Generation and characterization of a humanized PPARδ mouse model.

BACKGROUND AND PURPOSE: Humanized mice for the nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ), termed PPARδ knock-in (PPARδ KI) mice, were generated for the investigation of functional differences between mouse and human PPARδ and as tools for early drug efficacy assessment. EXPERIMENTAL APPROACH: Human PPARδ function in lipid metabolism was assessed at baseline, after fasting or when challenged with the GW0742 compound in mice fed a chow diet or high-fat diet (HFD). KEY RESULTS: Analysis of PPARδ mRNA levels revealed a hypomorph expression of human PPARδ in liver, macrophages, small intestine and heart, but not in soleus and quadriceps muscles, white adipose tissue and skin. PPARδ KI mice displayed a small decrease of high-density lipoprotein-cholesterol whereas other lipid parameters were unaltered. Plasma metabolic parameters were similar in wild-type and PPARδ KI mice when fed chow or HFD, and following physiological (fasting) and pharmacological (GW0742 compound) activation of PPARδ. Gene expression profiling in liver, soleus muscle and macrophages showed similar gene patterns regulated by mouse and human PPARδ. The anti-inflammatory potential of human PPARδ was also similar to mouse PPARδ in liver and isolated macrophages. CONCLUSIONS AND IMPLICATIONS: These data indicate that human PPARδ can compensate for mouse PPARδ in the regulation of lipid metabolism and inflammation. Overall, this novel PPARδ KI mouse model shows full responsiveness to pharmacological challenge and represents a useful tool for the preclinical assessment of PPARδ activators with species-specific activity.

Metabolic effects of rexinoids: tissue-specific regulation of lipoprotein lipase activity.

Hypertriglyceridemia is a frequent complication accompanying the treatment of patients with either retinoids or rexinoids, [retinoid X receptor (RXR)-selective retinoids]. To investigate the cellular and molecular basis for this observation, we have studied the effects of rexinoids on triglyceride metabolism in both normal and diabetic rodents. Administration of a rexinoid such as LG100268 (LG268) to normal or diabetic rats results in a rapid increase in serum triglyceride levels. LG268 has no effect on hepatic triglyceride production but suppresses post-heparin plasma lipoprotein lipase (LPL) activity suggesting that the hypertriglyceridemia results from diminished peripheral processing of plasma very low density lipoproteins particles. Treatment of diabetic rats with rexinoids suppresses skeletal and cardiac muscle but not adipose tissue LPL activity. This effect is independent of changes in LPL mRNA. In C2C12 myocytes, LG268 suppresses the level of cell surface (i.e., heparin-releasable) LPL activity without altering LPL mRNA. This effect is very rapid (t(1/2) = 2 h) and is blocked by the transcriptional inhibitor actinomycin D. These studies demonstrate that RXR ligands can have dramatic effects on the post-translational processing of LPL and suggest that skeletal muscle may be an important target of rexinoid action. In addition, these data underscore that the metabolic consequences of RXR activation are distinct from either retinoic acid receptor or peroxisome proliferator-activated receptor activation.

Increased levels of high-density lipoprotein cholesterol are ineffective in inhibiting the development of immune responses to oxidized low-density lipoprotein and atherosclerosis in transgenic rabbits expressing human apolipoprotein (apo) A-I with severe hypercholesterolaemia.

High levels of high-density lipoprotein (HDL) cholesterol have been reported to protect against the development of atherosclerosis in humans by increasing reverse cholesterol transport and inhibiting the oxidation of low-density lipoprotein (LDL) due to the paraoxonase content of HDL. The purpose of the present study was to assess if there are any relationships between in vivo increases in serum levels of immunological LDL oxidation markers [autoantibodies against oxidized LDL, autoantibodies against malondialdehyde-modified LDL, LDL immune complexes and anti-cardiolipin autoantibodies], paraoxonase activity and the development of atherosclerosis in control rabbits and in transgenic rabbits expressing human apolipoprotein (apo) A-I. A total of 13 apo A-I transgenic rabbits and 18 non-transgenic littermates were fed on a cholesterol-rich diet (0.4%, w/w) for 14 weeks, and were monitored at weeks 0, 2, 6, 10 and 14. Aortic atherosclerotic lesions were measured at the end of this period. Human apo A-I transgenic rabbits with high HDL cholesterol levels were not protected against the development of atherosclerosis when they were fed on a cholesterol-rich diet which induced dramatic hypercholesterolaemia. Immunological markers of LDL oxidation increased and serum paraoxonase activity decreased similarly in control and transgenic rabbits. In conclusion, the present study demonstrates that high HDL cholesterol levels are ineffective in inhibiting increases in immunological markers of LDL oxidation and the development of atherosclerosis in a mammal with severe hypercholesterolaemia.

