Jeffrey D. Covington

Effect of 8 Weeks of Overfeeding on Ectopic Fat Deposition and Insulin Sensitivity: Testing the “Adipose Tissue Expandability” Hypothesis

OBJECTIVE The presence of large subcutaneous adipocytes in obesity has been proposed to be linked with insulin resistance and type 2 diabetes through the “adipose tissue expandability” hypothesis, which holds that large adipocytes have a limited capacity for expansion, forcing lipids to be stored in nonadipose ectopic depots (skeletal muscle, liver), where they interfere with insulin signaling. This hypothesis has, however, been largely formulated by cross-sectional findings and to date has not been prospectively demonstrated in the development of insulin resistance in humans. RESEARCH DESIGN AND METHODS Twenty-nine men (26.8 ± 5.4 years old; BMI 25.5 ± 2.3 kg/m2) were fed 40% more than their baseline requirement for 8 weeks. Before and after overfeeding, insulin sensitivity was determined using a two-step hyperinsulinemic-euglycemic clamp. Intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) were measured by 1H-MRS and abdominal fat by MRI. Subcutaneous abdominal adipose and skeletal muscle tissues were collected to measure adipocyte size and markers of tissue inflammation. RESULTS Subjects gained 7.6 ± 2.1 kg (55% fat) and insulin sensitivity decreased 18% (P < 0.001) after overfeeding. IHL increased 46% from 1.5% to 2.2% (P = 0.002); however, IMCL did not change. There was no association between adipocyte size and ectopic lipid accumulation. Despite similar weight gain, subjects with smaller fat cells at baseline had a greater decrease in insulin sensitivity, which was linked with upregulated skeletal muscle tissue inflammation. CONCLUSIONS In experimental substantial weight gain, the presence of larger adipocytes did not promote ectopic lipid accumulation. In contrast, smaller fat cells were associated with a worsened metabolic response to overfeeding.

Ectopic lipid accumulation and reduced glucose tolerance in elderly adults are accompanied by altered skeletal muscle mitochondrial activity.

CONTEXT Aging is associated with insulin resistance and unfavorable changes in body composition including increased fat accumulation, particularly in visceral and ectopic depots. Recent studies suggest that skeletal muscle mitochondrial activity may underlie some age-associated metabolic abnormalities. OBJECTIVE Our objective was to measure mitochondrial capacity and coupling of the vastus lateralis muscle in elderly and young adults using novel in vivo approaches and relate mitochondrial activity to metabolic characteristics. DESIGN This was a cross-sectional study. PARTICIPANTS AND INTERVENTION Fourteen sedentary young (seven males and seven females, 20-34 yr of age) and 15 sedentary elderly (seven males and eight females, 70-84 yr of age) nonobese subjects selected for similar body weight underwent measures of body composition by magnetic resonance imaging and dual-energy x-ray absorptiometry, oral glucose tolerance, and in vivo mitochondrial activity by (31)P magnetic resonance and optical spectroscopy. Muscle biopsy was carried out in the same muscle to measure mitochondrial content, antioxidant activity, fiber type, and markers of mitochondrial biogenesis. RESULTS Elderly volunteers had reduced mitochondrial capacity (P = 0.05) and a trend for decreased coupling efficiency (P = 0.08) despite similar mitochondrial content and fiber type distribution. This was accompanied by greater whole-body oxidative stress (P = 0.007), less skeletal muscle mass (P < 0.001), more adipose tissue in all depots (P ≤ 0.002) except intramyocellular (P = 0.72), and lower glucose tolerance (P = 0.07). CONCLUSIONS Elderly adults show evidence of altered mitochondrial activity along with increased adiposity, oxidative stress, and reduced glucose tolerance, independent of obesity. We propose that mild uncoupling may be induced secondary to age-associated oxidative stress as a mechanism to dissipate the proton-motive force and protect against further reactive oxygen species production and damage.

