PPARgamma in the endothelium regulates metabolic responses to high-fat diet in mice

Abstract

Although endothelial dysfunction, defined as abnormal vasoreactivity, is a common early finding in individuals with type 2 diabetes, the endothelium has not been known to regulate metabolism. As PPARgamma, a transcriptional regulator of energy balance, is expressed in endothelial cells, we set out to investigate the role of endothelial cell PPARgamma in metabolism using mice that lack PPARgamma in the endothelium and BM (gammaEC/BM-KO). When gammaEC/BM-KO mice were fed a high-fat diet, they had decreased adiposity and increased insulin sensitivity compared with control mice, despite increased serum FFA and triglyceride (TG) levels. After fasting or olive oil gavage, gammaEC/BM-KO mice exhibited significant dyslipidemia and failed to respond to the FFA and TG lowering effects of the PPARgamma agonist rosiglitazone. BM transplantation studies, which reconstituted hematopoietic PPARgamma, established that these metabolic phenotypes were due to endothelial PPARgamma deficiency. We further found that the impairment in TG-rich lipoprotein metabolism in gammaEC/BM-KO mice was associated with fatty acid-mediated lipoprotein lipase inhibition and changes in a PPARgamma-regulated endothelial cell transcriptional program. Despite their metabolic improvements, high-fat diet-fed gammaEC/BM-KO mice had impaired vasoreactivity. Taken together, these data suggest that PPARgamma in the endothelium integrates metabolic and vascular responses and may contribute to the effects of PPARgamma agonists, thus expanding what endothelial function and dysfunction may entail.

Figure 1. PPARγ-floxed mice crossed with Tie2Cre⁺ transgenic mice (γEC/BM-KO) have decreased Pparg mRNA expression in endothelial and lymphocytes (hematopoietic cells) but not adipocytes, liver, or skeletal muscle as compared with PPARγ-floxed mice crossed to Tie2Cre^– mice (γEC/BM-WT).

Real-time quantitative PCR analysis of PPARγ expression in microvascular ECs, splenic lymphocytes, adipocytes, liver, and skeletal muscle isolated from Tie2Cre-expressing (γEC/BM-KO) or nonexpressing (γEC/BM-WT) mice (n = 3–5/group). *P < 0.05, **P < 0.01 γEC/BM-KO versus γEC/BM-WT mice.

Figure 2. Tie2Cre-mediated PPARγ deletion decreases white adipose tissue mass and adipocyte size after high-fat diet.

(A) Body weights of mice either with (γEC/BM-KO) or without (γEC/BM-WT) Tie2-mediated PPARγ deletion after standard chow or high-fat diet (12 weeks, here and throughout except as noted; n = 6). (B) Weight gain over time of γEC/BM-WT (gray squares) and γEC/BM-KO (black squares) mice on high-fat diet (n = 7–9/group). SEM < 8% for all measurements. (C) Tissue weight/total body weight of inguinal (ing), epididymal (epi), inguinal plus epididymal fat, liver, spleen, and kidney after standard chow or high-fat diet (12 weeks; n = 6). (D) Histology of epididymal adipose tissue from γEC/BM-KO and γEC/BM-WT mice on standard chow or high-fat diet as above. Scale bar: 200 μm. (E) Mean adipocyte size in γEC/BM-WT versus γEC/BM-KO mice (n = 5/group). *P < 0.05, **P < 0.01 versus γEC/BM-WT mice under the same diet.

Figure 3. Tie2Cre-mediated PPARγ deletion improves glucose control and insulin sensitivity in response to high-fat diet.

(A) Fasting glucose and (B) insulin levels and (C) glucose tolerance and (D) insulin tolerance testing in γEC/BM-KO and γEC/BM-WT mice after standard chow or high-fat diet (n = 5–6). (C) The AUC of glucose levels during glucose tolerance tests is shown. *P < 0.05, **P < 0.01 versus obese γEC/BM-WT mice. GTT, glucose tolerance test.

Figure 4. Tie2Cre-mediated PPARγ deletion increases serum FFA and TG levels but decreases TG deposition in skeletal muscle after high-fat diet feeding.

