Voluntary exercise improves insulin sensitivity and adipose tissue inflammation in diet-induced obese mice

Animals.

These studies were approved by the Beth Israel Deaconess Medical Center (BIDMC) Animal Care and Use Committee. Twelve-week-old male mice (29 male C57BL/6, Jackson Laboratory, Bar Harbor, ME) were purchased and divided into two groups: chow diet (n = 13) and HFD (n = 16). Chow diet purchased from Purina was Formulab Diet 5008 (3.71 kcal/g), which contains 6.5% fat by weight and provides 16.7% total calories from fat. HFD purchased from Research Diets, D12451 (4.7 kcal/g) contains 24% fat by weight and provides 45% calories from fat. Mice were housed in the BIDMC Animal Facility and maintained at 22°C under a 14:10-h alternating light-dark cycle, which is standard for this facility. After 4 wk, chow-fed and HFD-fed mice were further subdivided into no-exercise and exercise groups designated as chow-no exercise (n = 6), chow-exercise (n = 7), HFD-no exercise (n = 8), and HFD-exercise (n = 8), respectively. These four groups were monitored for an additional 6 wk. Mice were housed individually and received either a functional running wheel (Bio-Serv, Frenchtown, NJ) or an identical locked, nonfunctional running wheel. Body weights of animals were measured three times per week, and food intake was measured weekly.

Locomotor activity.

Exercise monitoring was performed between the fourth and fifth weeks after mice had been exposed to the running wheels. Total activity was assessed using two techniques. To assess wheel running activity, we used an infrared camera to record four mice running over a 6-h period from the beginning of the dark cycle and for a 6-h period from the beginning of the light cycle. The recordings were then reviewed, and total revolutions were recorded by an observer. A fluorescent marker was fixed to the wheel so that rotations could be counted as they were observed, using a hand-held manual counter. We also assessed relative locomotor activity over 24 h in mice in the absence of running wheels, using a monitoring system from Columbus Instruments (Columbus, OH) that reports activity as sequential beam breaks on an infrared grid.

Glucose tolerance tests.

Intraperitoneal glucose tolerance tests (GTT) were performed after mice had undergone 6 wk of exercise. Running wheels were removed from cages in the evening before metabolic testing. Mice were fasted overnight (1700–0800) and were subsequently injected with glucose (2 g/kg body wt ip). Tail blood was collected at −1, 15, 30, 60, and 120 min. Blood glucose concentrations were measured using a glucometer (Elite, Bayer, Mishawaka, IN).

Insulin tolerance tests.

Insulin tolerance tests (ITT) were also performed after 6 wk of exercise. Between 1400 and 1500, fed mice were injected with regular insulin (0.75 U/kg, Eli Lilly, Indianapolis, IN), and tail-blood samples were obtained at −1, 15, 30, 60, and 120 min after the insulin injection.

Quantitative real-time PCR.

Total RNA was extracted from liver and adipose tissues using the Ultraspec RNA Isolation System (Biotecx, Houston, TX) according to the manufacturer's instructions. cDNA was synthesized using oligo(dT) primers with the Advantage RT-for-PCR kit (BD Biosciences, San Jose, CA). Primers spanned intronic regions to generate 300- to 400-bp PCR products. PCR amplifications were quantified using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Primer sequences were as follows: TNF-α (Fwd: 5′-GACCCTCACACTCAGATCATCTTCT-3′, Rev: 5′-CCACTTGGTGGTTTGCTACGA-3′), MCP-1 (Fwd: 5′-GCTGACCCCAAGAAGGAATG-3′, Rev: 5′-GTGCTTGAGGTGGTTGTGGA-3′), and PAI-1 (Fwd: 5′-ACAGCCAACAAGAGCCAATC-3′, Rev: 5′-ATAGCCAGCACCGAGGACAC-3′). Mouse gene PCR primer sets (RT²) for IKKβ (Cat no. PPM03198A, Reference Sequence Accession no. NM_010546) were purchased from SuperArray Biosciences (Frederick, MD). Results were normalized against cyclophilin (Fwd: 5′-GGTGGAGAGCACCAAGACAGA-3′, Rev: 5′-GCCGGAGTCGACAATGATG-3′).

Plasma leptin, adiponectin, and insulin.

Plasma leptin and adiponectin were measured using commercially available ELISA kits (leptin and insulin: Crystal Chem, Downers Grove, IL; adiponectin: LINCO Research, St. Charles, MO) according to manufacturers' instructions. Plasma insulin was measured using a mouse insulin assay (Crystal Chem) as described by the manufacturer.

