PPARα deficiency reduces insulin resistance and atherosclerosis in apoE-null mice (original) (raw)
Effects of high-fat feeding on body weight. Mice were started on a high-fat, cholesterol-containing diet at the age of 8 weeks. In males, weight gain associated with high-fat feeding was not affected by PPARα genotype (Figure 1a). There was a genotype effect on weight gain in females (Figure 1b). PPARα–/–apoE–/– females (open circles) gained more weight than their PPARα+/+apoE–/– littermates with high-fat feeding. By week 10, female PPARα–/–apoE–/– weighed 27% more than female PPARα+/+apoE–/– mice (P < 0.0001). Food intake was unaffected by PPARα genotype. Single-knockout PPARα female (but not male) mice eating chow diets are known to develop obesity with aging (30).
Sex-specific effect of PPARα deficiency on weight gain after high-fat feeding. PPARα–/–apoE–/– (open symbols) and PPARα+/+apoE–/– (filled symbols) mice were started on a Western diet at the age of 2 months (week 0) and weighed every 2 weeks. (a) Triangles represent males and (b) circles represent females. A_P_ = 0.0002 vs. PPARα+/+apoE–/–. B_P_ < 0.0001 vs. PPARα+/+apoE–/–. For a, n = 19 at each time point for PPARα+/+apoE–/– males and n = 27 for PPARα–/–apoE–/– males. For b, n = 23 for PPARα+/+apoE–/– females and n = 27 for PPARα–/–apoE–/– females.
Fasting serum chemistries. For all serum measurements, values tended to be higher in males, but genotype effects were the same in both sexes so data from males and females are presented together. Fasting triglycerides (Figure 2a) were significantly higher in PPARα–/–apoE–/– mice than PPARα+/+apoE–/– mice at baseline (week 0) and remained significantly higher at 3, 6, and 10 weeks of high-fat feeding. Cholesterol levels (Figure 2b) were the same in PPARα–/–apoE–/– and PPARα+/+apoE–/– on a chow diet (week 0) and increased threefold in both genotypes with cholesterol feeding by 3 weeks. Cholesterol levels were 10% higher in PPARα–/–apoE–/– mice at 6 weeks and 20% higher at 10 weeks (P = 0.0019; n = 30 for PPARα–/–apoE–/– and n = 29 for PPARα+/+apoE–/–).
Fasting glucose (Figure 2c) was 18% lower in PPARα–/–apoE–/– mice compared with PPARα+/+apoE–/– mice at baseline (P = 0.0006). High-fat feeding increased glucose levels in both genotypes, but levels were significantly lower at each time point in PPARα–/– mice. After 10 weeks on the Western diet, glucose levels were 23% lower in PPARα–/–apoE–/– mice (P = 0.0022; n = 32 for PPARα–/– and n = 38 for PPARα+/+). Insulin levels were 0.25 ± 0.03 ng/ml in PPARα–/–apoE–/– mice (n = 15) and 0.55 ± 0.08 ng/ml in PPARα+/+apoE–/– mice (n = 17) at the 10-week time point (P = 0.0036), suggesting that lower glucose levels in PPARα-null mice reflect enhanced insulin sensitivity compared with their wild-type littermates.
Fasting NEFA levels (Figure 2d) were 39% higher in PPARα-null mice at baseline (week 0, P < 0.0001; n = 50 for PPARα–/–apoE–/– and n = 42 for PPARα+/+apoE–/–). This genotype-specific difference was lost with high-fat feeding (compare weeks 3, 6, and 10 with week 0 in Figure 2d).
Lipoprotein characterization and metabolism in PPARα+/+apoE–/– and PPARα–/– apoE–/– mice. Size exclusion chromatography of lipoproteins (Figure 3) from male mice fed a Western diet for 6 weeks showed that elevated triglycerides in PPARα–/–apoE–/– mice were due to elevated concentrations of VLDL (Figure 3a). PPARα–/–apoE–/– mice also tended to have higher levels of VLDL and IDL/LDL cholesterol (Figure 3b) that became more pronounced by 10 weeks on the Western diet (not shown). The same patterns were seen in female mice.
High-resolution size-exclusion chromatography profile of serum triglycerides (a) and cholesterol (b) from PPARα–/–apoE–/– mice (open symbols, broken line) and PPARα+/+apoE–/– littermates (filled symbols, solid line). Serum samples from eight males of each genotype eating a Western-type diet for 6 weeks were pooled, separated by chromatography, and individual fractions were assayed for lipid content. The profile is representative of four independent lipoprotein profile comparisons performed in different cohorts of mice.
VLDL clearance was the same in animals of each genotype (Figure 4a). For these studies, we used radiolabeled VLDL synthesized by PPARα+/+apoE–/– mice to focus on LPL and not particle composition. VLDL from PPARα–/–apoE–/– animals would be expected to have decreased clearance in part due to an elevated apoCIII content. Triglyceride production was increased in PPARα–/–apoE–/– animals (Figure 4b), providing an explanation for the elevated VLDL concentrations seen in these mice (Figure 3).
