Paradoxical coupling of triglyceride synthesis and fatty acid oxidation in skeletal muscle overexpressing DGAT1

Abstract

Objective: Transgenic expression of diacylglycerol acyltransferase-1 (DGAT1) in skeletal muscle leads to protection against fat-induced insulin resistance despite accumulation of intramuscular triglyceride, a phenomenon similar to what is known as the "athlete paradox." The primary objective of this study is to determine how DGAT1 affects muscle fatty acid oxidation in relation to whole-body energy metabolism and insulin sensitivity.

Research design and methods: We first quantified insulin sensitivity and the relative tissue contributions to the improved whole-body insulin sensitivity in muscle creatine kisase (MCK)-DGAT1 transgenic mice by hyperinsulinemic-euglycemic clamps. Metabolic consequences of DGAT1 overexpression in skeletal muscles were determined by quantifying triglyceride synthesis/storage (anabolic) and fatty acid oxidation (catabolic), in conjunction with gene expression levels of representative marker genes in fatty acid metabolism. Whole-body energy metabolism including food consumption, body weights, oxygen consumption, locomotor activity, and respiration exchange ratios were determined at steady states.

Results: MCK-DGAT1 mice were protected against muscle lipoptoxicity, although they remain susceptible to hepatic lipotoxicity. While augmenting triglyceride synthesis, DGAT1 overexpression also led to increased muscle mitochondrial fatty acid oxidation efficiency, as compared with wild-type muscles. On a high-fat diet, MCK-DGAT1 mice displayed higher basal metabolic rates and 5-10% lower body weights compared with wild-type littermates, whereas food consumption was not different.

Conclusions: DGAT1 overexpression in skeletal muscle led to parallel increases in triglyceride synthesis and fatty acid oxidation. Seemingly paradoxical, this phenomenon is characteristic of insulin-sensitive myofibers and suggests that DGAT1 plays an active role in metabolic "remodeling" of skeletal muscle coupled with insulin sensitization.

FIG. 1.

Whole-body, hepatic, and muscle insulin sensitivity assessed by hyperinsulinemic-euglycemic clamp. A: Steady-state whole-body glucose infusion rates in age-, sex-, genetic background–, and breeding environment–matched wild-type mice on standard rat diet (WT-NC), wild-type mice pretreated with ad lib HFD feeding for 8 weeks (WT-HF), and MCK-DGAT1 mice pretreated with ad lib HFD feeding for 8 weeks (DGAT1-HF). B: Steady-state HGO before (basal HGO) and after (clamp HGO) insulin infusion at the rate of 3 mU · kg⁻¹ · min⁻¹ in WT-NC, WT-HF, and DGAT1-HF mice under the same clamp conditions as in A. C: 2-Deoxyglucose uptake in skeletal muscle after bolus injection of the [¹⁴C]-labeled 2-deoxyglucose under the same clamp conditions as in A and B. Data are presented as means ± SE; P and n are as indicated; *P < 0.05. 2-DG, 2-deoxyglucose.

FIG. 2.

Assessment of anabolic and catabolic fatty acid metabolism in skeletal muscle of MCK-DGAT1 and control wild-type mice. Male mice pretreated with 8-week HFD as described in Fig. 1. A: DGAT activity in soleus muscles isolated from the 4-month-old wild-type and MCK-DGAT1 mice (n = 4, each group). B: Triglyceride content in soleus muscle from the wild-type (n = 5) and MCK-DGAT1 (n = 6) mice. C: Mitochondrial copy numbers as determined by PCR quantification of ND1 or ND4 as mitochondrial DNA (using β-actin as reference for nuclear DNA) in anterior tibial and soleus muscles of the wild-type (n = 7) and MCK-DGAT1 (n = 6) mice. D: MAPR (determined in isolated mitochondria) and citrate synthase activity (measured in muscle homogenates) of the anterior tibial and soleus muscles from the wild-type (n = 7) and MCK-DGAT1 (n = 6) mice. E: β-HAD activity (measured in muscle homogenates) and CPT-I activity (measured in isolated mitochondria) of the soleus muscles from the wild-type and MCK-DGAT1 mice (n = 6, each group). F and G: Representative low- and high-power (inserts) views of the electron microscopic fields of the soleus muscle from the wild-type (F) and MCK-DGAT1 (G) mice. Quantification of 6000× EM micrographs using an image analysis system (Imagine-Pro Plus 5.0, Media Cybernetics) showed that wild-type and DGAT1 mice have 149 ± 3.6 and 98 ± 7.7 mitochondria per 10 × 10 micron muscle area, respectively (P < 0.001). The average mitochondrial size is 34,432 ± 4,246 pixels for wild-type and 62,418 ± 938 pixels for DGAT1 mice (P = 0.014). H: Maximal mitochondrial fatty acid oxidation rates measured in isolated mitochondria of the soleus muscles from the wild-type and MCK-DGAT1 mice (n = 6, each group). Values are expressed as means ± SE; NS, no statistical significance (P > 0.05); *P < 0.05, **P < 0.01 (n as indicated). CS, citrate synthase; FA, fatty acid; 28S, 28S rRNA; TG, triglyceride; WT, wild type.

FIG. 3.

Effects of DGAT1 overexpression and exercise on relative levels of gene expression in soleus muscle. A: Gene expression levels were measured by RT-PCR in soleus muscles isolated from wild-type and MCK-DGAT1 mice pretreated with 8-week HFD as described in Fig. 1, using primer sets listed in suppl. Table 1. Gene abbreviations are the same as listed in suppl. Table 1. *P < 0.05 (n = 5–7 in each group). B: Relative gene expression levels of PDK4 in soleus muscles from HFD-pretreated wild-type versus MCK-DGAT1 mice. C: Relative gene expression levels of PDK4 in soleus muscles from sedentary (Sed) versus exercised (Exe) wild-type mice. The swimming exercise regimen is as previously described (3); P values and n are as indicated. D: Western blot analysis of CD36, UCP3, and PDK4 in soleus muscle from the 8-week HFD–pretreated wild-type and MCK-DGAT1 mice. WT, wild type.

FIG. 4.

Physical activity and whole-body oxygen consumption. Two-month-old male wild-type and MCK-DGAT1 mice were treated with 8-week HFD as in Fig. 1 before indirect calorimetry study. Weight-matched mice were used in this study to avoid the need to correct for differences in weight and body composition across the groups (wild-type vs. MCK-DGAT1 mice). A: Body composition by magnetic resonance imaging (MRI). B. locomotor activity. C: Twenty-four hour oxygen consumption (Vo₂). D: RER, the above were measured during the study period while the mice had ad lib access to HFD. In a separate experiment, Vo₂ was measured in the same wild-type and MCK-DGAT1 mice during a 24-h fasting period (E), followed by a 24-h period of ad lib refeeding (F). Food intake during the 24-h period of ad lib feeding and during the 24-h refeeding period were also measured (G). Data are expressed as means ± SE; P values are as indicated or denoted by “NS” for no statistical significance or **P < 0.01. WT, wild type.

FIG. 5.

Food consumption and growth curves. Food intake per day was averaged weekly and plotted over time in separate groups of age-matched male wild-type and MCK-DGAT1 mice either on standard rat diet (A) or on HFD (B) starting at age of 2 months. The growth curve is plotted using weekly measured body weights in wild-type and MCK-DGAT1 mice on HFD for 10 weeks starting at age of 2 months (C). P values are denoted by “NS” for no statistical significance, *P < 0.05, and **P < 0.01 (n as indicated). WT, wild type.