O'Keefe, Lannie ORCID: 0000-0002-0264-0007 (2017) Determining the Influence of Endocannabinoids in Skeletal Muscle Adiponectin sensitivity in Diet Induced Obesity and Diabetes. PhD thesis, Victoria University.

Abstract

Obesity kills more than 2.8 million people globally each year regardless of economic status or age. The endocannabinoid system is a widely distributed lipid signalling system that regulates appetite, fatty acid oxidation, glucose metabolism and inflammation. Adiponectin plays a protective role against metabolic disorders. Skeletal muscle plays a leading role in fatty acid oxidation and glucose metabolism. Activation of Cannabinoids in adipose tissue results in a decrease in adiponectin and an increase in inflammation causing ER stress. Blocking cannabinoids causes a decrease in inflammation and increase in adiponectin. This suggests a direct yet to be determined relationship between the Endocannabinoid system and adiponectin resistance, which was the focus of this candidature. Sprague Dawley rats were fed a HFD (22%) for 12 weeks to elicit DIO, then injected daily (IP) with CB1 Antagonist (AM251- 3mg/kg) for 6 wks. Animals were anesthetised, and skeletal muscles (Red /White fibre types) surgically removed. Skeletal muscle was immediately placed in organ bath (37C -95%O25%) with Adiponectin for 30 minutes. Plasma analysis determined that chronic CB1 Antagonism in these rats resulted in a significant reduction in food intake, weight reduction, a reduction in Peri-renal and brown adipose tissue weight, and a reduction in plasma leptin and Glucagon. There was an increase in inflammatory plasma cytokines (Il-1α, IL-2, Il-4, Il-5, Il-17α, Il-18, RANTES, IL12p70). Muscle analysis found that CB1 Antagonism on whole muscle resulted in no changes in mitochondrial markers. Incubation of the soleus muscle with adiponectin showed a significant decrease in AdipoR1 expression. There was a decrease in markers of fatty acid oxidation in white skeletal muscle Sprague Dawley rats were fed HFD (22%) for 12 weeks then injected daily (IP) with either CB2 Agonist (AM1241-3mg/kg or CB2 Antagonist (AM630- 0.3mg/kg) for 6 wks. Animals were anesthetised, and muscles (Red and White fibre types) were surgically removed. Muscle was placed in an organ bath (37C -95%O2-5%) with Adiponectin for 30 minutes. Plasma analysis determined that CB2 modulation resulted in an initial decrease in food intake. CB2 stimulation caused an increase in IL12p70 and a decrease in Leptin in plasma. CB2 Antagonism caused a decrease in plasma Leptin, GLP-1, Ghrelin. Muscle analysis showed that blocking CB2 caused an increase in mitochondrial activity in red fibres via elevated concentrations of citrate synthase. Adiponectin exposure resulted in CB2 agonism causing a down regulation of the mRNA expression of both AMPK and PGC1α in the Extensor Digitorum Longus muscle. Human skeletal muscle (rectus abdominus) were sourced from Obese and Diabetic individuals undergoing routine lap band surgery. Myotubes were treated for a 24-hour period with either CB1 antagonist (AM251), CB2 antagonist (AM630) in isolation or in combination with Adiponectin. Results showed significant increase of AdipoR2 in combination with Adiponectin in Diabetic tissue. Blocking CB2 caused an increase in both AdipoR1 and AdipoR2 expression in diabetic tissue. The results of this thesis are the first to support the hypothesis of synergistic mechanisms at play between Endocannabinoids and Adiponectin in the skeletal muscle of Obese and Diabetic skeletal muscle tissue.

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