Myeloid cell-specific ABCA1 deletion does not worsen insulin resistance in HF diet-induced or genetically obese mouse models

Obesity-associated low-grade chronic inflammation plays an important role in the development of insulin resistance. The membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) promotes formation of nascent HDL particles. ABCA1 also dampens macrophage inflammation by reducing cellular membrane cholesterol and lipid raft content. We tested the hypothesis that myeloid-specific ABCA1 deletion may exacerbate insulin resistance by increasing the obesity-associated chronic low-grade inflammation. Myeloid cell-specific ABCA1 knockout (MSKO) and wild-type (WT) mice developed obesity, insulin resistance, mild hypercholesterolemia, and hepatic steatosis to a similar extent with a 45% high-fat (HF) diet feeding or after crossing into the ob/ob background. Resident peritoneal macrophages and stromal vascular cells from obese MSKO mice accumulated significantly more cholesterol. Relative to chow, HF diet markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissue of WT and MSKO mice. Among pro-inflammatory cytokines examined, only IL-6 was highly upregulated in MSKO-ob/ob versus ob/ob mouse peritoneal macrophages, indicating a nonsignificant effect of myeloid ABCA1 deficiency on obesity-associated chronic inflammation. In conclusion, myeloid-specific ABCA1 deficiency does not exacerbate obesity-associated low-grade chronic inflammation and has minimal impact on the pathogenesis of insulin resistance in both HF diet-induced and genetically obese mouse models.     

Lipocalin 13 protein protects against hepatic steatosis by both inhibiting lipogenesis and stimulating fatty acid β-oxidation

Obesity is associated with hepatic steatosis, partially due to increased lipogenesis and decreased fatty acid β-oxidation in the liver; however, the underlying mechanism of abnormal lipid metabolism is not fully understood. We reported previously that obesity is associated with LCN13 (lipocalin 13) deficiency. LCN13 is a lipocalin family member involved in glucose metabolism, and LCN13 deficiency appears to contribute to hyperglycemia in obese mice. Here, we show that LCN13 is also an important regulator of lipogenesis and β-oxidation in the liver. In primary hepatocytes, recombinant LCN13 directly suppressed lipogenesis and increased fatty acid β-oxidation, whereas neutralization of endogenous LCN13 had an opposite effect. Transgenic overexpression of LCN13 protected against hepatic steatosis in mice with either dietary or genetic (ob/ob) obesity. LCN13 transgenic overexpression also improved hyperglycemia, glucose intolerance, and insulin resistance in ob/ob mice. Short-term LCN13 overexpression via an adenovirus-mediated gene transfer similarly attenuated hepatic steatosis in db/db mice. LCN13 inhibited the expression of important lipogenic genes and stimulated the genes that promote β-oxidation. These results suggest that LCN13 decreases liver lipid levels by both inhibiting hepatic lipogenesis and stimulating β-oxidation. LCN13 deficiency is likely to contribute to fatty liver disease in obese mice.     

Effect of metformin on adipose tissue resistin expression in db/db mice

Resistin, a novel adipose-derived protein, has been proposed to cause insulin-resistant states in obesity. To evaluate whether an insulin-sensitizing drug, metformin, regulates adipose tissue resistin expression, murine models of obesity and diabetes, db/db mice, were treated with metformin (metformin group), insulin (insulin group), and vehicle (control group) for 4 weeks, followed by analyzing resistin protein expression in their adipose tissues. Unexpectedly, resistin protein expression was increased by 66% in the metformin group relative to the control group, while it did not differ between the insulin and control groups. Hyperinsulinemia was improved in the metformin group, while the insulin group exhibited severe hyperinsulinemia, similar to the control group. Furthermore, in comparison between obese mice (db/db mice) and age-matched lean controls, resistin protein expression was reduced by 58% in the obese mice with severe hyperinsulinemia. These data collectively suggest that resistin expression may be suppressed by hyperinsulinemia and that metformin may upregulate resistin expression via the improvement of hyperinsulinemia in obesity.     

Stearoyl-CoA desaturase-1 deficiency attenuates obesity and insulin resistance in leptin-resistant obese mice

Obesity and adiposity greatly increase the risk for secondary conditions such as insulin resistance. Mice deficient in the enzyme stearoyl-CoA desaturase-1 (SCD1) are lean and protected from diet-induced obesity and insulin resistance. In order to determine the effect of SCD1 deficiency on various mouse models of obesity, we introduced a global deletion of the Scd1 gene into leptin-deficient ob/ob mice, leptin-resistant Agouti (A^y/a) mice, and high-fat diet-fed obese (DIO) mice. SCD1 deficiency lowered body weight, adiposity, hepatic lipid accumulation, and hepatic lipogenic gene expression in all three mouse models. However, glucose tolerance, insulin, and leptin sensitivity were improved by SCD1 deficiency only in A^y/a and DIO mice, but not ob/ob mice. These data uncouple the effects of SCD1 deficiency on weight loss from those on insulin sensitivity and suggest a beneficial effect of SCD1 inhibition on insulin sensitivity in obese mice that express a functional leptin gene.     

