Cellular thermogenic mechanisms : a comparative study of brown adipose tissue and skeletal muscle

Thermogenic mechanisms in homeotherms appear to be largely controlled by norepinephrine and the sympathetic nervous system. The thermogenic mechanism of brown adipose tissue (BAT) has been well characterized by other researchers and recent data from our laboratory have suggested a role for the vasculature in skeletal muscle thermogenesis. The aim of this thesis was to compare the thermogenic mechanisms of BAT and skeletal muscle. The contribution of the vasculature to BAT thermogenesis and the possibility of a mitochondria' uncoupling mechanism in skeletal muscle were investigated. To examine the vascular contribution to BAT thermogenesis a technique for the isolation and constant flow perfusion of the periaortic BAT deposit was developed and characterized. In this preparation, the thermogenic response was found to be entirely 0-adrenoreceptor mediated. Similar increases in oxygen consumption were induced by norepinephrine, isoproterenol and BRL 35135A (a specific 133-agonist). These induced responses were completely inhibited by propranolol, but unaffected by either phentolamine or nitroprusside (a nitrovasodilator). The lack of an a-mediated or nitroprusside-sensitive effect suggested that the vasculature did not contribute significantly either directly (e.g. "hot pipes") or indirectly (through alterations in blood flow) to BAT thermogenesis. Initially, perfused rat hindlimbs were used in our laboratory to address the question of uncoupling in skeletal muscle. When infused the rate of conversion of a redox dye (MTT) was decreased by the addition of norepinephrine in association with a stimulation of oxygen consumption. Similarly, measurement of muscle cell mitochondria' membrane potential, using [311]-TPMT, suggested decreases were occurring in the presence of norepinephrine. Together, these data implied that norepinephrine could be causing uncoupling in skeletal muscle mitochondria. Thus, a search for the mechanism responsible for this thermogenic process was conducted using isolated subsarcolemmal skeletal muscle mitochondria. Potential candidates that led to increased oxygen consumption and decreased MTT conversion by subsarcolenunal skeletal muscle mitochondria were assessed in conjunction with measurements of mitochondrial membrane potential (using Rhodatnine 123). Mitochondria exhibited little respiratory control when succinate was used as the substrate, suggesting that it may be able to act as an uncoupler. With succinate as the substrate, respiration was maximal despite an inhibition of the rate of MTT conversion. The inhibition of MTT conversion was overcome by the transition of the mitochondria from state IV to state III, and this ADP-regulated, succinateinduced uncoupling appears specific for skeletal muscle mitochondria. Although succinate seems a viable candidate for the uncoupling seen in skeletal muscle, it does not decrease the mitochondrial membrane potential and this eliminates it as the uncoupler which is acting in the perfused rat hindlimb. As glycerol release has been noted from the perfused rat hindlimb on addition of norepinephrine, fatty acids and their uncoupling action were investigated in the isolated mitochondria. Fatty acids gave responses with isolated mitochondria similar to that seen on the addition of uncouplers such as FCCP. Respiration was increased, while both membrane potential and MTT conversion were decreased. In conclusion, the development and examination of a novel perfused BAT preparation suggested that the vasculature does not contribute significantly to thermogenesis in a manner similar to that seen in skeletal muscle. In skeletal muscle, however, fatty acids are a candidate for an uncoupler that can act in a manner similar to that seen in BAT. With other researchers having shown that the induced loss of mitochondrial membrane potential can be prevented if ADP and Mg2+ are present, fatty acids may be able to induce uncoupling that is readily reversed with the onset of exercise