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Impaired visceral white adipose tissue (WAT) metabolism has been implicated in the pathogenesis of several lifestyle-related disease states, with diminished expression of several WAT mitochondrial genes reported in both insulin-resistant humans and rodents. We have used rat models selectively bred for low- (LCR) or high-intrinsic running capacity (HCR) that present simultaneously with divergent metabolic phenotypes to test the hypothesis that oxidative enzyme expression is reduced in epididymal WAT from LCR animals.

Based on this assumption, we further hypothesized that short-term exercise training (6 wk of treadmill running) would ameliorate this deficit. Approximately 22-wk-old rats (generation 22) were studied. In untrained rats, the abundance of mitochondrial respiratory complexes I–V, citrate synthase (CS), and PGC-1 was similar for both phenotypes, although CS activity was greater than 50% in HCR ( P = 0.09). Exercise training increased CS activity in both phenotypes but did not alter mitochondrial protein content.

Training increased the expression and phosphorylation of proteins with roles in β-adrenergic signaling, including β 3-adrenergic receptor (16% increase in LCR; P. White adipose tissue (WAT) mass is linked to metabolic health and plays a critical role in the maintenance of whole body energy homeostasis (, ). Increased WAT mass, especially in visceral storage depots, is associated with a greater risk of metabolic disease and mortality (, ), whereas enlarged adipocyte cell size due to increased lipid content is linked to intrinsic cellular metabolic defects (, ). Conversely, small adipocytes may play a protective role against the increased risk of metabolic disease because compared with larger adipocytes these cells have enhanced rates of glucose transport (, ). Although excessive lipid storage in visceral WAT depots is linked to metabolic abnormalities such as insulin resistance and impaired lipolysis (, ), the metabolic characteristics of WAT in differing metabolic phenotypes have not been well characterized. Compared with other metabolically active tissues, the oxidative capacity of WAT is relatively low , but essential cellular activities such as adipogenesis, lipogenesis, lipolysis, and fatty acid (FA) oxidation require large amounts of ATP (, ). Given that WAT metabolism is altered in obesity and insulin resistance (, ) and the metabolic activity of most cells is highly dependent on mitochondrial content, impairments in the regulation of the adipocyte mitochondrial network may lead to dysfunctional WAT metabolism.

Indeed, diminished mitochondrial gene expression has been observed in WAT from insulin-resistant humans (, ) and rodents (, ). Through two-way artificial selection, we have generated animal models of high high-intrinsic running capacity (HCR) and low aerobic treadmill running capacity low-intrinsic running capacity (LCR) in the absence of exercise training. Such selection has produced rats that simultaneously present with different metabolic and cardiovascular disease risk factors without the necessity for any environmental intervention. Previously, we have shown that the metabolic characteristics of the skeletal muscle from these rats diverge substantially (, ) and that exercise training ameliorates many of the adverse health features observed in the LCR rats. Since exercise training is capable of reducing visceral WAT lipid content and adipocyte cell size (, ) while increasing the WAT expression of a number of mitochondrial proteins (, ), we hypothesized that the oxidative profile of visceral WAT would be lower in untrained LCR compared with HCR rats but that short-term exercise training would ameliorate this impairment. In line with this hypothesis, we aimed to determine whether differences in intrinsic running capacity or training state would affect the expression and activity of a number of proteins with important roles in WAT metabolism.

Animal model. This study was undertaken with the combined approval of the animal ethics committees from both the University of Michigan (Ann Arbor, MI) and California State University (Northridge, CA). Rat models for LCR and HCR were derived from genetically heterogeneous N:NIH stock (National Institutes of Health) rats by artificial selection for treadmill running capacity, as described previously. Rats were housed in pairs in a temperature-controlled environment that provided a reverse 12:12-h light-dark cycle. Throughout the study, rats were given ad libitum access to standard rodent chow and water. Prior to the commencement of any experimental procedures, rats were allowed to acclimate to laboratory conditions for 1 wk. Experimental design.

Age-matched pairs of ∼20-wk-old male LCR and HCR rats were randomly assigned to two groups: sedentary LCR-SED ( n = 10) and HCR-SED ( n = 10) or exercise trained LCR-EX ( n = 10) and HCR-EX ( n = 10). Rats assigned to undergo exercise training completed a 6-wk (4 days/wk) incremental treadmill running protocol where all rats completed the same absolute cumulative running distance (∼10 km) (, ).

Trained rats undertook their final exercise bout 48 h before the commencement of any experimental procedures. Tissue collection and blood analyses. Following a 5-h fast, blood samples were taken for the analysis of fasting blood glucose concentrations using a hand-held glucometer (Roche Diagnostics, Castle Hill, New South Wales, Australia).