Adiponectin is stimulated by adrenalectomy in ob/ob mice and is highly correlated with resistin mRNA

Plasma levels of the adipocyte product adiponectin, a putative insulin-sensitizing agent, are reduced in obesity, whereas plasma levels of resistin, an agent that some believe to confer insulin resistance, are thought to increase with obesity. Because adrenalectomy can increase insulin sensitivity, we hypothesized that adrenalectomy would increase expression of adiponectin and decrease expression of resistin. Therefore, we measured adiponectin mRNA, adiponectin peptide, and resistin mRNA in adrenalectomized ob/ob mice. Adrenalectomy restored adiponectin expression in ob/ob mice to wild-type levels and stimulated adiponectin peptide to above wild-type levels. Surprisingly, expression of adiponectin and resistin was highly positively correlated even after statistical removal of effects of insulin, glucose, and adiposity. In addition, adiponectin and resistin expression were also highly correlated in diet-induced obese mice. The data support a role for adiponectin in mediating some effects of adrenalectomy on insulin sensitivity.     

Partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice

Partial leptin deficiency is not uncommon in the general population. We hypothesized that leptin insufficiency could favor obesity, nonalcoholic steatohepatitis (NASH), and other metabolic abnormalities, particularly under high calorie intake. Thus, mice partially deficient in leptin (ob/+) and their wild-type (+/+) littermates were fed for 4 mo with a standard-calorie (SC) or a high-calorie (HC) diet. Some ob/+ mice fed the HC diet were also treated weekly with leptin. Our results showed that, when fed the SC diet, ob/+ mice did not present significant metabolic abnormalities except for elevated levels of plasma adiponectin. Under high-fat feeding, increased body fat mass, hepatic steatosis, higher plasma total cholesterol, and glucose intolerance were observed in +/+ mice, and these abnormalities were further enhanced in ob/+ mice. Furthermore, some metabolic disturbances, such as blunted plasma levels of leptin and adiponectin, reduced UCP1 expression in brown adipose tissue, increased plasma liver enzymes, beta-hydroxybutyrate and triglycerides, and slight insulin resistance, were observed only in ob/+ mice fed the HC diet. Whereas de novo fatty acid synthesis in liver was decreased in +/+ mice fed the HC diet, it was disinhibited in ob/+ mice along with the restoration of the expression of several lipogenic genes. Enhanced expression of several genes involved in fatty acid oxidation was also observed only in ob/+ animals. Leptin supplementation alleviated most of the metabolic abnormalities observed in ob/+ fed the HC diet. Hence, leptin insufficiency could increase the risk of obesity, NASH, glucose intolerance, and hyperlipidemia in a context of calorie overconsumption.     

Obesity causes very low density lipoprotein clearance defects in low-density lipoprotein receptor-deficient mice

We have reported that obese leptin-deficient mice (ob/ob) lacking the low-density lipoprotein receptor (LDLR(-/-)) develop severe hyperlipidemia and spontaneous atherosclerosis. In the present study, we show that obese leptin receptor-deficient mice (db/db) lacking LDLR have a similar phenotype, even in the presence of elevated plasma leptin levels. We investigated the mechanism for the hyperlipidemia in obese LDLR(-/-) mice by comparing lipoprotein production and clearance rates in C57BL/6, ob/ob, LDLR(-/-) and ob/ob;LDLR(-/-) mice. Hepatic triglyceride production rates were equally increased ( approximately 1.4-fold, P<.05) in both LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob mice. LDL clearance was decreased ( approximately 1.3- fold, P<.01) to a similar extent in LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob controls. While VLDL clearance was delayed in LDLR(-/-) compared to C57BL/6 and ob/ob mice (2-fold, P<.001), this delay was exaggerated in ob/ob;LDLR(-/-) mice (3.8-fold, P<001). The VLDL clearance defects were due to decreased hepatic uptake compared to C57BL/6 (54% and 26% for LDLR(-/-) and ob/ob;LDLR(-/-), respectively, P<.001). When VLDL was collected from C57BL/6, ob/ob, LDLR(-/-), and ob/ob;LDLR(-/-) donors and injected into LDLR(-/-) recipient mice, counts remaining in the liver were 1.4-fold elevated in mice receiving LDLR(-/-) VLDL and 2-fold increased in mice receiving ob/ob;LDLR(-/-) VLDL compared to controls receiving C57BL/6 VLDL (P<.01). Thus, the increase in plasma lipoproteins in ob/ob;LDLR(-/-) mice is caused by delayed VLDL clearance. This appears to be due to defects in both the liver and the lipoproteins themselves in these obese mice.     

