Application of dynamic metabolomics to examine in vivo skeletal muscle glucose metabolism in the chronically high-fat fed mouse

https://doi.org/10.1016/j.bbrc.2015.04.096Get rights and content

Highlights

  • Dynamic metabolomics was used to investigate muscle glucose metabolism in vivo.

  • Mitochondrial TCA cycle metabolism is altered in muscle of HFD mice.

  • This defect was not pyruvate dehydrogenase mediated, as has been previously thought.

  • Mitochondrial TCA cycle anaplerosis in muscle is virtually absent during the OGTT.

Abstract

Rationale

Defects in muscle glucose metabolism are linked to type 2 diabetes. Mechanistic studies examining these defects rely on the use of high fat-fed rodent models and typically involve the determination of muscle glucose uptake under insulin-stimulated conditions. While insightful, they do not necessarily reflect the physiology of the postprandial state. In addition, most studies do not examine aspects of glucose metabolism beyond the uptake process. Here we present an approach to study rodent muscle glucose and intermediary metabolism under the dynamic and physiologically relevant setting of the oral glucose tolerance test (OGTT).

Methods and results

In vivo muscle glucose and intermediary metabolism was investigated following oral administration of [U-13C] glucose. Quadriceps muscles were collected 15 and 60 min after glucose administration and metabolite flux profiling was determined by measuring 13C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates via gas chromatography–mass spectrometry. While no dietary effects were noted in the glycolytic pathway, muscle from mice fed a high fat diet (HFD) exhibited a reduction in labelling in TCA intermediates. Interestingly, this appeared to be independent of alterations in flux through pyruvate dehydrogenase. In addition, our findings suggest that TCA cycle anaplerosis is negligible in muscle during an OGTT.

Conclusions

Under the dynamic physiologically relevant conditions of the OGTT, skeletal muscle from HFD fed mice exhibits alterations in glucose metabolism at the level of the TCA cycle.

Introduction

Defects in muscle glucose metabolism, particularly insulin resistance, are a characteristic feature of obesity and type 2 diabetes [1]. Our current understanding of the mechanisms underlying muscle insulin resistance has been aided by rodent models, especially the high-fat diet (HFD) fed mouse. Accordingly, techniques to determine insulin sensitivity in humans, such as the ‘gold standard’ euglycemic hyperinsulinemic clamp, have been adapted to allow for detailed assessment of mouse glucose homeostasis [2], [3], [4]. The application of the clamp to rodent studies has yielded significant insight into the regulation of muscle insulin action, however, there are limitations associated with this method, including the fact that the clamp does not replicate the dynamic changes in circulating insulin, glucose and other hormones and metabolites that occur following nutrient ingestion. During a typical mouse clamp, glucose and insulin are infused directly into the systemic circulation via a peripheral vein, not the hepatic portal vein which is their natural route of entry into the body [5]. Moreover, studies largely focus on determining radioactive tracer-derived measures of muscle glucose uptake as the sole readout of glucose metabolism [3], [4], without examining the metabolic fate of glucose once it has entered the myocyte. Studies are therefore required to examine how the presence of insulin resistance impacts on the pathways of glucose metabolism in muscle under conditions that better reflect the physiology of the postprandial state. The oral glucose tolerance test (OGTT) is used in clinical and laboratory investigations to assess whole-body glucose metabolism. The OGTT has also been adapted for rodent studies and is now one of the most widely used physiological tests in mice [2]. However, unlike the clamp, the OGTT not only takes into account insulin sensitivity, but also other factors critical for regulating glucose homeostasis including insulin secretion and glucose effectiveness, and therefore more closely replicates physiological conditions [5]. Given the lack of data on muscle glucose metabolism in mice under these physiologically relevant conditions, the aim of this study was to integrate stable isotope methodology and targeted metabolomics to develop an approach to track the fate of glucose derived carbon through key metabolic pathways in muscle during an OGTT. Furthermore, we aimed to examine whether skeletal muscle glucose metabolism is altered in the obese, glucose intolerant HFD fed mouse.

Section snippets

Animals

All experiments were approved by the Monash University Animal Research Platform Animal Ethics Committee and were in accordance with the National Health and Medical Research Council of Australia Guidelines on Animal Experimentation. Mice were maintained at 22 ± 1 °C on a 12 h light/dark cycle, with free access to food and water. Eight week old male C57BL/6 mice (Monash Animal Research Platform) were maintained on a standard chow control diet (9% energy as fat, Barastoc Rat and Mouse, Ridley

HFD induced obesity, insulin resistance and glucose intolerance

As expected, body mass was elevated in mice fed the HFD (Fig. 1A). Glucose tolerance was assessed following administration of [U-13C] glucose, allowing us to track the movement of glucose derived carbon through pathways of intermediary metabolism in muscle via GC–MS. Blood glucose (Fig. 1B) and plasma insulin (Fig. 1C) were higher in HFD mice under fasting conditions, at 15 and 60 min after glucose administration, confirming the presence glucose intolerance and insulin resistance. Prior to [U-13

Sources of funding

This work was supported by a grant from the Diabetes Australia Research Trust. MJM is supported by a fellowship from the National Health and Medical Research Council (APP1059530).

Conflict of interest

None.

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