Liver-restricted PPARδ expression improves glucose homeostasis in mice fed a high fat diet. A, adenovirus mediated hepatic PPARδ expression increases the respiratory exchange ratio at the resting state. High fat-fed C57BL/6 male mice were injected with adenoviral GFP or PPARδ through the tail vein. Three days after viral injection, the mice (n = 5) were placed in metabolic cages to determine the RER. The mice were 20 weeks old and had been on a high fat diet for 10 weeks. active, average RER during the dark cycle; rest, average RER during the light cycle. B and C, GTT (B) and ITT (C) showing improved glucose handling and insulin sensitivity in adenoviral PPARδ-infected mice compared with control animals (n = 7). GTT (overnight fasted) and ITT (6 h fasted) were performed 4 and 5 days after virus injection, respectively. GFP and PPARδ indicate mice receiving adenoviral GFP and PPARδ, respectively. D, histological analyses of liver sections (200×) from GFP and PPARδ adenovirus-injected mice. Liver samples were collected 7 days following virus injection after an overnight fast. Hematoxylin and eosin (H&E) staining was conducted for morphological assessment, and periodic acid-Schiff (PAS) staining (counterstained with hematoxylin) was performed to identify glycogen, which stained purple. Hepatic glycogen and lipid contents were quantified by enzymatic assays. TG, triglyceride; FFA, free fatty acid. E, PPARδ regulates the expression of genes in glucose and lipid metabolism. Liver samples were harvested from control (GFP) or adPPARδ (PPARδ) mice after an overnight fast, and gene expression was determined by real time qPCR. LDH, lactate dehydrogenase; ChREBP, carbohydrate response element-binding protein; AOX, acyl-CoA oxidase; CPT1, carnitine palmitoyl-coA transferase 1; PEPCK, phosphoenolpyruvate carboxykinase. *, p < 0.05.

PPARδ increases glucose utilization in primary hepatocytes. A–C, PPARδ increases glucose flux to glycogen synthesis, lipogenesis, and glycolysis determined by radioactive tracers. Hepatocytes infected with GFP or PPARδ virus were labeled with [¹⁴C]glucose without or with 100 nm insulin. The conversion of radioactive glucose to glycogen, fatty acid, and CO₂ (to estimate glycolysis) was determined and normalized to protein content. D, fatty acid β-oxidation assay determined by [³H]palmitate. E, the expression of glucokinase (GK), GLUT2, and lipogenic genes is up-regulated in hepatocytes infected with adenoviral PPARδ. Gene expression was determined by real time qPCR 48 h post-infection. F, assessment of target gene regulation by endogenous PPARδ. Primary hepatocytes from wild type (wt) and liver-specific PPARδ^−/− (ko) mice were given 0.1 μm GW501516 for 6 h, and gene expression was examined by real time qPCR. *, p < 0.05.

Direct and indirect transcriptional mechanisms by PPARδ in the control of hepatic gene expression. A–E, promoter analyses to determine PPARδ direct target genes. Promoter regions of potential target genes were cloned into a luciferase reporter. The resulting constructs were co-transfected with combinations of expression vectors for PPARδ/RXRα, SREBP-1c, PGC-1β, and PGC-1α (for E only) into HepG2 cells, together with a β-galactosidase reporter internal control. PPARδ/RXRα-transfected cells were cultured in the presence or absence of GW501516 (0.1 μm, PPARδ agonist) for 24 h. The reporter luciferase activity was normalized to β-galactosidase activity to obtain relative luciferase unit (RLU). mGK-2kb, mouse GK 2 kb promoter; hACC2-PI and -PII, human ACC2 promoter I and II; mFAS, mouse FAS promoter; mMCAD, mouse MCAD promoter.

