Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) cholesterol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the relevance of SR-BI in the enterohepatic circulation of cholesterol and bile salts, we studied biliary lipid secretion, hepatic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol absorption in SR-BI knockout mice. SR-BI deficiency selectively impaired biliary cholesterol secretion, without concomitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol contents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal cholesterol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice relative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In contrast, SR-BI expression is not essential for intestinal cholesterol absorption.     

Constitutive and inducible expression of hepatic CYP2E1 in leptin-deficient ob/ob mice

In this study we have analyzed the inducible as well as constitutive hepatic expression of Cyp2e1 in a genetic model of obesity and non-insulin dependent (type II) diabetes, the leptin-deficient ob/ob mouse. In obese mice, Cyp2e1 levels were decreased compared to lean littermates. Treatment with leptin increased hepatic Cyp2e1 in obese mice to the levels observed in lean animals, but failed to alter Cyp2e1 expression in lean animals. As expected, leptin also reduced food intake in treated mice compared to saline-treated controls. In obese mice pair-fed the reduced amount of food, there was a significant increase in Cyp2e1 mRNA but no increase in Cyp2e1 protein or enzyme activity. Fasting and administration of acetone and 4-methylpyrazole increased Cyp2e1 mRNA as well as protein and activity in both obese and lean mice. The present data indicate that while Cyp2e1 is still inducible in obese mice by xenobiotics and fasting, full constitutive expression of Cyp2e1 requires leptin to be present. This effect of leptin appears to be at least partly independent of the hypothalamic control of food intake.     

Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts

Leptin administration to obese C57BL/6J (ob/ob) mice results in weight loss by reducing body fat. Because adipose tissue is an important storage depot for cholesterol, we explored evidence that leptin-induced weight loss in ob/ob mice was accompanied by transport of cholesterol to the liver and its elimination via bile. Consistent with mobilization of stored cholesterol, cholesterol concentrations in adipose tissue remained unchanged during weight loss. Plasma cholesterol levels fell sharply, and microscopic analyses of gallbladder bile revealed cholesterol crystals as well as cholesterol gallstones. Surprisingly, leptin reduced biliary cholesterol secretion rates without affecting secretion rates of bile salts or phospholipids. Instead, cholesterol supersaturation of gallbladder bile was due to marked decreases in bile salt hydrophobicity and not to hypersecretion of biliary cholesterol per se, such as occurs in humans during weight loss. In addition to regulating bile salt composition, leptin treatment decreased bile salt pool size. The smaller, more hydrophilic bile salt pool was associated with substantial decreases in intestinal cholesterol absorption. Within the liver, leptin treatment reduced the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, but it did not change activities of cholesterol 7alpha-hydroxylase or acyl-CoA:cholesterol acyltransferase. These data suggest that leptin regulates biliary lipid metabolism to promote efficient elimination of excess cholesterol stored in adipose tissue. Cholesterol gallstone formation during weight loss in ob/ob mice appears to represent a pathologic consequence of an adaptive response that prevents absorption of biliary and dietary cholesterol.     

Atherosclerotic lesion formation and triglyceride storage in obese apolipoprotein AI-deficient mice

Obese leptin-deficient (ob/ob) mice have increased levels of high-density lipoprotein (HDL) and a unique lipoprotein referred to as low-density lipoprotein (LDL)/HDL1. When crossed onto an apolipoprotein AI (apoAI)-deficient (-/-) background, ob/ob;apoAI-/- mice accumulate LDL/HDL1 in the absence of traditional HDL. To determine the role of LDL/HDL1 in atherosclerosis, C57BL/6, apoAI-/-, ob/ob and ob/ob;apoAI-/- mice were placed on butterfat diet. After 20 weeks, all four groups had a significant increase in total cholesterol levels. The cholesterol in C57BL/6 mice was carried on very low-density lipoprotein (VLDL) and LDL and, in ob/ob and ob/ob;apoAI-/- mice, on HDL and LDL/HDL1. Atherosclerotic lesion area was similar among C57BL/6, ob/ob and ob/ob;apoAI-/- groups despite their dissimilar lipoprotein profiles. Hepatic triglyceride production and VLDL clearance rates were similar among the four groups. The ob/ob;apoAI-/- group had a significant decrease in liver weight and an increase in white adipose tissue (WAT) weight compared to the ob/ob group. Hepatic scavenger receptor class B type I (SR-BI) levels were decreased in both liver and WAT in ob/ob;apoAI-/- compared to ob/ob mice. Conclusions regarding the atherogenicity of LDL/HDL1 were confounded by the differences in lipoprotein profiles among the four groups. However, our studies provide support for the concept that apoAI and SR-BI assist in the partitioning of lipid from adipose tissue to the liver.     

