The effect of obesity-induced cytokine elevation on the molecular regulation of protein turnover and carbohydrate metabolism in human skeletal muscle

Obesity in humans has been shown to result in the increased release of small inflammatory-inducing proteins, called cytokines, from the fat cells of the body. We are interested in the effects of these cytokines on the mechanisms that control muscle mass and metabolism in the obese human. Previous research from work in cells and animals has shown the cytokines reduce the synthesis of muscle proteins and simultaneously enhance their rate of breakdown, resulting in a loss of muscle mass. Furthermore, research suggests that the same cytokines may inhibit carbohydrate oxidation, a pivotal step in muscle metabolism. However, despite these potential negative consequences for skeletal muscle function, the effect of low-level and persistent inflammation as seen in obese humans, remains largely unknown. Our animal based work demonstrated, for the first time, that the molecular events that occur in muscle to reduce muscle mass and inhibit carbohydrate oxidation operate, at least following statin-induced myopathy and sepsis, in unison. Moreover, these events appear to be dependent on the inactivation of a single enzyme, called AKT. Interestingly, the molecular events that result in increased activity of AKT also appear inhibited by the cytokines. Given the findings from cell and animal research, we believe that in obese individuals, where levels of cytokines in the blood are elevated, synthesis of muscle proteins and carbohydrate oxidation may be reduced and breakdown of muscle proteins increased, and that these observations are due to cytokine inactivation of the AKT enzyme. In the current application, we therefore wish to measure the rates of synthesis and breakdown of muscle proteins in conjunction with rates of carbohydrate oxidation in obese individuals, and compare them to rates determined in healthy non-obese individuals. We intend to make these measurements by utilising stable isotope techniques and determining O2 uptake and CO2 production by the subject. Furthermore, we intend to obtain tissue samples from the thigh muscles of volunteers, allowing us to examine the molecular signalling events that underpin these processes and thus determine for the first time the effect of obesity associated low-dose chronic inflammation on essential muscle events. Following these initial determinations, we will start the subjects on a 12-week course of either pioglitazone, an insulin-sensitiser often prescribed to type II diabetics, or a placebo. Pioglitazone and similar compounds have been shown to increase the activity of AKT in obese and diabetic patients and normalise the levels of cytokines in the blood of the former. By repeating the initial measurements described above, and by accurately determining the levels of the cytokines, we hope to elucidate their role in the instigation of any molecular or functional effect in the obese individual. Furthermore, we wish to determine if an insulin sensitising agent can reverse purported effects of obesity on molecular events in muscle and moreover, lead to functional improvements in synthesis and breakdown of muscle proteins and muscle carbohydrate metabolism. The proposed experiments will be performed in the Centre for Integrated Systems Biology and Medicine (CISBM; www.nottingham.ac.uk/cisbm) at the University of Nottingham Medical School, where integrated human physiology is a major research focus. This work is essential because it will further our understanding of the health consequences of obesity which is all the more important given the purported obesity epidemic threatening to face the Western World in future years. Furthermore, this work may have implications for other disease states characterised by low-grade chronic inflammation. This work therefore falls within the strategic plan of the Diet and Health theme of the BBSRC Agri-Foods Committee.

Obesity is associated with chronic low-grade inflammation and elevated circulating levels of the cytokines TNFa and IL-6. In vitro and animal based evidence has shown TNFa and IL6 to negatively impact upon protein synthesis, protein degradation and fuel metabolism in skeletal muscle. Interestingly, despite the increasing incidence of obesity and the potential negative connotations on muscle function, the effect of obesity-induced elevations of the cytokines on muscle protein turnover and carbohydrate oxidation in humans is largely unknown. Utilising contemporary isotope and respiratory gas exchange methodologies during insulin clamp conditions in obese and non-obese individuals, we will determine the consequences of obesity on muscle protein turnover and carbohydrate metabolism. The taking of muscle samples will enable us to examine by RT-PCR and Western blot the transcriptional and translational changes to the signalling pathways thought to underpin these processes, namely the AKT/mTOR pathway (protein synthesis), the ubiquitin proteasome system, including the MAFbx and MuRF1 ubiquitin ligases (protein degradation), and PDC activity and PDK expression (carbohydrate metabolism). Furthermore, recent evidence suggests that these three processes may be intrinsically linked via the activity of AKT, whose own activity is known to be negatively impacted by the action of the cytokines. To investigate this further, we will administer to the subjects a course of pioglitazone, a compound known to normalise cytokine levels and increase muscle AKT activity in the obese, and follow up by repeating our initial functional and molecular measurements, thereby allowing us to determine whether any effect of pioglitazone on protein turnover and carbohydrate metabolism occurs in a fashion that is consistent with the proposed central role of AKT in the regulation of these processes, and whether this leads to functional improvements in muscle protein turnover and carbohydrate oxidation.