Decreased susceptibility to diet-induced atherosclerosis in human apolipoprotein A-II transgenic mice.

Studies performed in vivo have been controversial regarding the implication of human apolipoprotein (apo)A-II in the atherogenic process. Expression of human apoA-II in transgenic mice fed a chow diet leads to (1) a bimodal distribution of high density lipoprotein (HDL) size as in humans, (2) a reduction in total cholesterol concentration that is mainly due to a reduction in non-HDL cholesterol level, and (3) a dramatic reduction in mouse endogenous apoA-I and apoA-II. After 20 weeks on an atherogenic diet, transgenic mice had reduced total cholesterol concentrations because of a reduction in cholesterol associated with all lipoprotein classes. Endogenous apoA-I and apoA-II were also dramatically decreased in transgenic mice. The mean area of atherosclerotic lesions was drastically decreased in transgenic mice (-44%, P=0.0027) compared with control mice. The amount of aortic surface covered by lesions was positively correlated with very low density lipoprotein cholesterol (P<0.01) and intermediate density lipoprotein cholesterol levels (P<0.05). Transgenic mice were protected against the development of atherosclerosis despite a marked decrease in HDL cholesterol and apoA-I concentrations. This protection may be related to the marked reduction in circulating low density lipoprotein (very low density and intermediate density lipoprotein) levels in transgenic mice.

Activation of PPARdelta alters lipid metabolism in db/db mice.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARalpha and PPARgamma, relatively little is known about the most widely expressed PPAR subtype, PPARdelta. Here we show that treatment of insulin resistant db/db mice with the PPARdelta agonist L-165041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L-165041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARdelta ligand, but was increased by a PPARgamma agonist. These data suggest both that PPARdelta is involved in the regulation of cholesterol metabolism in db/db mice and that PPARdelta ligands could potentially have therapeutic value.

Induction of the fatty acid transport protein 1 and acyl-CoA synthase genes by dimer-selective rexinoids suggests that the peroxisome proliferator-activated receptor-retinoid X receptor heterodimer is their molecular target.

The intracellular fatty acid content of insulin-sensitive target tissues determines in part their insulin sensitivity. Uptake of fatty acids into cells is a controlled process determined in part by a regulated import/export system that is controlled at least by two key groups of proteins, i.e. the fatty acid transport protein (FATP) and acyl-CoA synthetase (ACS), which facilitate, respectively, the transport of fatty acids across the cell membrane and catalyze their esterification to prevent their efflux. Previously it was shown that the expression of the FATP-1 and ACS genes was controlled by insulin and by peroxisome proliferator-activated receptor (PPAR) agonists in liver or in adipose tissue. The aim of this investigation was to determine the effects of retinoic acid derivatives on the expression of FATP-1 and ACS. In several cultured cell lines, it was shown that the expression of both the FATP-1 and ACS mRNAs was specifically induced at the transcriptional level by selective retinoid X receptor (RXR) but not by retinoic acid receptor (RAR) ligands. This effect was most pronounced in hepatoma cell lines. A similar induction of FATP-1 and ACS mRNA levels was also observed in vivo in Zucker diabetic fatty rats treated with the RXR agonist, LGD1069 (4-[1-(3,5,5,8,8-pentamethyl-5,6,7, 8-tetrahydro-2-naphthyl)ethenyl]benzoic acid). Through the use of heterodimer-selective compounds, it was demonstrated that the modulatory effect of these rexinoids on FATP-1 and ACS gene expression was mediated through activation of RXR in the context of the PPAR-RXR heterodimer. The observation that both RXR and PPAR agonists can stimulate the transcription of genes implicated in lipid metabolism, suggest that rexinoids may also act as lipid-modifying agents and support a role of the permissive PPAR-RXR heterodimer in the control of insulin sensitivity.

Paraoxonase activity is reduced by a pro-atherosclerotic diet in rabbits.