Serum was assessed for fasting insulin concentrations using a rat-specific ELISA (ALPCO diagnostics, Salem, NH) and for nonesterified FAs (NEFA) using a commercially available kit (WAKO Pure Diagnostics, Osaka, Japan). Rats were weighed and anesthetized using pentobarbital sodium (1 ml/kg body mass). Hindlimb skeletal muscles and epididymal fat pads were surgically excised, weighed, freeze-clamped in liquid nitrogen, and stored at −80°C for later analyses. Muscle data has been reported previously. Citrate synthase activity. Approximately 100 mg of epididymal adipose tissue was visibly cleared of blood vessels and connective tissue and then mechanically homogenized in buffer 175 mM KCl and 2 mM EDTA (pH 7.4), 1:2 dilution and centrifuged at 20,000 g for 15 min at 4°C. The infranatant was collected and assayed for citrate synthase activity, as described previously.

Protein concentration of the infranatant was determined using the bicinchoninic method (Pierce). Activity is expressed in nMmin −1μg protein −1.

Approximately 250–300 mg of epididymal adipose tissue was visibly cleared of blood vessels and connective tissue and then mechanically homogenized in buffer (50 mM TrisHCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 50 mM NaF, 5 mM Na pyrophosphate, 10% glycerol, 1% Triton X-100, 10 μg/ml trypsin inhibitor, 2 μg/ml aprotinin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1:8 dilution) and centrifuged at 20,000 g for 30 min at 4°C. Protein concentration of the infranatant was determined using the bicinchoninic method (Pierce). Adipose tissue lysates containing 10 μg of protein were prepared in 4× Laemmli buffer, subjected to SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. A pooled lysate sample was prepared and included in each gel as an internal control for normalizing the data. Ponceau staining was used to confirm equal protein transfer. Membranes were then washed and blocked (5% nonfat dry milk or 5% BSA) for 1 h at room temperature prior to incubation with the appropriate antibodies.

Membranes were incubated overnight at 4°C with primary antibodies specific for citrate synthase (CS; ∼52 kDa; Abcam, no. Ab96600), mitochondrial respiratory complexes I, II, III, IV (subunit 4; COX-IV), and V of the electron transport chain (∼18, ∼25, ∼45, ∼15, and ∼52 kDa respectively; MitoSciences, nos. MA604 and MS407), uncoupling protein 1 (UCP1; Santa Cruz Biotechnology, no. Sc6529), peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1; ∼100 kDa; Chemicon, no. Ab3242), hormone-sensitive lipase (HSL; ∼88 kDa; Cell Signaling Technology, no. 4107), phospho-HSL S660 , adipose triglyceride lipase (ATGL; ∼54 kDa; Cell Signaling Technology, no.

2138), phospho-ATGL S406 ∼54 kDa , β 3-adrenergic receptor (β 3-AR; ∼68 kDa; Santa Cruz Biotechnology, no. Sc50436), perilipin 1 (PLIN1; ∼68 kDa; Sigma, P1873), comparative gene identification-58 (CGI-58; ∼42 kDa), FA-binding protein 4 (FABP4; ∼15 kDa; Abcam, no.

Ab37458), neuron-derived clone 77 (NUR77; ∼48 kDa; Santa Cruz Biotechnology, no. Sc5569), neuron-derived orphan receptor 1 (NOR1; ∼68 kDa; Abcam, no.92777), GLUT4 (∼45 kDa; Abcam, no. Ab654), AMP-activated protein kinase (AMPK)α (∼62 kDa; Cell Signaling Technology, no. 2532), AMPK phospho-Thr 172 (∼62 kDa; Cell Signaling Technology, no. 2535), extracellular regulated kinase 1/2 (ERK1/2; ∼46 and ∼42 kDa; Cell Signaling Technology, no. 9102), phospho-ERK1/2 T202/Y204 (∼46 and ∼42 kDa; Cell Signaling Technology, no. 9101), p38 mitotgen-activated protein kinase (p38 MAPK; ∼44 kDa; Cell Signaling Technology, no.

9212), phospho-p38 MAPK T180/Y182 (∼44 kDa; Cell Signaling Technology, no. 9211), c-Jun NH 2-terminal ninase 1/2 (JNK1/2; ∼50 and ∼46 kDa; Cell Signaling Technology, no.

9252), or phospho-JNK1/2 T183/Y185 (∼50 and ∼46 kDa; Cell Signaling Technology, no. Membranes were also probed with anti-α-tubulin (∼50 kDa; Cell Signaling Technology, no. 2144) or β-actin (∼42 kDa; Sigma-Aldrich) to confirm equal protein loading. After 1-h room temperature incubation in the appropriate secondary antibody, protein expression was detected using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) and quantified by densitometry.

Physiological parameters. Data for body mass (BM) and fat pad mass have been reported previously , with new statistical analyses presented here. Adipose tissue protein content is reported as an indirect marker of changes in adipocyte cellularity. Intrinsic running capacity and training status were main effects for BM ( P.