Weight Gain Reveals Dramatic Increases in Skeletal Muscle Extracellular Matrix Remodeling

CONTEXT In animal models of obesity, chronic inflammation and dysregulated extracellular matrix remodeling in adipose tissue leads to insulin resistance. Whether similar pathophysiology occurs in humans is not clear. OBJECTIVE The aim of this study was to test whether 10% weight gain induced by overfeeding triggers inflammation and extracellular matrix remodeling (gene expression, protein, histology) in skeletal muscle and sc adipose tissue in humans. We also investigated whether such remodeling was associated with an impaired metabolic response (hyperinsulinemic-euglycemic clamp). DESIGN, SETTING, PARTICIPANTS, AND INTERVENTION Twenty-nine free-living males were fed 40% over their baseline energy requirements for 8 weeks. RESULTS Ten percent body weight gain prompted dramatic up-regulation of a repertoire of extracellular matrix remodeling genes in muscle and to a lesser degree in adipose tissue. The amount of extracellular matrix genes in the muscle were directly associated with the amount of lean tissue deposited during overfeeding. Despite weight gain and impaired insulin sensitivity, there was no change in local adipose tissue or systemic inflammation, but there was a slight increase in skeletal muscle inflammation. CONCLUSION We propose that skeletal muscle extracellular matrix remodeling is another feature of the pathogenic milieu associated with energy excess and obesity, which, if disrupted, may contribute to the development of metabolic dysfunction.

Caveolin-1 Expression and Cavin Stability Regulate Caveolae Dynamics in Adipocyte Lipid Store Fluctuation

Adipocytes specialized in the storage of energy as fat are among the most caveolae-enriched cell types. Loss of caveolae produces lipodystrophic diabetes in humans, which cannot be reversed by endothelial rescue of caveolin expression in mice, indicating major importance of adipocyte caveolae. However, how caveolae participate in fat cell functions is poorly understood. We investigated dynamic conditions of lipid store fluctuations and demonstrate reciprocal regulation of caveolae density and fat cell lipid droplet storage. We identified caveolin-1 expression as a crucial step in adipose cell lines and in mice to raise the density of caveolae, to increase adipocyte ability to accommodate larger lipid droplets, and to promote cell expansion by increased glucose utilization. In human subjects enrolled in a trial of 8 weeks of overfeeding to promote fattening, adipocyte expansion response correlated with initial caveolin-1 expression. Conversely, lipid mobilization in cultured adipocytes to induce lipid droplet shrinkage led to biphasic response of cavin-1 with ultimate loss of expression of cavin-1 and -3 and EHD2 by protein degradation, coincident with caveolae disassembly. We have identified the key steps in cavin/caveolin interplay regulating adipocyte caveolae dynamics. Our data establish that caveolae participate in a unique cell response connected to lipid store fluctuation, suggesting lipid-induced mechanotension in adipocytes.

Skeletal muscle perilipin 3 and coatomer proteins are increased following exercise and are associated with fat oxidation

Lipid droplet-associated proteins such as perilipin 3 (PLIN3) and coatomer GTPase proteins (GBF1, ARF1, Sec23a, and ARFRP1) are expressed in skeletal muscle but little is known so far as to their regulation of lipolysis. We aimed here to explore the effects of lipolytic stimulation in vitro in primary human myotubes as well as in vivo following an acute exercise bout. In vitro lipolytic stimulation by epinephrine (100 μM) or by a lipolytic cocktail (30 μM palmitate, 4 μM forskolin, and 0.5 μM ionomycin, PFI) resulted in increases in PLIN3 protein content. Coatomer GTPases such as GBF1, ARF1, Sec23a, and ARFRP1 also increased in response to lipolytic stimuli. Furthermore, a long duration endurance exercise bout (20 males; age 24.0±4.5 y; BMI 23.6±1.8 kg/m2) increased PLIN3 protein in human skeletal muscle (p = 0.03) in proportion to ex vivo palmitate oxidation (r = 0.45, p = 0.04) and whole body in vivo fat oxidation (r = 0.52, p = 0.03). Protein content of ARF1 was increased (p = 0.04) while mRNA expression was increased for several other coatomers (GBF1, ARF1, and Sec23a, all p<0.05). These data provide novel observational insight into the possible relationships between lipolysis and PLIN3 along with these coatomoer GTPase proteins in human skeletal muscle.