Serum paraoxonase (PON1) is believed to protect against the development of atherosclerosis because of its ability to retard the oxidation of low-density lipoprotein (LDL) by hydrolysing LDL-associated phospholipid and cholesteryl-ester hydroperoxides. We have examined the relationship between PON1 and atherosclerosis development in transgenic rabbits overexpressing human apolipoprotein (apo) A-I and nontransgenic littermates fed a pro-atherogenic diet. PON1 activity was higher in transgenic (4006.1 +/- 716.7 nmol/min/ml) compared to control (3078.5 +/- 623.3 nmol/min/ml) rabbits (P < 0.01) while high-density lipoprotein (HDL) cholesterol was 1.84 +/- 0.54 mmol/L in transgenic rabbits and 0.57 +/- 0.21 mmol/L in control rabbits (P = 0.0001). After feeding rabbits a high-cholesterol diet for 14 weeks HDL-cholesterol fell by 70% in both transgenic and control rabbits (P < 0.001 compared to week 0) PON1 activity fell by 50% in both groups of rabbits (P < 0. 01 compared to week 0). The amount of thoracic aortic surface area covered by lesions was 29 +/- 16% in the control group and 26 +/- 15% in the transgenic group (P = NS). A pro-atherosclerotic diet reduces PON1 which may exaggerate the effects of the diet on the development of atherosclerosis.

Beneficial effects of fibrates on apolipoprotein A-I metabolism occur independently of any peroxisome proliferative response.

BACKGROUND: In humans, fibrates are frequently used normolipidemic drugs. Fibrates act by regulating genes involved in lipoprotein metabolism via activation of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in liver. In rodents, however, fibrates induce a peroxisome proliferation, leading to hepatomegaly and possibly hepatocarcinogenesis. Although this peroxisome proliferative response appears not to occur in humans, it remains controversial whether the beneficial effects of fibrates on lipoprotein metabolism can occur dissociated from such undesirable peroxisomal response. Here, we assessed the influence of fenofibrate on lipoprotein metabolism and peroxisome proliferation in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. METHODS AND RESULTS: First, we demonstrate that in normal rabbits, fenofibrate given at a high dose for 2 weeks does not influence serum concentrations or intestinal mRNA levels of the HDL apolipoprotein apoA-I. Therefore, the study was continued with human apoA-I transgenic rabbits that overexpress the human apoA-I gene under control of its homologous promoter, including its PPAR-response elements. In these animals, fenofibrate increases serum human apoA-I concentrations via an increased expression of the human apoA-I gene in liver. Interestingly, liver weight or mRNA levels and activity of fatty acyl-CoA oxidase, a rate-limiting and marker enzyme of peroxisomal beta-oxidation, remain unchanged after fenofibrate. CONCLUSIONS: Expression of the human apoA-I transgene in rabbit liver suffices to confer fibrate-mediated induction of serum apoA-I. Furthermore, these data provide in vivo evidence that the beneficial effects of fibrates on lipoprotein metabolism occur mechanistically dissociated from any deleterious activity on peroxisome proliferation and possibly hepatocarcinogenesis.

Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice.

The peroxisome proliferator-activated receptor-alpha (PPARalpha) controls gene expression in response to a diverse class of compounds collectively referred to as peroxisome proliferators. Whereas most known peroxisome proliferators are of exogenous origin and include hypolipidemic drugs and other industrial chemicals, several endogenous PPARalpha activators have been identified such as fatty acids and steroids. The latter finding and the fact that PPARalpha modulates target genes encoding enzymes involved in lipid metabolism suggest a role for PPARalpha in lipid metabolism. This was investigated in the PPARalpha-deficient mouse model. Basal levels of total serum cholesterol, high density lipoprotein cholesterol, hepatic apolipoprotein A-I mRNA, and serum apolipoprotein A-I in PPARalpha-deficient mice are significantly higher compared with wild-type controls. Treatment with the fibrate Wy 14,643 decreased apoA-I serum levels and hepatic mRNA levels in wild-type mice, whereas no effect was detected in the PPARalpha-deficient mice. Administration of the fibrate Wy 14,643 to wild-type mice results in marked depression of hepatic apolipoprotein C-III mRNA and serum triglycerides compared with untreated controls. In contrast, PPARalpha-deficient mice were unaffected by Wy 14,643 treatment. These studies demonstrate that PPARalpha modulates basal levels of serum cholesterol, in particular high density lipoprotein cholesterol, and establish that fibrate-induced modulation in hepatic apolipoprotein A-I, C-III mRNA, and serum triglycerides observed in wild-type mice is mediated by PPARalpha.