In vivo cardiac glucose metabolism in the high-fat fed mouse: Comparison of euglycemic–hyperinsulinemic clamp derived measures of glucose uptake with a dynamic metabolomic flux profiling approach

Highlights

• Insulin clamp was used to determine the evolution of cardiac insulin resistance.

• Clamp measures were compared to a dynamic metabolomics approach.

• The clamp revealed the presence of cardiac insulin resistance after 3 weeks of HFD.

• Cardiac glucose metabolism was not affected by HFD during an oral glucose challenge.

Abstract

Rationale

Cardiac metabolism is thought to be altered in insulin resistance and type 2 diabetes (T2D). Our understanding of the regulation of cardiac substrate metabolism and insulin sensitivity has largely been derived from ex vivo preparations which are not subject to the same metabolic regulation as in the intact heart in vivo. Studies are therefore required to examine in vivo cardiac glucose metabolism under physiologically relevant conditions.

Objective

To determine the temporal pattern of the development of cardiac insulin resistance and to compare with dynamic approaches to interrogate cardiac glucose and intermediary metabolism in vivo.

Methods and results

Studies were conducted to determine the evolution of cardiac insulin resistance in C57Bl/6 mice fed a high-fat diet (HFD) for between 1 and 16 weeks. Dynamic in vivo cardiac glucose metabolism was determined following oral administration of [U-¹³C] glucose. Hearts were collected after 15 and 60 min and flux profiling was determined by measuring ¹³C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Cardiac insulin resistance, determined by euglycemic–hyperinsulinemic clamp, was evident after 3 weeks of HFD. Despite the presence of insulin resistance, in vivo cardiac glucose metabolism following oral glucose administration was not compromised in HFD mice. This contrasts our recent findings in skeletal muscle, where TCA cycle activity was reduced in mice fed a HFD. Similar to our report in muscle, glucose derived pyruvate entry into the TCA cycle in the heart was almost exclusively via pyruvate dehydrogenase, with pyruvate carboxylase mediated anaplerosis being negligible after oral glucose administration.

Conclusions

Under experimental conditions which closely mimic the postprandial state, the insulin resistant mouse heart retains the ability to stimulate glucose metabolism.

Introduction

Cardiac insulin resistance, as demonstrated by a reduction in insulin stimulated glucose uptake into the myocardium, is often present in patients with whole-body insulin resistance and type 2 diabetes (T2D) [1], [2], [3], [4], [5]. However, developing an understanding of the mechanisms responsible for cardiac insulin resistance has been challenging due to the obvious limitations in obtaining fresh heart tissue from humans. Thus, is not surprising that together with the emergence of genetic engineering techniques to manipulate the mouse genome, the field of cardio-metabolic research has become increasingly reliant on mouse models. One of the most commonly used models in this field is the chronically high-fat diet (HFD) fed C57Bl/6 mouse, which shows important metabolic changes that resemble the human ‘pre-diabetic’ condition, including obesity, hyperinsulinemia, insulin resistance and glucose intolerance [6]. We have previously performed detailed time course studies in C57Bl/6 mice investigating the evolution of HFD-induced whole-body and tissue specific (liver, skeletal muscle and adipose) insulin resistance via the use of the ‘gold standard’ euglycemic–hyperinsulinemic clamp [6]. We found that HFD-induced whole body insulin resistance developed rapidly, within 1 week of HFD feeding, with this being entirely mediated by impaired hepatic insulin action [6]. Extending the HFD to 3 weeks exacerbated the insulin resistance due to the induction of skeletal muscle insulin resistance, however beyond this point, even after 16 weeks of HFD, did not further deteriorate whole-body or organ specific insulin resistance [6]. However, our previous report did not document the temporal development of cardiac insulin resistance [6]. While the clamp remains the ‘gold-standard’ approach to assess insulin sensitivity, it fails to adequately simulate the dynamic changes in hormones and substrates which occur in the postprandial state. We have recently developed an innovative method to asses intracellular glucose metabolism under the physiologically and clinically relevant condition of the oral glucose tolerance test (OGTT) using dynamic metabolomics [7]. Therefore we aimed to determine the evolution of HFD induced cardiac insulin resistance in the C57Bl/6 mouse using the euglycemic–hyperinsulinemic clamp and to examine how the presence of cardiac insulin resistance impacts on intermediary metabolism under the dynamic physiological conditions of an oral glucose challenge.