(A) Serum levels of adiponectin, leptin, retinol binding protein 4 (RBP4), resistin, FFA, and TG in γEC/BM-KO and γEC/BM-WT mice after standard chow or high-fat diet (n = 4–9). (B) Skeletal muscle TG content in γEC/BM-KO and γEC/BM-WT mice after high-fat diet is shown (left panel; n = 5). Western blotting and quantification of insulin-stimulated AKT serine 473 phosphorylation in skeletal muscle is shown (right panel; n = 3–6). (C) Liver TG content in γEC/BM-KO and γEC/BM-WT mice after high-fat diet is shown (left panel; n = 5). Western blotting and quantification of insulin-stimulated AKT serine 473 phosphorylation in liver is shown (right panel; n = 3–6). Real-time quantitative PCR analysis of Pparg2 and Cd36 expression in liver in γEC/BM-KO and γEC/BM-WT mice after high-fat diet (n = 5). *P < 0.05, **P < 0.01 versus obese γEC/BM-WT mice.

Figure 6. Tie2Cre-mediated PPARγ endothelial deletion decreases adiposity, adipocyte size, and insulin resistance after high-fat diet in a manner dependent on endothelial but not BM PPARγ expression.

(A) Ratio of inguinal, epididymal, liver, and spleen weight/body weight after high-fat diet in BMT mice (n = 4–7). Histology of epididymal adipose tissue from BMT mice on high-fat diet as above. Scale bar: 200 μm. Mean adipocyte size in BMT mice on high-fat diet (n = 4–7/group). *P < 0.05, **P < 0.01 versus γEC-WT/BM-WT mice. (B) Glucose tolerance and insulin tolerance testing in BMT mice on high-fat diet (n = 4–7). AUC of glucose levels during glucose tolerance tests is shown. *P < 0.05 γBM-WT or γBM-KO transplanted into EC-KO mice versus γBM-WT or γBM-KO into γEC-WT mice, respectively.

Figure 5. Tie2Cre-mediated PPARγ endothelial deletion decreases adiposity and adipocyte size in response to rosiglitazone treatment in a manner dependent on endothelial but not BM PPARγ expression.

(A) Ratio of inguinal, epididymal, and brown adipose tissue weight/body weight in γEC/BM-KO and γEC/BM-WT mice after rosiglitazone (Rosi) treatment when fed with a standard chow diet (n = 5–8). *P < 0.05, **P < 0.01 versus same genotype mice. ^††P < 0.01 versus γEC/BM-WT mice treated with rosiglitazone. (B) Histology of inguinal adipose tissue from γEC/BM-WT and γEC/BM-KO mice from A treated with or without rosiglitazone. Scale bar: 200 μm. (C) Adipocyte size of inguinal adipose tissue from γEC/BM-KO and γEC/BM-WT mice from A treated with or without rosiglitazone. *P < 0.05 versus γEC/BM-WT with same treatment. (D) Real-time quantitative PCR analysis of PPARγ expression in splenic lymphocytes isolated from mice after BMT (n = 4–5/group). Ratio of epididymal, inguinal, inguinal plus epididymal and brown adipose tissue weight/body weight after rosiglitazone treatment in BMT mice (n = 5–9). *P < 0.05, **P < 0.01 versus γEC-WT/BM-WT mice.

Figure 7. Endothelial PPARγ deletion increases TG and FFA levels after fasting.

(A) FFA and TG concentrations were determined in fed and fasted (24 hours, throughout) γEC/BM-WT and γEC/BM-KO mice (n = 6/genotype). *P < 0.05; **P < 0.01 versus same genotype in fed state. ^††P < 0.01 versus fasted γEC/BM-WT mice. (B) Lipoprotein profiles after fasting (n = 4/genotype). (C) Total cholesterol concentration in fasted γEC/BM-WT and γEC/BM-KO mice (n = 10/genotype). **P < 0.01 versus γEC/BM-WT mice. Lipoprotein profiles of cholesterol after fasting (n = 4/genotype).

Figure 8. Endothelial PPARγ deletion increases TG and FFA levels after olive oil gavage independent of hematopoietic PPARγ expression.