Lipoprotein metabolism studies in PPARα–/–apoE–/– mice (open symbols, broken line) and PPARα+/+apoE–/– littermates (filled symbols, solid line). (a) For the VLDL clearance data high-fat fed mice were injected with radiolabeled VLDL that was synthesized in vivo by administering [9,10-3H] palmitic acid in corn oil to PPARα+/+apoE–/– mice. After injection of radiolabeled VLDL at time 0, mice underwent venipuncture at 0.5, 1, 2, 5, and 20 minutes. Data are expressed as the percentage of radioactivity at the 0.5-minute time point. Data from 20 minutes were identical to those at 5 minutes and are excluded for clarity; n = 4 for each genotype. The same results were seen in two independent experiments. (b) For the triglyceride production data, fasting triglyceride levels were determined at time 0, mice were injected with Triton WR 1339 to inhibit systemic lipolysis, and venipuncture was performed at 15 and 30 minutes. Data are expressed as the percentage of the fasting triglyceride levels (mean triglycerides at time 0 were 129 mg/dl for PPARα+/+apoE–/– and 236 mg/dl for PPARα–/–apoE–/– in this experiment). A_P_ = 0.0165 vs. PPARα+/+apoE–/–; n = 4 for each genotype. The same results were seen in two independent experiments.
Lesion extent in Western diet–fed mice. Atherosclerosis was quantified by pinning aortas en face and measuring the percentage of intimal surface affected by atheroma at three different sites in each animal: aortic arch, thoracic aorta, and abdominal aorta. Figure 5 shows lesion extent in 54 PPARα+/+apoE–/– mice and 53 PPARα–/–apoE–/– mice studied after either 6 weeks (Figure 5, a and b) or 10 weeks (Figure 5c) on the Western diet. Littermates with the same C57Bl/6 background of approximately 75% were used for Figure 5, b and c. Figure 5a shows data from mice with a C57Bl/6 background of approximately 50%.
Despite having 20% higher serum cholesterol than PPARα+/+apoE–/– littermates, PPARα–/–apoE–/– mice fed the Western diet for 10 weeks had less atherosclerosis (Figure 5c). Median lesion area was 25% lower at the arch (P < 0.0001), 54% lower at the thoracic aorta (P < 0.0001), and 65% lower at the abdominal aorta (P < 0.0001) in PPARα–/–apoE–/– compared with PPARα+/+apoE–/– mice. As expected, overall lesion extent was less after 6 weeks on the Western diet, but PPAR genotype effects were the same (Figure 5b). Lesion area at 6 weeks was 31% lower at the arch (P = 0.0038), 60% lower at the thoracic aorta (P < 0.0001), and 100% lower at the abdominal aorta (P = 0.0385) in the PPARα-null animals. The same genotype effects on atherosclerosis were seen in mice with a lower admixture of C57Bl/6 background genes (Figure 5a). At 6 weeks, lesion area was 34% lower at the arch (P = 0.0017), 34% lower at the thoracic aorta (P < 0.0001), and 29% lower at the abdominal aorta (P = 0.0316) in PPARα-null animals.
Immunocytochemistry of vascular lesions. To confirm that PPARα was present in the atherosclerotic lesions of PPARα+/+apoE–/– mice, immunocytochemical studies were performed (Figure 6). Immunostaining for PPARα was prominent in nuclei throughout the lipid-filled neointima of PPARα+/+apoE–/– lesions. Some endothelial cells showed staining for PPARα. In addition, nuclear staining was detected in the media within elongated, spindle-shaped cells (arrows, Figure 6) resembling smooth muscle cells. Parallel staining (not shown) using an anti-macrophage Ab (MAC-3; BD Pharmingen, San Diego, California, USA) and an anti-smooth muscle α actin Ab (Zymed, South San Francisco, California, USA) confirmed that the PPARα+/+-positive cells in the neointima and media were macrophages and smooth muscle cells, respectively.
Detection of PPARα protein by immunocytochemistry in atherosclerotic lesions of PPARα+/+apoE–/– mice (left) but not PPARα–/–apoE–/– mice (right). Nuclear staining was detected throughout the neointima. In other experiments (not shown), serial sections showed these PPARα-positive cells to also react with an anti-macrophage Ab. Arrows indicate additional positive staining in elongated cells associated with the media. In other experiments (not shown), serial sections showed these PPARα-positive cells to react also with an anti–smooth muscle α actin Ab. These sections were from mice fed a Western diet for 6 weeks. The same results were seen in multiple sections from three different PPARα+/+ and four different PPARα–/– mice. No signals were seen in PPARα+/+ lesions when slides were subjected to the immunocytochemical protocol with the omission of the primary Ab (not shown).