Low Macrophage Accumulation in Skeletal Muscle of Obese Type 2 Diabetics and Elderly Subjects

In addition to adipose tissue, recent studies suggest that skeletal muscle may also be a source of low‐grade inflammation, particularly in inactive and/or overweight individuals. The aim of this study was to examine the presence of macrophages in skeletal muscle from obese subjects with type 2 diabetes (T2D) before and after a 9‐month exercise program (vs. a non‐exercising control group) (Study 1) and in young vs. elderly subjects (Study 2). In both studies, CD68+ macrophages in vastus lateralis biopsies were determined by immunohistochemistry and inflammation gene expression measured. Macrophage content (%) was calculated by the number of macrophages per 100 muscle fibers. In Study 1, we found relatively low numbers (2–3%) of CD68+ macrophages in skeletal muscle in obese T2D subjects (BMI = 37.3 ± 5.2 kg/m2), which were unchanged after a 9‐month exercise program (P = 0.42). Similarly, in Study 2 (BMI = 27.1 ± 2.5 kg/m2), CD68+ macrophages were relatively low in muscle (4–5%) and were not different between young and elderly individuals (P = 0.42). However, elderly subjects had twofold higher CD68 and CD206 gene expression (both P < 0.002) than young participants. In both studies, CD68+ muscle macrophages were not associated with BMI. In conclusion, we found little evidence of macrophage accumulation in skeletal muscle in obese T2D subjects or in elderly individuals. A 9‐month exercise program was not associated with a decrease in macrophage content.

Ten nights of moderate hypoxia improves insulin sensitivity in obese humans.

Hypoxia in obese adipose tissue (AT) plays an important role in the development of whole-body insulin resistance by inducing local inflammation and the release of proinflammatory cytokines (1). Yet, living at high altitude is associated with a lower prevalence of impaired fasting glucose and type 2 diabetes compared with living at low altitude (2). Furthermore, exposure to hypoxic environments increases whole-body glucose fluxes in healthy males and glucose uptake in human and murine skeletal muscle (3). In addition, exercising under hypoxic conditions improves glucose tolerance more than exercising under normoxia (4), strongly suggesting an insulin-sensitizing effect of hypoxia. Therefore, we hypothesized that exposing obese men to 10 consecutive nights of moderate hypoxia (15 ± 0.5% O2, ∼2,400 m elevation) would improve insulin sensitivity. Eight healthy obese men (4 Caucasians, 3 African Americans, and 1 Hispanic of mean ± SEM age 28 ± 1 years, weight 96.5 ± 5.3 kg, and BMI 32.7 ± 1.3 kg/m2) without evidence of chronic disease or sleep apnea and taking no medication participated in this study. The protocol was approved by the institutional review board at Pennington Biomedical Research Center (Baton Rouge, LA). Subjects slept for 10 consecutive nights (∼10 h/night, ≥100 h in total) in a hypoxic tent (Hypoxico Inc., New York, NY) maintained at ∼15% O2 (range 14.5–15.5% O2, ∼2,400 m above sea level) using nitrogen dilution. Biopsies of abdominal subcutaneous AT and skeletal muscle were obtained at baseline and on day 11 under …

Effect of protein overfeeding on energy expenditure measured in a metabolic chamber

BACKGROUND Energy expenditure (EE) increases with overfeeding, but it is unclear how rapidly this is related to changes in body composition, increased body weight, or diet. OBJECTIVE The objective was to quantify the effects of excess energy from fat or protein on energy expenditure of men and women living in a metabolic chamber. DESIGN We conducted a randomized controlled trial in 25 participants who ate ∼40% excess energy for 56 d from 5%, 15%, or 25% protein diets. Twenty-four-hour EE (24EE) and sleeping EE (SleepEE) were measured on days 1, 14, and 56 of overfeeding and on day 57 while consuming the baseline diet (usually day 57). Metabolic and molecular markers of muscle metabolism were measured in skeletal muscle biopsy specimens. RESULTS In the low-protein diet group whose excess energy was fat, the 24EE and SleepEE did not increase during the first day of overfeeding. When extra energy contained protein, both 24EE and SleepEE increased in relation to protein intake (r = 0.50, P = 0.02). The 24EE over 8 wk in all 3 groups was correlated with protein intake (r = 0.60, P = 0.004) but not energy intake (r = 0.16; P = 0.70). SleepEE was unchanged by overfeeding in the low-protein diet group, and baseline surface area predicted increased 24EE in this group. Protein and fat oxidation were reciprocally related during overfeeding. Observed 24EE was higher than predicted on days 1 (P ≤ 0.05), 14 (P = 0.0001), and 56 (P = 0.0007). There was no relation between change in fat mass and change in EE. CONCLUSIONS Excess energy, as fat, does not acutely increase 24EE, which rises slowly as body weight increases. Excess energy as protein acutely stimulates 24EE and SleepEE. The strongest relation with change in 24EE was the change in energy expenditure in tissue other than muscle or fat-free mass.