Restoration of gallstone susceptibility by leptin in C57BL/6J ob/ob mice

The absence of leptin due to the ob mutation leads to obesity and confers resistance to diet-induced cholesterol gallstone formation in otherwise susceptible C57BL/6J mice. To investigate contributions of obesity and leptin to gallstone susceptibility, C57BL/6J ob/ob mice were treated daily with i.p. saline or recombinant murine leptin at low (1 microgram/g bw) or high (10 microgram/g bw) doses and were pair-fed a lithogenic diet (15% dairy fat, 1.25% cholesterol, 0.5% cholic acid). Weight loss in ob/ob mice increased in proportion to leptin dose, indicating that the lithogenic diet did not impair leptin sensitivity. In a dose-dependent manner, leptin promoted cholesterol crystallization and gallstone formation, which did not occur in saline-treated mice. Notwithstanding, leptin decreased biliary lipid secretion rates without enriching cholesterol in bile. Leptin did not affect bile salt hydrophobicity, but did increase the biliary content of the most abundant molecular species of phosphatidylcholine, 16:0-18:2. Treatment with leptin down-regulated 3-hydroxy-3-methylglutaryl CoA reductase and prevented cholesterol from accumulating in liver. Consistent with increased hepatic clearance, leptin decreased plasma HDL cholesterol concentrations. This was accommodated in liver without up-regulation of cholesterol 7alpha-hydroxylase or Acat. These data suggest that despite the lithogenic diet, endogenous sources constitute a significant proportion of biliary cholesterol during leptin-induced weight loss. Kinetic factors related to cholesterol nucleation, gallbladder contractility, or mucin secretion may have accounted for leptin-induced gallstone formation.     

Scavenger receptor BI plays a role in facilitating chylomicron metabolism

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of high density lipoprotein (HDL) cholesterol esters is well established. However, the potential role of SR-BI in chylomicron and chylomicron remnant metabolism is largely unknown. In the present investigation, we report that the cell association of 160 nm-sized triglyceride-rich chylomicron-like emulsion particles to freshly isolated hepatocytes from SR-BI-deficient mice is greatly reduced (>70%), as compared with wild-type littermate mice. Competition experiments show that the association of emulsion particles with isolated hepatocytes is efficiently competed for (>70%) by the well established SR-BI ligands, HDL and oxidized low density lipoprotein (LDL), whereas LDL is ineffective. Upon injection into SR-BI-deficient mice the hepatic association of emulsion particles is markedly decreased ( approximately 80%) as compared with wild-type mice. The relevance of these findings for in vivo chylomicron (remnant) metabolism was further evaluated by studying the effect of SR-BI deficiency on the intragastric fat load-induced postprandial triglyceride response. The postprandial triglyceride response is 2-fold higher in SR-BI-deficient mice as compared with wild-type littermates (area-under-the-curve 39.6 +/- 1.2 versus 21.1 +/- 3.6; p < 0.005), with a 4-fold increased accumulation of chylomicron (remnant)-associated triglycerides in plasma at 6 h after intragastric fat load. We conclude that SR-BI is important in facilitating chylomicron (remnant) metabolism and might function as an initial recognition site for chylomicron remnants whereby the subsequent internalization can be exerted by additional receptor systems like the LDL receptor and LDL receptor-related protein.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI "att" mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether approximately 50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.     

Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

High density lipoprotein cholesterol is thought to represent a preferred source of sterols secreted into bile following hepatic uptake by scavenger receptor class B type I (SR-BI). The present study aimed to determine the metabolic effects of an endothelial lipase (EL)–mediated stimulation of HDL cholesterol uptake on liver lipid metabolism and biliary cholesterol secretion in wild-type, SR-BI knockout, and SR-BI overexpressing mice. In each model, injection of an EL expressing adenovirus decreased plasma HDL cholesterol (P < 0.001) whereas hepatic cholesterol content increased (P < 0.05), translating into decreased expression of sterol-regulatory element binding protein 2 (SREBP2) and its target genes HMG-CoA reductase and LDL receptor (each P < 0.01). Biliary cholesterol secretion was dependent on hepatic SR-BI expression, being decreased in SR-BI knockouts (P < 0.001) and increased following hepatic SR-BI overexpression (P < 0.001). However, in each model, biliary secretion of cholesterol, bile acids, and phospholipids as well as fecal bile acid and neutral sterol content, remained unchanged in response to EL overexpression. Importantly, hepatic ABCG5/G8 expression did not correlate with biliary cholesterol secretion rates under these conditions. These results demonstrate that an acute decrease of plasma HDL cholesterol levels by overexpressing EL increases hepatic cholesterol content but leaves biliary sterol secretion unaltered. Instead, biliary cholesterol secretion rates are related to the hepatic expression level of SR-BI. These data stress the importance of SR-BI for biliary cholesterol secretion and might have relevance for concepts of reverse cholesterol transport.     