Body mass ( A), epididymal fat pad mass ( B), and white adipose tissue (WAT) total protein ( C) for low- (LCR; open bars) and high-capacity running rats (HCR; filled bars) with or without exercise training. Values are means ± SE; n = 8–10/group.

Intrinsic running capacity improved fasting blood glucose (main effect, P = 0.0002) and serum NEFA concentrations (main effect, P = 0.02; ), but not fasting serum insulin levels. Compared with HCR-SED, LCR-SED rats had 8% higher blood glucose ( P = 0.0008) and 37% higher NEFA concentrations ( P = 0.003). Exercise training was a main effect for fasting serum NEFAs ( P. Intracellular regulators of lipolysis. Β 3-AR expression was increased with exercise training (main effect, P = 0.03; ), whereas running capacity showed a tendency to increase this parameter ( P = 0.09). There was a significant interaction between running capacity and exercise training ( P = 0.006). The expression of the β 3-AR was 17% greater in HCR-SED compared with LCR-SED rats ( P = 0.006) and 18% greater in LCR-EX compared with LCR-SED rats ( P = 0.004).The phosphorylation of ATGL at Ser 406 and HSL at Ser 660 was assessed as surrogate markers of their activity (, ).

ATGL Ser 406 phosphorylatation and total ATGL protein content were increased by both running capacity ( P = 0.02 and P = 0.003, respectively) and exercise training status ( P = 0.0005 and P = 0.03, respectively;, B and C). Post hoc analyses revealed a 25% increase in ATGL Ser 406 phosphorylation in HCR-EX compared with HCR-SED rats ( P = 0.01; ). Total ATGL expression was 17% greater in HCR-EX compared with LCR-EX rats ( P = 0.04; ) and the ratio of ATGL Ser 406 to total ATGL was not different. Total HSL protein remained similar for all groups, although there was a tendency for HSL Ser 660 phosphorylation to be decreased in both LCR-EX and HCR-EX rats with training (main effect, P = 0.09). There was no difference in the ratio of HSL Ser 660 phosphorylation to total HSL protein (data not shown). The content of PLIN1 (which controls lipolysis by regulating protein-protein interactions at the surface of lipid droplets, thereby facilitating access of lipases to their substrates) was increased by training ( P = 0.02; ), and an interaction was observed between training and running capacity ( P = 0.003). Post hoc analyses revealed that PLIN1 expression was 19% greater in LCR-SED compared with HCR-SED rats ( P = 0.03) and 25% greater in HCR-EX compared with HCR-SED rats ( P = 0.002).

The protein content of CGI-58 (which binds to and activates ATGL triglyceride lipase activity) was increased by 15% in HCR-SED compared with LCR-SED rats and 15% greater in LCR-EX compared with LCR-SED rats, although neither of these values attained statistical significance ( P = 0.08 and P = 0.1, respectively; ). There were main effects for both running capacity and exercise training for CGI-58 protein content ( P = 0.04 and P = 0.03, respectively; ). No difference in FABP4 expression was observed between groups (data not shown).

NOR1, NUR77, and GLUT4 expression. A main effect of exercise training was observed for NOR1 expression ( P.

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Stress kinase activation. We investigated several stress-activated kinases to determine their involvement in the adaptive response of WAT metabolism to exercise training. Total p38 MAPK expression was reduced with training ( P = 0.03; ), whereas there was a tendency for phosporylation of p38 MAPK on the Thr 180 and Tyr 182 residues to be reduced by training ( P = 0.07). There was a main effect of exercise training on the ratio of phospho-p38 MAPK T180/Y182 to total p38 MAPK ( P = 0.03). No differences were observed in total JNK1/2 expression; however, training increased phospho-JNK1/2 T183/Y185 ( P = 0.02) and the ratio of phospho-JNK1/2 T183/Y185 to total JNK1/2 ( P = 0.002; ). No differences were observed in total ERK1/2 expression , although a significant main effect of running capacity was observed for phospho-ERK1/2 T202/Y204 ( P = 0.03). There was also a tendency for the ratio of phospho-ERK1/2 T202/Y204 to total ERK1/2 to be affected by running capacity (LCR HCR, P = 0.06).

No differences were observed in total or phospho-AMPK T172 expression, or the ratio of phospho-AMPK T172 to total AMPK expression (data not shown). AUTHOR CONTRIBUTIONS E.J.S., S.J.L., D.A.R., M.J.W., B.B.Y.I., L.G.K., S.L.B., and J.A.H. Contributed to the conception and design of the research; E.J.S., S.J.L., D.A.R., and B.B.Y.I.

Performed the experiments; E.J.S. Analyzed the data; E.J.S. Interpreted the results of the experiments; E.J.S. Prepared the figures; E.J.S.

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Drafted the manuscript; E.J.S., M.J.W., L.G.K., S.L.B., and J.A.H. Edited and revised the manuscript; E.J.S., S.J.L., D.A.R., M.J.W., L.G.K., S.L.B., and J.A.H.

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Approved the final version of the manuscript.