Little evidence of systemic and adipose tissue inflammation in overweight individuals

Context: The effect of weight loss by diet alone or diet in conjunction with exercise on low-grade inflammation in non-obese (overweight) individuals is not known. Objective: Test the hypothesis that 24 weeks of moderate calorie restriction (CR; 25%) by diet only or with aerobic exercise would reduce markers of systemic inflammation and attenuate inflammation gene expression in subcutaneous adipose tissue. Design: Randomized controlled trial. Setting: Institutional Research Center. Participants: Thirty-five overweight (body mass index: 27.8 ± 0.7 kg/m2) but otherwise healthy participants (16M/19F) completed the study. Intervention: Participants were randomized to either CR (25% reduction in energy intake, n = 12), caloric restriction + exercise (CR + EX: 12.5% reduction in energy intake + 12.5% increase in exercise energy expenditure, n = 12), or control (healthy weight-maintenance diet, n = 11) for 6 months. Main outcome measures: Fasting serum markers of inflammation [leptin, highly sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), adiponectin] and inflammation-related genes [CD68, IL-6, TNF-α, macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein-1 (MCP-1), adiponectin, plasminogen activator inhibitor-1 (PAI-1)] in subcutaneous adipose tissue. Results: CR and CR + EX lost similar amounts of body weight (–10 ± 1%), fat mass (–24 ± 3%), visceral fat (–27 ± 3%), and had increased insulin sensitivity (CR: 40 ± 20%, CR + EX: 66 ± 22%). Leptin was significantly decreased from baseline (p < 0.001) in both groups however TNF-α and IL-6 were not changed. hsCRP was decreased in CR + EX. There was no change in the expression of genes involved in macrophage infiltration (CD68, MIF MCP-1, PAI-1) or inflammation (IL-6, TNF-α, adiponectin) in either CR or CR + EX. Conclusion: A 10% weight loss with a 25% CR diet alone or with exercise did not impact markers of systemic inflammation or the expression of inflammation-related adipose genes in overweight individuals.

Effect of Short-Term Thyroxine Administration on Energy Metabolism and Mitochondrial Efficiency in Humans

The physiologic effects of triiodothyronine (T3) on metabolic rate are well-documented; however, the effects of thyroxine (T4) are less clear despite its wide-spread use to treat thyroid-related disorders and other non-thyroidal conditions. Here, we investigated the effects of acute (3-day) T4 supplementation on energy expenditure at rest and during incremental exercise. Furthermore, we used a combination of in situ and in vitro approaches to measure skeletal muscle metabolism before and after T4 treatment. Ten healthy, euthyroid males were given 200 µg T4 (levothyroxine) per day for 3 days. Energy expenditure was measured at rest and during exercise by indirect calorimetry, and skeletal muscle mitochondrial function was assessed by in situ ATP flux (31P MRS) and in vitro respiratory control ratio (RCR, state 3/state 4 rate of oxygen uptake using a Clark-type electrode) before and after acute T4 treatment. Thyroxine had a subtle effect on resting metabolic rate, increasing it by 4% (p = 0.059) without a change in resting ATP demand (i.e., ATP flux) of the vastus lateralis. Exercise efficiency did not change with T4 treatment. The maximal capacity to produce ATP (state 3 respiration) and the coupled state of the mitochondria (RCR) were reduced by approximately 30% with T4 (p = 0.057 and p = 0.04, respectively). Together, the results suggest that T4, although less metabolically active than T3, reduces skeletal muscle efficiency and modestly increases resting metabolism even after short-term supplementation. Our findings may be clinically relevant given the expanding application of T4 to treat non-thyroidal conditions such as obesity and weight loss.