Retinol-binding protein-4.

Serum retinol-binding protein-4 (RBP4) was measured by quantitative Western blotting standardized to full-length recombinant RBP4, as described previously (15). Signals were quantified with GeneSnap software (Synoptics/Syngene, Frederick, MD).

Body composition.

Fat and lean body mass were assessed using dual-energy X-ray absorptiometry (DEXA; Lunar PIXImus2 mouse densitometer, GE Medical Systems, Madison, WI) as described by the manufacturer. Mice were anesthetized by intraperitoneal injection of a (1:1) mixture of tribromoethanol and tert-amyl alcohol, 0.015 ml/g body wt. The animals were then scanned, and total body fat and lean body mass were determined using an analysis program provided by the manufacturer.

Statistical analysis.

Values are reported as group means ± SE. Interactions between diet and exercise on physiological parameters were analyzed by two-way ANOVA. One-way ANOVA and independent t-test were also used. A probability value of <0.05 was considered statistically significant. Statistical comparisons were made using StatView (Abacus Concepts, Berkley, CA).

Voluntary exercise mitigates diet-induced obesity.

Mice that had access to running wheels ran a total of 3,402 ± 1,822 m during the first 6 h of the dark cycle and 135 ± 75 m during the first 6 h of the light cycle. This is consistent with other reports, which showed that male C57BL/6 mice will run on an average of 10 km over 24 h (5). Relative activity of mice that had exercised was increased over control mice, even in the absence of running wheels. When monitored in cages using infrared beam breaks, mice with previous access to running wheels had 1,319 ± 239 beam breaks/h during the dark cycle, whereas mice housed with fixed running wheels had only 638 ± 124 beam breaks/h during the dark cycle. During the light cycle, exercising mice were also more active and had 279 ± 31 beam breaks/h compared with nonexercised mice, which had an average of 129 ± 14 beam breaks/h.

To validate the effects of HFD on body weight and body composition, 12-wk-old male C57BL/6 mice were divided into two groups, a chow-fed group (n = 13) and a HFD-fed group (n = 16). As expected, the HFD group gained substantially more body weight. After 4 wk of ad libitum feeding, HFD mice gained 6.8 ± 0.5 g compared with 2.3 ± 0.3 g for the chow group. HFD mice weighed 33 ± 1.3 g vs. 28.5 ± 0.8 g for chow-fed animals (Fig. 1A).

After 4 wk, chow-fed and HFD-fed mice were further subdivided into chow-no exercise (n = 6), chow-exercise (n = 7), HFD-no exercise (n = 8), and HFD-exercise (n = 8) groups, respectively. These four groups were monitored for an additional 6 wk. As shown in Fig. 1B, both chow-no exercise and HFD-no exercise mice continued to gain weight during this period, whereas no further increase in body weight occurred in chow-exercise and HFD-exercise mice. Over the course of 6 wk, chow-no exercise and HFD-no exercise mice gained an average of 1.2 ± 0.3 and 3.3 ± 0.5 g, respectively. Body composition analysis by DEXA (Fig. 2A) showed that the chow-no exercise and HFD-no exercise groups had significantly greater fat mass (6 ± 0.7 and 12 ± 0.8 g, respectively) compared with chow-exercise and HFD-exercise groups (4 ± 0.2 and 9 ± 0.7 g, respectively). By comparison, lean body mass was unchanged among the four groups (Fig. 2A). Furthermore, the reduced fat body mass in chow-exercise and HFD-exercise mice persisted despite an ∼12% increase in cumulative food intake in both these groups (Fig. 2B).

Voluntary exercise reverses HFD-induced insulin resistance.

Numerous studies have shown a strong correlation between consumption of HFD and the onset of insulin resistance in rodents and humans (30). To evaluate the effects of exercise on HFD-induced insulin resistance, GTT and ITT were performed in chow-no exercise, chow-exercise, HFD-no exercise, and HFD-exercise mice. Glucose tolerance was assessed by calculating the incremental area under the curve (AUC). GTT showed that chow-exercise mice had slightly better glucose tolerance (6% decrease in glucose AUC) than chow-no exercise mice (Fig. 3A). Compared with chow-no exercise mice, HFD-no exercise mice had significantly worse glucose tolerance, as evidenced by a 25% increase in their glucose AUC (P < 0.05). Indeed, among the four groups, HFD-no exercise animals had the worst glucose tolerance. However, voluntary exercise (HFD-exercise group) markedly improved this impaired glucose tolerance. Overall, a 20% reduction in glucose AUC was seen in HFD-exercise mice compared with HFD-no exercise mice (P < 0.05).