Attenuation of obesity-induced hyperlipidemia reduces tumor growth

Accumulating evidence suggests that hyperlipidemia is associated with obesity and cancer mortality in humans. We tested the hypotheses that inhibition of microsomal triglyceride transfer protein (MTP) would attenuate obesity-induced hyperlipidemia and reduce tumor growth by treating BCR-ABL B cell tumor-bearing hyperlipidemic obese ob/ob obese mice with a MTP inhibitor. MTP inhibition in tumor-bearing mice reduced concentrations of plasma apoB100 5-fold together with a corresponding decrease in VLDL triacylglycerol (TG) and cholesterol. Inhibition of MTP decreased tumor volume by 50%. MTP inhibitor did not alter tumor cell viability in vitro, suggesting that the in vivo tumor shrinkage effect was related to altered circulating lipids. Tumor volume reduction occurred without change in the protein expression of LDLR, FASN and HMGCR in the tumor, suggesting a lack of compensatory mechanisms in response to decreased hyperlipidemia. Expression of genes encoding GLUT4 and PEPCK was increased 6- and 10-fold, respectively, but no change in the expression of genes encoding regulatory enzymes of glycolysis was observed, suggesting that the tumors were not dependent on or switching to carbohydrates for energy requirement to support their growth. No change of proliferative signaling PI3K/AKT and ERK pathways after MTP inhibition was observed in the tumors. In conclusion, MTP inhibition decreased dyslipidemia and tumor growth in obese, insulin resistant mice. Therefore, decreasing VLDL secretion could be further explored as an adjuvant therapeutic intervention together with standard care to reduce tumor growth in obese patients.     

Hepatic insulin resistance in ob/ob mice involves increases in ceramide,aPKC activity,and selective impairment of Akt-dependent FoxO1 phosphorylation

Pathogenesis of insulin resistance in leptin-deficient ob/ob mice is obscure. In another form of diet-dependent obesity, high-fat-fed mice, hepatic insulin resistance involves ceramide-induced activation of atypical protein kinase C (aPKC), which selectively impairs protein kinase B (Akt)-dependent forkhead box O1 protein (FoxO1) phosphorylation on scaffolding protein, 40 kDa WD(tryp-x-x-asp)-repeat propeller/FYVE protein (WD40/ProF), thereby increasing gluconeogenesis. Resultant hyperinsulinemia activates hepatic Akt and mammalian target of rapamycin C1, and further activates aPKC; consequently, lipogenic enzyme expression increases, and insulin signaling in muscle is secondarily impaired. Here, in obese minimally-diabetic ob/ob mice, hepatic ceramide and aPKC activity and its association with WD40/ProF were increased. Hepatic Akt activity was also increased, but Akt associated with WD40/ProF was diminished and accounted for reduced FoxO1 phosphorylation and increased gluconeogenic enzyme expression. Most importantly, liver-selective inhibition of aPKC decreased aPKC and increased Akt association with WD40/ProF, thereby restoring FoxO1 phosphorylation and reducing gluconeogenic enzyme expression. Additionally, lipogenic enzyme expression diminished, and insulin signaling in muscle, glucose tolerance, obesity, hepatosteatosis, and hyperlipidemia improved. In conclusion, hepatic ceramide accumulates in response to CNS-dependent dietary excess irrespective of fat content; hepatic insulin resistance is prominent in ob/ob mice and involves aPKC-dependent displacement of Akt fromWD40/ProF and subsequent impairment of FoxO1 phosphorylation and increased expression of hepatic gluconeogenic and lipogenic enzymes; and hepatic alterations diminish insulin signaling in muscle.     

Increased Hepatic Insulin Action in Diet-Induced Obese Mice Following Inhibition of Glucosylceramide Synthase

Background

Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder.

Methodology/Principal Findings

DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity.

Conclusions/Significance

These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity.     