Plasma and macrophage LPL enzyme activity. PPARα may regulate LPL expression, and LPL in the vasculature can promote atherosclerosis. The lack of a difference in VLDL clearance (Figure 4a) suggests that PPARα genotype does not have a major effect on LPL activity in these mice. Heparin-releasable activity was identical in thioglycolate-elicited peritoneal macrophages from PPARα+/+apoE–/– mice and PPARα–/–apoE–/– mice (Table 1). There was also no significant difference in postheparin plasma LPL activity between these animals (Table 1). However, these animals were studied in the fasting state and natural PPARα agonists provided by feeding might alter LPL expression.
LPL enzyme activity in Western diet–fed mice
To determine if PPAR activation can affect LPL in these animals, mice were fed Western diet containing 0.1% WY-14,643, a potent PPARα agonist. After 1 week, postheparin plasma LPL activity was significantly higher in PPARα+/+ mice (Table 1). However, VLDL clearance after 1 week of dietary supplementation with WY-14,643 was virtually identical in PPARα+/+apoE–/– and PPARα–/–apoE–/– mice (n = 4 per genotype, data not shown).
Aortic gene expression. Consistent with our finding of greater postheparin LPL enzyme activity in PPARα agonist–treated PPARα+/+ mice, LPL mRNA levels (normalized to GAPDH) were significantly higher in the aortas of PPARα+/+apoE–/– as compared with PPARα–/–apoE–/– mice after 1 week of agonist treatment (Figure 7a). At the same time point, aortic message levels for CD36 and MCP-1 were also higher in agonist-treated PPARα+/+ mice (Figure 7, b and c). This difference was sustained at 4 weeks of agonist treatment for MCP-1 (Figure 7f). However, CD36 mRNA levels were the same in both genotypes (Figure 7e), and LPL mRNA levels were significantly lower in PPARα+/+ mice after 4 weeks of WY-14,643 (Figure 7d). In the same animals, hepatic ACO (a known PPARα-responsive gene) mRNA levels were 10.7-fold higher in PPARα+/+apoE–/– as compared with PPARα–/–apoE–/– mice (P = 0.0004) at 1 week and 4.3-fold higher in PPARα+/+apoE–/– as compared with PPARα–/–apoE–/– mice (P = 0.0002) at 4 weeks.
Aortic expression of LPL, CD36, and MCP-1. PPARα+/+apoE–/– (filled bars) and PPARα–/–apoE–/– (open bars) mice were fed a Western diet plus 0.1% WY-14,643 for 1 week or 4 weeks, followed by isolation of RNA from the entire aorta and analysis by quantitative RT-PCR. Data are presented relative to GAPDH mRNA levels in the same samples. (a) A_P_ = 0.0002, (b) A_P_ = 0.0437, (c) A_P_ = 0.0002, (d) A_P_ = 0.0041, (f) A_P_ = 0.0006, all versus PPARα–/–apoE–/–. Hepatic ACO mRNA in the same animals was 10.7-fold and 4.3-fold higher in PPARα+/+apoE–/– as compared with PPARα–/–apoE–/– mice at 1 and 4 weeks, respectively (both P < 0.001).
Glucose metabolism. To determine if a difference in insulin sensitivity is present before the time points showing differences in atherosclerosis, we performed glucose-tolerance and insulin-tolerance tests in separate cohorts of animals after 4 weeks of high-fat feeding. PPARα–/–apoE–/– mice had lower glycemic excursions than PPARα+/+apoE–/– mice at 30 minutes (P = 0.0003) and 60 minutes (P = 0.0162) after a glucose challenge (Figure 8a). PPARα–/–apoE–/– mice became more hypoglycemic than PPARα+/+apoE–/– mice in response to exogenous insulin (Figure 8b). Hyperinsulinemic clamp experiments demonstrated that the lower glucose levels were due to the effects of insulin on endogenous glucose production (Table 2).
Endogenous glucose production and insulin responsiveness in Western diet–fed mice
Blood pressure determinations. Since hypertension is associated with insulin resistance and promotes atherosclerosis, we measured blood pressures in these mice. There was no effect of PPARα on systolic blood pressure at baseline (Figure 9a). Blood pressure increased in both genotypes after 4 weeks of high-fat feeding (Figure 9b), but were 10% lower in PPARα–/–apoE–/– mice as compared with PPARα+/+apoE–/– mice (P = 0.0336; n = 29 for PPARα–/–apoE–/– and n = 17 for PPARα+/+apoE–/–).
Lower blood pressures in PPARα-deficient mice fed a Western diet. PPARα+/+apoE–/– mice (filled bars) and PPARα–/–apoE–/– mice (open bars) were started on a Western diet at the age of 8 weeks, and systolic blood pressures were measured at the tail 4 weeks later. Five to eight determinations were performed for each mouse at each session after habituating each animal to the blood pressure apparatus for several days. The final pressure was recorded as the mean of two or three sessions for each mouse. Data represent measurements for both sexes. (a) n = 17 for PPARα+/+ and n = 25 for PPARα–/–. (b) n = 17 for PPARα+/+ and n = 29 for PPARα–/–. A_P_ = 0.0336 vs. PPARα+/+.