects of ezetimibe were identical between the two groups. After treatment, mean LDL cholesterol was decreased by 58% (174-72 mg/dL), total cholesterol by 42% (241-138 mg/dL), and PCSK9 levels were increased by 137% (147-314 ng/mL). PCSK9 levels on regular diet before and after HFD were also inversely correlated to LDL cholesterol. CONCLUSIONS In a cynomolgus dyslipidemia model, PCSK9 levels are inversely correlated with LDL cholesterol in the absence of statin treatment, regardless whether lipid changes are modulated by diet or ezetimibe treatment.

Efficacy and safety of ezetimibe plus atorvastatin therapy

Abstract Statin therapy is highly effective in reducing low-density lipoprotein cholesterol (LDL-C) and cardiovascular events; however, many high-risk patients on statin monotherapy do not achieve sufficient LDL-C lowering. Statin-dose uptitration, switching to more potent statins and/or addition of complementary lipid-lowering drugs has been recommended for these patients. Numerous clinical trials have shown that coadministration of statins with cholesterol absorption inhibitor, ezetimibe, improves LDL-C lowering and other lipid parameters more than statins alone, including doubling the statin dose. In clinical outcome trials, ezetimibe combined with simvastatin reduced ischemic events in high-risk patients with chronic kidney disease and aortic stenosis; the incremental benefit of LDL-C lowering when ezetimibe is added to statin therapy on cardiovascular risk reduction compared with statin monotherapy is currently being evaluated in an ongoing clinical trial. Recently, a fixed-dose combination of ezetimibe plus atorvastatin, a statin with greater potency than most other statins, was approved by the US FDA and offers a therapeutic option for high-risk patients who do not achieve recommended LDL-C levels on statin monotherapy. This article provides an update on the safety and efficacy of ezetimibe plus atorvastatin therapy.