Endocannabinoids and skeletal muscle glucose uptake.

Abstract

Obesity is a risk factor for type 2 diabetes mellitus and cardiovascular disease. Obesity, in particular when the fat is predominantly visceral, is associated with insulin resistance and a reduced ability to increase the rate of fat oxidation in response to an increase in dietary fat intake. Skeletal muscle is a primary site for insulin-stimulated glucose uptake. Insulin responsiveness in skeletal muscle is regulated by a number of factors including growth hormone, cortisol, sex steroids, cytokines secreted by inflammatory cells and adipocytes, fatty acids, and fatty acid derivatives such as the endocannabinoids. The most abundant endocannabinoids, anandamide (AEA) and 2- arachidonoylglycerol (2-AG) are synthesised from arachidonic acid. They have autocrine or paracrine mechanisms of action which are rapidly terminated by cellular uptake and subsequent metabolism by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) which degrades AEA and 2-AG, respectively. AEA and 2-AG are ligands for the cannabinoid receptor type 1 (CB₁) and the cannabinoid receptor type 2 (CB₂ ); both are 7 transmembrane domain G-protein coupled receptors. AEA and 2-AG also bind to the transient receptor potential channel-vanilloid sub-family member 1 (TRPV1). TRPV1 is a putative sixtransmembrane domain protein with a pore region between segments five and six and cytoplasmic N and C termini. TRPV1 was initially discovered as a receptor for capsaicin, the main pungent component of hot chilli. Activation of TRPV1 leads to an increase in intracellular calcium either by entry through the plasma membrane or through calcium release of intracellular stores. Endocannabinoids and their receptors form part of an endogenous system that regulates a number of homeostatic functions, including food intake (appetite and motivation to eat via effects in the hypothalamus and nucleus accumbens shell), the regulation of fat mass and intermediary metabolism. An overactivity of the endocannabinoid system in obesity may serve to maintain fat mass and may also underlie some of the associated metabolic consequences. Several studies have shown that inhibition of CB₁ in obese animal models improved the metabolic profile and reversed the deleterious effects of obesity on metabolism. The majority of this data was based on the effects of endocannabinoids on adipose tissue and liver. The studies that form the basis of this thesis examined the effect of endocannabinoids on glucose uptake and metabolism in skeletal muscle. It was initially shown that CB₁ inhibition improves basal glucose uptake in primary cultures obtained from obese, but not lean humans. This is consistent with the notion of an “overactive endocannabinoid system” apparent even in the ex-vivo system of primary culture (Chapter 3). These data could not however all be explained by the presence of a single type of endocannabinoid receptor in skeletal muscle. In a series of studies messenger RNA for CB₁, CB₂, TRPV1 and the enzyme FAAH was shown to be present in human and rat skeletal muscle biopsies, primary cultures of human skeletal muscle and a rat skeletal muscle cell line (L6) (Chapter 4). Subsequent experiments to determine the effect of endocannabinoids on basal and insulin-stimulated glucose uptake and receptors mediating these effects were performed in L6 cells (Chapter 5). Chronic (24 h), but not acute (30 min) exposure to AEA and 2-AG increased insulin-stimulated glucose uptake and the effect of 2-AG was greater than that of AEA. 2-AG was used in subsequent studies. 2-AG-mediated glucose uptake was ameliorated by inhibition of CB₁ (SR141716), CB₂ (SR144528) or TRPV1 (SB366791) with no additional effect when more than one receptor was blocked concurrently. These studies are the first to demonstrate the presence of TRPV1 in skeletal muscle and that it has a role in glucose regulation. To investigate a role for TRPV1 on glucose metabolism in vivo, targeted mutant mice with a deletion of the TRPV1 gene were utilised. The studies described in Chapter 6 measured glucose tolerance in TRPV1[superscript]-/- mice in comparison to wild-type mice in response to a standard or high fat diet (HFD) via intraperitoneal glucose tolerance testing. At baseline the TRPV1[superscript]-/- mice were able to clear a glucose load more efficiently than their wild-type counterparts. After 18 weeks of high fat feeding, body weight of the wild-type mice increased significantly and glucose tolerance was impaired. In contrast, the TRPV1[superscript]-/- mice were resistant to diet induced obesity, but their glucose tolerance was similar to that of the wild-type mice. The reason for the discrepancy between adiposity and glucose tolerance is unknown, however, in vitro studies describing an effect of endocannabinoids to increase insulin-stimulated glucose uptake via TRPV1 suggests a role for this receptor in the regulation of glucose utilisation. The novel observations relating to TRPV1 offer a new perspective on endocannabinoid mediated effects on peripheral metabolism with potential therapeutic implications. Further studies are required to determine the relationship between the effects of endocannabinoids on peripheral metabolism and the emerging role of TRPV1 in diabetes and obesity.