Defects in the leptin axis reduce abundance of the ABCG5-ABCG8 sterol transporter in liver

ABGG5 (G5) and ABCG8 (G8) are ABC half-transporters that dimerize within the endoplasmic reticulum, traffic to the cell surface, and mediate cholesterol excretion into bile. Mice harboring defects in the leptin axis (db/db and ob/ob) have reduced biliary cholesterol concentrations. Rapid weight loss brought about by administration of leptin or dietary restriction increases biliary cholesterol excretion. We hypothesized that the reduction in biliary cholesterol in mice harboring defects in the leptin axis is associated with a reduction in G5G8 transporters and that levels of the transporter would increase with leptin administration and dietary restriction. We examined mRNA and protein levels for G5 and G8 in db/db and ob/ob mice. In both models G5 and G8 protein levels were reduced. In ob/ob mice, both leptin administration and dietary restriction increased G5 and G8 protein and biliary cholesterol concentrations. Finally, we examined the effects of tauroursodeoxycholate, which has been shown to increase biliary cholesterol excretion and function as a molecular chaperone. Tauroursodeoxycholate increased G5 and G8 protein and biliary cholesterol concentrations in both wild-type and db/db mice. Our results indicate that the mechanism for reduced biliary cholesterol excretion in db/db and ob/ob mice involves reductions in G5 and G8 protein levels and that this may occur at the level of G5G8 heterodimer assembly within the endoplasmic reticulum.     

Altered expression of hepatic CYP2E1 and CYP4A in obese, diabetic ob/ob mice, and fa/fa Zucker rats

Hepatic levels of the cytochrome P450 (CYP) proteins 2E1 and 4A are often increased in obesity, diabetes and fasting. In such states of nutritional imbalance, CYPs 2E1 and 4A may play a more significant role in fatty acid oxidation. In order to more fully characterize the regulation of CYP2E1 and CYP4A in obesity and obesity-related (type II) diabetes, we analyzed the hepatic expression of CYP2E1 and CYP4A in ob/ob mice which are leptin deficient, and fa/fa Zucker rats which have defective leptin receptor function. CYP2E1 protein and mRNA were either unchanged or reduced in both models. Conversely, expression of murine Cyp4a10 and 4a14 in the obese mice, and 4A2 in the male fatty Zucker rat, were greatly increased. The levels of other CYP4As were either unchanged or reduced. These results show that CYP2E1 is not inevitably increased by obesity and diabetes and indicate differential regulation of CYP4A subfamily genes in rodent models. Further, they implicate leptin receptor signaling as a factor that may modulate expression of CYP gene products involved in fatty acid oxidation.     

降脂益生菌调节胆固醇代谢 改善小鼠非酒精性脂肪肝

目的研究一种由鼠李糖乳杆菌DM9054和植物乳杆菌86066构成的降脂益生菌组合对非酒精性脂肪性肝病(NAFLD)小鼠胆固醇代谢的影响及其可能机制。方法 24只雄性LDLR-/-小鼠随机分为对照组、模型组和益生菌干预组。高脂饮食(HFD)15周建立小鼠NAFLD模型,造模同时干预组给予鼠李糖乳杆菌DM9054联合植物乳杆菌86066灌胃,对照组和模型组给予等量生理盐水灌胃。实验过程中监测各组小鼠体重变化。实验结束后,检测小鼠血清甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL)和高密度脂蛋白胆固醇(HDL)的水平差异。检测小鼠肝脏组织病理变化。使用Realtime PCR检测小鼠肠道内法尼脂受体(FXR)mRNA、顶端膜钠依赖的胆汁酸转运体(ASBT)mRNA、纤维生长因子15(FGF-15)mRNA和三磷酸腺苷结合盒转运体G5(ABCG-5)mRNA表达水平。Western blot检测小鼠肝脏胆固醇7α-羟化酶(CYP7A1)、FXR、三磷酸腺苷结合盒转运体G8(ABCG-8)、清道夫受体BI(SR-BI)、3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGCR)、胆盐输出泵(ABCB-11)、纤维生长因子受体4(FGFR-4)和胆固醇调节元件结合蛋白-2(SREBP-2)蛋白表达水平。结果与模型组相比,降脂益生菌干预组小鼠体重减轻(P0.05);小鼠血清TC、TG、LDL水平降低,HDL水平升高(P0.05);小鼠肝脏脂肪变性和炎性细胞浸润的现象显著减少;小鼠肠道ASBT mRNA和ABCG-5mRNA表达水平明显降低(Ps0.05),FGF-15mRNA表达水平明显升高(P0.05),FXR mRNA表达水平差异无统计学意义(P0.05);小鼠肝脏FGFR-4蛋白表达水平升高(P0.05),SREBP-2和HMGCR蛋白表达水平降低(Ps0.05),FXR、CYP7A1、SR-BI、ABCG-8和ABCB-11蛋白表达水平差异无统计学意义(Ps0.05)。结论降脂益生菌可能通过激活FXR-FGF15通路调节胆汁酸代谢;通过下调SREBP-2表达水平,抑制HMGCR表达,减少胆固醇的生成,从而起到改善非酒精性脂肪肝的作用。