Low resistin levels in adipose tissues and serum in high-fat fed mice and genetically obese mice: development of an ELISA system for quantification of resistin

Obesity is a major risk factor for insulin resistance. Resistin, an adipocyte-derived hormone-like molecule, is considered to serve as an important link between obesity and insulin resistance. However, the physiological role of resistin and the mechanism by which it neutralizes insulin action are still unclear. There are also conflicting reports that cast doubt on the cause of insulin resistance. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) system for quantification of mouse resistin levels, analyzed in relation to insulin resistance. C57BL/6J mice fed high-fat diet compared with normal diet had low resistin levels (by 70%, P<0.01) in epididymal adipose tissues. Genetically obese mice, db/db and KK-A(y), had hyperinsulinemia and hyperglycemia but low resistin levels (decreases by 83 and 90%, both P<0.01) compared with C57/BL6J mice in epididymal adipose tissues. Serum resistin levels determined by Western blotting showed a similar pattern to those in adipose tissues. Resistin levels in adipose tissues correlated with serum adiponectin concentrations positively (r=0.49). Our results indicate that the novel ELISA system is suitable for measurement of resistin levels in adipose tissues. The results do not support a role for resistin in insulin resistance.     

Conditioned medium obtained from in vitro differentiated adipocytes and resistin induce insulin resistance in human hepatocytes

Adipocyte-derived factors might play a role in the development of hepatic insulin resistance. Resistin was identified as an adipokine linking obesity and insulin resistance. Resistin is secreted from adipocytes in rodents but in humans it was proposed to originate from macrophages and its impact for insulin resistance has remained elusive. To analyze the role of adipokines in general and resistin as a special adipokine, we cultured the human liver cell line HepG2 with adipocyte-conditioned medium (CM) containing various adipokines such as IL-6 and MCP-1, and resistin. CM and resistin both induce insulin resistance with a robust decrease in insulin-stimulated phosphorylation of Akt and GSK3. Insulin resistance could be prevented by co-treatment with troglitazone but not by co-stimulation with adiponectin. As human adipocytes do not secrete resistin, HepG2 cells were also treated with resistin added into CM. CM with resistin addition induced stronger insulin resistance than CM alone pointing to a specific role of resistin in the initiation of hepatic insulin resistance in humans.     

Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor.

Leptin-deficient mice (ob/ob) are an excellent murine model for obesity, insulin resistance, and diabetes, all of which are components of a multiple risk factor syndrome that, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis. In the current study, we show an unexpectedly severe hyperlipidemia in ob/ob mice on a background of low density lipoprotein receptor (LDLR) deficiency (-/-). Doubly mutant mice (LDLR-/-;ob/ob) exhibited striking elevations in both total plasma cholesterol (TC) and triglyceride (TG) levels (1715 +/- 87 and 1016 +/- 172 mg/dl, respectively), at age 3-4 months, resulting in extensive atherosclerotic lesions throughout the aorta by 6 months. Lipoprotein analyses revealed the elevated TC and TG levels to be due to a large increase in an apoB-containing broad-beta remnant lipoprotein fraction. While fasting, diet restriction, and low level leptin treatment significantly lowered TG levels, they caused only slight changes in TC levels. Hepatic cholesterol and triglyceride contents as well as mRNA levels of cholesterologenic and lipogenic enzymes suggest that leptin deficiency increased hepatic triglyceride production but did not change cholesterol production in ob/ob mice regardless of their LDLR genotype. These data provide evidence that the hypertriglyceridemia and hypercholesterolemia in the doubly mutant mice are caused by distinct mechanisms and point to the possibility that leptin might have some impact on plasma cholesterol metabolism, possibly through an LDLR-independent pathway. This model will be an excellent tool for future studies on the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis.     

Association of resistin gene polymorphisms with insulin resistance in Egyptian obese patients

Background

Obesity associated insulin resistance is a major risk factor for type 2 diabetes mellitus. Resistin is recently reported to provide a link between obesity, insulin resistance and type 2 diabetes mellitus. We aimed to investigate the possible associations of resistin gene (RETN) polymorphisms with obesity, and to detect whether these polymorphisms are associated with glucose intolerance and type 2 diabetes mellitus in obese patients.

Methods

One hundred and forty-five Egyptian obese patients with or without glucose intolerance and 155 unrelated healthy controls were enrolled in this study. Polymorphisms of RETN + 299G>A and RETN –420 C>G gene were detected by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP). Serum resistin was measured by ELISA.