These findings were consistent with fasting plasma insulin levels (Fig. 3B), which were lower in chow-fed than in HFD-mice and were reduced in both groups by voluntary exercise (chow-no exercise: 0.7 ± 0.05 ng/ml; chow-exercise: 0.53 ± 0.02 ng/ml; HFD-no exercise 1.5 ± 0.06 ng/ml; HFD-exercise: 1.0 ± 0.04 ng/ml). Among the four groups, chow-exercise had the lowest insulin levels and HFD-no exercise had the highest insulin levels. In addition, as illustrated in Fig. 3C, ITT showed that among the four groups chow-exercise mice and HFD-exercise mice were significantly more insulin sensitive than their sedentary counterparts (P < 0.05). Furthermore, despite continued consumption of HFD, HFD-exercise mice demonstrated similar insulin sensitivity to chow-exercise mice.

Effects of exercise on two key mediators of energy balance and insulin sensitivity, the adipocyte-derived hormones leptin and adiponectin, were also evaluated. As shown in Fig. 4A, and consistent with their increased adiposity, plasma leptin levels were increased sevenfold in HFD-no exercise mice compared with chow-no exercise animals. Furthermore, exercise attenuated this increase in leptin in HFD mice. Plasma leptin levels were reduced by 58% in HFD-exercise mice compared with HFD-no exercise mice. In addition, plasma leptin levels were also reduced by 33% in chow-exercise mice compared with chow-no exercise mice. In contrast, no significant differences were seen in adiponectin levels among the four groups (Fig. 4B). We also measured circulating levels of another potential mediator of insulin sensitivity, serum RBP4 but saw no significant differences in mean RBP4 levels among the four groups: (chow-no exercise: 20.1 ± 5.6 μg/ml; chow-exercise: 23.4 ± 6.0 μg/ml; HFD-no exercise 21.1 ± 7.2 μg/ml and HFD-exercise: 20.9 ± 7.9 μg/ml).

Effects of voluntary exercise on expression of adipose tissue and liver inflammatory markers.

To begin characterizing the effects of voluntary exercise on HFD-induced inflammation, we examined expression of several key inflammatory markers in adipose tissue and liver by using real-time PCR. Inflammatory markers evaluated included TNF-α, PAI-1, MCP-1, and IKKβ. Consumption of HFD markedly increased expression of these inflammatory markers in adipose tissue. As shown in Fig. 5, A–D, relative to chow-no exercise animals, HFD-no exercise mice showed increased expression of each of these inflammatory markers in perigonadal fat. TNF-α and IKKβ expression increased twofold and 38% respectively, and there was a fourfold increase in expression of both MCP-1 and PAI-1. The increased expression of TNF-α and IKKβ was reversed by voluntary exercise. HFD-exercise mice showed a 40% reduction in TNF-α mRNA levels and a 50% decrease in IKKβ expression compared with HFD-no exercise mice. Similarly, MCP-1 and PAI-1 gene expression were decreased in HFD-exercise mice by 35 and 77%, respectively, compared with HFD-no exercise mice. Furthermore, voluntary exercise reduced expression of these inflammatory markers in chow-exercise compared with chow-no exercise mice. TNF-α and IKKβ mRNAs were reduced by 42 and 49%, respectively, whereas MCP-1 and PAI-1 mRNAs were reduced by 73 and 64%, respectively.

On the other hand, mesenteric fat showed a differential expression of these inflammatory markers compared with perigonadal fat. In mesenteric fat, HFD-no exercise mice showed a sixfold and a 21% increase in PAI-1 and MCP-1 mRNA, respectively, compared with chow-no exercise mice (Fig. 6, A and B). Exercise reduced expression of both markers in HFD mice by 31 and 53%, respectively. Similarly, expression of PAI-1 and MCP-1 were reduced in chow-exercise compared with chow-no exercise mice (40 and 36%, respectively). However, in contrast to the changes seen in perigonadal fat, expression of TNF-α and IKKβ in mesenteric fat was unchanged among the four animal groups (not shown). Further indication of the tissue-specific expression pattern of inflammatory markers in response to the HFD/exercise paradigm was seen in liver, where expression of TNF-α, IKKβ, PAI-1, and MCP-1 was not significantly different among the four groups (Fig. 7, A–D).