Increased hepatic AMPK activity in adPPARδ mice. A, Western blot analyses showing increased phospho-AMPK (p-AMPK, Thr-172) in adPPARδ livers. Liver lysates were collected from four individual GFP or adPPARδ mice. B, adenine nucleotide concentrations of liver lysates from control or adPPARδ mice (n = 4) determined by HPLC assays. *, p < 0.05; +, p = 0.08. C, real time qPCR analyses demonstrating up-regulation of adiponectin receptor 2 (AdipoR2) in adPPARδ livers. The difference in adiponectin receptor 1 (AdipoR1) expression was not significant. D, circulating adiponectin concentrations in control (GFP) and adPPARδ mice determined by ELISA. E and F, PPARδ expression increases AMPK phosphorylation. Hepatocytes were infected with GFP or PPARδ virus for 24 h in William's E, 5% FBS. The cells were washed and cultured in DMEM for 2 h. In E, hepatocytes were incubated in DMEM for 4 more hours before harvesting. The results from two representative samples were shown. In F, hepatocytes were treated with metformin (met, 2 mm) and harvested at different time points. The basal phospho-AMPK was higher at 6 h (E) than at 3 h (F, minus metformin) after replacing medium to DMEM in PPARδ-expressing hepatocytes. G, PPARδ reduces glucose production through AMPK activation in primary hepatocytes. Hepatocytes were treated as described above. The cells were then cultured in glucose free DMEM containing 1 mm pyruvate and 10 mm lactate, without or with 20 μm compound C (AMPK inhibitor, AMPKi) or 2 mm metformin (AMPK activator) for 2 h. Supernatant was collected to determine glucose concentration. Metformin was included as a control for AMPK-mediated suppression of glucose production. *, p < 0.05.

Assessment of the effect of hepatic PPARδ expression on glycogen synthesis and lipogenesis in chow-fed mice. A and B, histological, glycogen, and lipid analyses of liver samples from GFP and PPARδ adenovirus-injected mice on a chow diet. Liver samples were collected from ad libitum fed animals 2 weeks following virus injection. Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) (counterstained with hematoxylin) staining (A) as well as enzymatic assays (B) were conducted to determine glycogen and triglyceride content. C, levels of liver glycogen synthase (GS) and ACC determined by Western blotting. The samples were collected from four individual animals from GFP and PPARδ adenovirus-injected mice. Actin was included as the loading control. D, hepatic gene expression determined by real time qPCR. Liver samples were harvested from control (GFP) or adPPARδ (PPARδ) mice under ad libitum feeding condition. *, p < 0.05.

Increased monounsaturated to saturated fatty acid ratios in adPPARδ livers. A and B, triglycerides were isolated from GFP or PPARδ adenovirus-infected livers of normal chow-fed (NC) or high fat diet-fed (HF) mice. Fatty acid compositions in triglycerides were determined by gas-liquid chromatography. The ratios of monounsaturated to saturated fatty acids were shown in the tables. C, PPARδ regulates SCD1 promoter. Luciferase reporters driven by 5.3- or 1.5-kb mouse SCD1 promoter were co-transfected with expression vectors for PPARδ/RXRα into HepG2 cells, together with a β-galactosidase reporter internal control ± GW501516 (0.1 μm, PPARδ agonist) for 24 h. The reporter luciferase activity was normalized to β-galactosidase activity to obtain relative luciferase unit (RLU). D, triglyceride (TG) production determined by administration of a lipoprotein lipase inhibitor, Triton WR1339. Serum triglyceride concentrations were measured at the indicated time course after Triton injection. *, p < 0.05.

Reduced stress signaling and inflammatory gene expression in the liver of adPPARδ mice. A, assessment of liver damage in GFP or PPARδ adenovirus-infected mice on normal chow (NC) or high fat diet (HF) diets by serum AST and ALT activities. B, Western blot analyses demonstrating decreased JNK activity in livers of adPPARδ mice. Liver lysates were harvested from four individual mice/group of the high fat-fed cohort. p-JNK, phospho-JNK; t-JNK, total JNK; p-Erk1/2, phospho-Erk1/2. C, PPARδ inhibits phospho-JNK and increases insulin-stimulated phospho-Akt in primary hepatocytes. Hepatocytes were infected with GFP or PPARδ virus for 24 h in William's E, 5% FBS. The cells were washed and maintained in the same medium ± 100 μm palmitate (albumin-bound) overnight. Hepatocytes were serum-starved for 2 h, followed by insulin stimulation (100 nm) for 30 min. Left panel, JNK and Akt signaling was determined by Western blotting in GFP or PPARδ adenovirus-infected hepatocytes without (control) or with fatty acid treatment (FA loading). Right panel, normalized cellular triglyceride content. D, PPARδ suppresses the expression of pro-inflammatory genes. Liver samples were harvested from control (GFP) or adPPARδ (PPARδ) mice on normal chow (NC) or high fat (HF) diets, and gene expression was determined by real time qPCR. *, p < 0.05.