Investigations of Altered Proteome Dynamics in Calorie Restriction, Insulin Resistance, and Type 2 Diabetes

Proteins are the ultimate laborers in biological systems. The behavior of a cell is governed by the dynamic interactions between thousands of proteins; a tissue is governed by the interactions of thousands of cells; and an organism is governed by the interaction of multiple tissues. The maintenance of protein homeostasis (proteostasis) is therefore a critical component of proper cellular function and the response to constantly changing environmental signals./nThe dynamic proteomic approach used in these rodent studies involves the administration of 2H2O in the drinking water to achieve a target body water enrichment of 5%. During the labeling period, in vivo protein synthesis occurs, resulting in the creation of "heavy" proteins identical to their "light" counterparts in function, differing only in the incorporation of deuterium atoms at specific positions in the constituent amino acids from which they are comprised. These peptides are then resolved by high-affinity liquid chromatography mass spectrometry (LC-MS), resulting in the identification of hundreds to thousands of proteins from a single tissue sample. Using this experimental approach, we investigated the effect of calorie restriction (CR), insulin resistance, and diabetes on dynamic protein synthesis./nTo date, CR has taken center stage as the most effective intervention in lifespan extension. Despite this, the biological mechanisms underlying increased health and longevity have yet to be fully described. While a growing body of evidence suggests that CR promotes significant gain-of-function attributes in mitochondria, a verdict on whether CR promotes mitochondrial biogenesis has not been established. In order to elucidate the controversy in the field regarding the response of mitochondria to reduced energy intake, we designed an experiment to study the effect of long-term CR on hepatic protein turnover. Our data provide conclusive evidence that mitochondrial protein turnover, concentration, and overall flux are reduced in response to CR, and may play central a role in mediating the health and longevity benefits of reduced energy intake./nInsulin resistance and islet cell failure are the two fundamental processes underlying type 2 diabetes. Alterations in mitochondrial protein turnover have been implicated in the pathology of type 2 diabetes, however the specific effect of insulin resistance and diabetes on the intracellular dynamic islet proteome have yet to be described. We investigated the effects of insulin resistance and diabetes on the synthesis of proteins from isolated rat islets for the first time, using both 2H2O (heavy water) labeling and SILAM quantitative proteomics. Using this approach, we measured fractional and absolute synthesis rates of cytoskeletal, glycolytic, mitochondrial, ER, and ribosomal proteins, the principal pathways responsible for glucose stimulated insulin secretion (GSIS). We found that insulin resistance increased the fractional synthesis rates (FSR) of 97% of all measured islet proteins, and the subsequent transition to diabetes resulted in the selective impairment of ribosomal protein synthesis. Our findings suggest that the rapid rate of islet cell proliferation due to insulin resistance is accompanied by increased fractional and absolute synthesis of critical GSIS proteins, and that the failure of islets results mainly in impaired ribosomal pathway flux, independent of alterations in mitochondrial metabolism. Our data suggest that the rapid rate of islet cell proliferation due to insulin resistance is accompanied by increased fractional and absolute synthesis of critical GSIS components, and that the failure of islet cells in diabetes results mainly in impaired ribosomal pathway flux, independent of alterations in mitochondrial protein metabolism.

Advisor: Hellerstein, Marc K