(A) Representative blood samples and FFA and TG concentrations in standard chow–fed γEC/BM-WT and γEC/BM-KO mice after overnight fasting, followed by olive oil gavage (n = 6–8/genotype). **P < 0.01 versus γEC/BM-WT. (B) Lipoprotein profiles 3 hours after olive oil gavage (n = 3/genotype). (C) FFA and TG concentration after olive oil feeding in mice after γBM-WT or γBM-KO BMT into EC-KO or EC-WT mice (n = 5–7/genotype). *P < 0.05, **P < 0.01 over time course versus γBM-WT into γEC-WT mice. (D) Uptake of a fluorescently labeled long-chain FA (BODIPY-dodecanoic acid) by primary microvascular ECs from γEC/BM-KO and γEC/BM-WT mice measured without or with rosiglitazone stimulation (n = 5,6/genotype). **P < 0.01 versus ECs from γEC/BM-WT without rosiglitazone. (E) Cd36 mRNA expression (real-time quantitative PCR) and protein levels in microvascular ECs (n = 3–5/genotype). One representative Western blot is shown at right. *P < 0.05, **P < 0.01 versus γEC/BM-WT mice. (F) Expression of genes in endothelial cells of γEC/BM-KO versus γEC/BM-WT mice. (n = 5,6/genotype) *P < 0.05, **P < 0.01 versus γEC/BM-WT mice.

Figure 9. Tie2Cre-mediated PPARγ deletion increases VLDL production rate and inhibits LPL activity.

(A) TG concentrations and calculated VLDL production rate after triton injection in fasted (24 hours) γEC/BM-WT and γEC/BM-KO mice (n = 5/genotype). *P < 0.05 versus γEC/BM-WT. (B) Correlation between serum FFA levels and TG levels in fasted γEC/BM-WT (white circles) and γEC/BM-KO (black circles) mice (n = 6–8/genotype) 3 hours after olive oil gavage. (C) Real-time quantitative PCR analysis of Lpl mRNA expression in white adipose tissue and skeletal muscle from γEC/BM-WT and γEC/BM-KO mice when fed with a standard chow diet. (D) Post-heparin LPL and HL activity 3 hours after olive oil feeding, in the absence (left panel) or presence (right panel) of excess FFA-free BSA (n = 6–8). *P < 0.05 γEC/BM-KO versus γEC/BM-WT mice.

Figure 10. Rosiglitazone fails to lower FA and TG levels after lipid challenge in γEC/BM-KO mice as compared with γEC/BM-WT mice.

(A) FFA and (B) TG concentrations after olive oil gavage in γEC/BM-WT (left panels) and γEC/BM-KO (right panels) mice on standard chow diet with or without rosiglitazone treatment (n = 5–8/genotype). *P < 0.05, **P < 0.01 for entire curve, versus same genotype without rosiglitazone.

Figure 11. Tie2Cre-mediated PPARγ deletion impairs vasodilation in arteries from mice fed high-fat diet.

Vascular responses were measured in arterial preparations isolated from γEC/BM-WT and γEC/BM-KO mice fed standard chow (A) or high-fat diet (B). Percent dilation of left common carotid arteries after phenylephrine preconstriction (10^–5 M), followed by increasing doses of carbachol (n = 4–5). *P < 0.05, **P < 0.01 versus γEC/BM-WT mice under similar dietary conditions.

Figure 12. PPARγ in the endothelium integrates metabolic and vascular phenotypes.

Studies in mice lacking PPARγ in the endothelium identify endothelial PPARγ as controlling specific metabolic and vascular responses to high-fat diet, as summarized. After high-fat diet feeding, mice lacking endothelial PPARγ manifest increased plasma TG and FFA levels, decreased adiposity, less skeletal muscle TG accumulation, and decreased insulin resistance (IR). This phenotype derives from endothelial PPARγ regulation of target genes involved in TG metabolism as well as FA uptake and handling, including Cd36, aP2, CRBP-III, and Gpihbp1. In contrast, livers in endothelial PPARγ-deficient mice have greater TG accumulation, increased VLDL production, and decreased AKT phosphorylation. In the liver, the endothelium is fenestrated, which fosters FFA uptake, while in skeletal muscle and adipose tissue, a nonfenestrated endothelium is found. The dyslipidemia seen in endothelial PPARγ-deficient mice after high-fat diet and acute lipid loading involves both increased VLDL production and inhibition of LPL function by elevated FFA levels. The metabolic improvements evident in endothelial PPARγ-deficient mice contrast with their impaired arterial vasodilation, highlighting the tissue-specific actions of endothelial PPARγ, the role of the endothelium in directing metabolic responses, and the concept of metabolic endothelial function/dysfunction.