Results

RETN + 299 AA and RETN − 420 GG genotypes were significantly associated with obesity in Egyptian population. Moreover, the mutant alleles or genotypes of both examined polymorphisms were associated with impaired glucose tolerance and diabetes mellitus compared to normal glucose tolerant obese patients. Furthermore, our results revealed elevated waist/hip ratio, BMI, blood pressure, fasting blood glucose level, HOMA-IR, triglycerides, total cholesterol, resistin level, and decreased HDL cholesterol level in homozygote mutant genotypes carriers of both RETN polymorphisms among obese patients.

Conclusion

Resistin gene polymorphisms may play an important role in pathogenesis and susceptibility to obesity, impaired glucose tolerance, and type 2 diabetes mellitus in Egyptian population.     

p53 involvement in the pathogenesis of fatty liver disease

Obesity is a major health problem in industrialized societies, and fatty liver disease (hepatic steatosis) is common in obese individuals. Oxidative stress originating from increased intracellular levels of fatty acids has been implicated as a cause of hepatocellular injury in steatosis, although the precise mechanisms remain to be elucidated. p53, widely known as a tumor suppressor, has been shown often to be activated in stressed cells, inducing cell cycle arrest or death. Here we demonstrate that p53 is involved in the molecular mechanisms of hepatocellular injury associated with steatosis. We found that p53 in the nucleus is induced in the liver from two mouse models of fatty liver disease, ob/ob and a transgenic mouse model that overexpresses an active form of sterol regulatory element-binding protein-1 in the liver (TgSREBP-1), the one with obesity and the other without obesity. This activation of the p53 pathway leads to the elevation of p21 mRNA expression, which can be considered an indicator of p53 activity, because ob/ob mice lacking p53 generated by targeting gene disruption exhibited the complete restoration of the p21 elevation to wild type levels. Consistent with these results, the amelioration of hepatic steatosis caused by Srebp-1 gene disruption in ob/ob mice lowered the p21 expression in a triglyceride content-dependent manner. Moreover, p53 deficiency in ob/ob mice resulted in a marked improvement of plasma alanine aminotransferase levels, demonstrating that p53 is involved in the mechanisms of hepatocellular injury. In conclusion, we revealed that p53 plays an important role in the pathogenesis of fatty liver disease.     

iPla2β deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes

Group VIA calcium-independent phospholipase A2 (iPla2β) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2β deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2β-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2β-null mice. However, iPla2β deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptorγ, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2β deficiency under MCD did not have any effects on inflammasomes and pro-inflammatory markers but exacerbated hepatic expression of myofibroblast α-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2β inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2β mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types.     

Generation and characterization of two novel mouse models exhibiting the phenotypes of the metabolic syndrome: Apob48-/-Lepob/ob mice devoid of ApoE or Ldlr

The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E (Apoe(-/-)) or low-density lipoprotein receptor (Ldlr(-/-)) and express no leptin (Lep(ob/ob)) or apolipoprotein B-48 but exclusively apolipoprotein B-100 (Apob(100/100)). These two lines are referred to as Apoe triple-knockout-Apoe 3KO (Apoe(-/-)Apob(100/100)Lep(ob/ob)) and Ldlr triple-knockout-Ldlr 3KO (Ldlr(-/-)Apob(100/100)Lep(ob/ob)) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition.     

Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice

Conjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 x 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-alpha mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice.     

Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice fed a Western diet exhibit severe hyperlipidemia and develop significant atherosclerosis. Apolipoprotein E (apoE) is a multifunctional protein synthesized by hepatocytes and macrophages. We sought to determine effect of macrophage apoE deficiency on severe hyperlipidemia and atherosclerosis. Female LDLR(-/-) mice were lethally irradiated and reconstituted with bone marrow from either apoE(-/-) or apoE(+/+) mice. Four weeks after transplantation, recipient mice were fed a Western diet for 8 weeks. Reconstitution of LDLR(-/-) mice with apoE(-/-) bone marrow resulted in a slight reduction in plasma apoE levels and a dramatic reduction in accumulation of apoE and apoB in the aortic wall. Plasma lipid levels were unaffected when mice had mild hyperlipidemia on a chow diet, whereas IDL/LDL cholesterol levels were significantly reduced when mice developed severe hyperlipidemia on the Western diet. The hepatic VLDL production rate of mice on the Western diet was decreased by 46% as determined by injection of Triton WR1339 to block VLDL clearance. Atherosclerotic lesions in the proximal aorta were significantly reduced, partially due to reduction in plasma total cholesterol levels (r=0.56; P<0.0001). Thus, macrophage apoE-deficiency alleviates severe hyperlipidemia by slowing hepatic VLDL production and consequently reduces atherosclerosis in LDLR(-/-) mice.