TODD LEFF - US grants

Apo CIII is one of ten apolipoproteins that form the lipoprotein particles responsible for transporting cholesterol and triglyceride throughout the body. Apolipoproteins function to regulate the synthesis and catabolism of the different classes of lipoproteins, and as such are important determinants of plasma cholesterol and triglyceride levels. Elevated plasma lipids are a significant risk factor for atherosclerosis, the leading cause of heart disease. Apo CIII is associated primarily with triglyceride rich lipoprotein particles, and physiological evidence indicates that it plays an important role in regulating plasma triglyceride levels. Recent studies with transgenic mice that over-express CIII, demonstrate that CIII production rate is an important determinant of plasma triglyceride levels. These results present the possibility that the transcriptional regulation of the CIII gene could play a significant role in modulating plasma triglyceride levels. Several lines of evidence suggest that the CIII gene is regulated in response to physiological signals that are also associated with changes in lipoprotein levels. Preliminary results demonstrate that several intercellular messengers known to have an effect on plasma triglyceride metabolism (glucocorticoids and cytokines that mediate cellular responses to inflammation), after CIII transcriptional activity in cultured hepatocytes. In addition, the CIII promoter contains several transcription regulatory elements that have been implicated in the induction of gene expression by a variety of stimuli. Taken together, these results present the possibility that CIII gene expression is regulated by physiological signals and that this regulation plays a significant role in the modulation of plasma triglyceride levels. The overall goal of the proposed research is to understand the molecular mechanisms that regulate apo CIII gene expression, and to elucidate the role that this regulation plays in modulating lipoprotein metabolism. The specific aims are to: 1) Identify physiological signals (hormones and cytokines) that modulate CIII gene transcription in tissue culture cells, 2) Characterize the transcriptional regulatory elements and protein factors that are responsible for the modulation of CIII gene expression by cytokines and hormones, and 3)Analyze the regulation of CIII gene expression and its relationship to lipoprotein metabolism in normal and transgenic mice. Results generated from these experiments will provide valuable insights into the potentially crucial role of CIII transcriptional regulation in the modulation of plasma triglyceride levels, and will increase our understanding of how physiological signals like hormones and cytokines modulate transcription.

DESCRIPTION (provided by applicant): AMP activated protein kinase (AMPK) is the central component of a signaling system that functions to maintain cellular energy balance in response to stresses that deplete intracellular ATP. It is often referred to as a "metabolic master switch" because of the central role that it plays in the maintenance of metabolic homeostasis. AMPK activity has a strong influence on the regulation of whole body glucose metabolism and it may also play a role in the development of type-2 diabetes. It has recently been reported that some of the antidiabetic activities of the drug metformin and the hormone adiponectin may be mediated by activation of AMPK in the liver, suggesting a potential link between AMPK and hepatic glucose metabolism. We have recently demonstrated that the transcription factor HNF4alpha, a well-characterized regulator of metabolic gene expression in the liver, is inhibited by AMPK mediated phosphorylation on serine-304. HNF4alpha is known to play an important role in the expression of liver genes involved in regulating hepatic glucose production, a key contributor to normal and diabetic whole body glucose homeostasis. The central hypothesis of the work proposed here is that an AMPK-HNF4alpha signaling pathway in the liver mediates important metabolic effects of AMPK. The overall goals of this proposal are to characterize the molecular details of this proposed AMPK-HNF4alpha signaling pathway and to determine the extent to which it influences gene expression and metabolism in normal and diabetic liver. In addition, the possibility that AMPK mediated phosphorylation of HNF4alpha contributes to the hepatic effects of metformin and adiponectin will be explored. Results from these studies will provide a clearer understanding of the functional relationship between HNF4alpha and AMPK and a more complete picture of the physiological role that these two important proteins play in the liver.

Project Summary/Abstract Age-related disorders including diabetes have replaced infectious diseases as the leading cause of death in developed countries. There is an urgent need to identify new molecular targets that could lead to better therapeutic and diagnostic strategies for treatment of these disorders. Numerous studies have demonstrated that a highly predictive metabolic characteristic of insulin resistant and diabetic states is a reproducible and significant reduction in the level of glycine in circulation. These observations raise the intriguing possibility that there may be a causal relationship between aberrant glycine metabolism and the development of diabetes, and that the molecular pathways underling glycine homeostasis represent novel molecular targets for diabetes intervention. Glycine plays a key role in multiple cellular processes that, if dysregulated, could influence metabolic health and diabetes susceptibility. In particular, glycine is a required substrate for the biosynthesis of the cellular antioxidant glutathione. Compromised protection against oxidative stress due to a decline in the level of glutathione occurs during aging and is thought to play a key role in the development of many age-related disorders including type 2 diabetes. Glycine levels are controlled primarily by the hepatic glycine cleavage system (GCS), which degrades glycine. The overall activity of the GCS is elevated in diabetes and our preliminary observations indicate that the gene for the rate-limiting enzyme of the GCS, glycine decarboxylase (GLDC), is over-expressed in diabetic animals. Additional preliminary data demonstrate that GLDC gene transcription is regulated by the transcription factor SREBP1c, which is known to be activated by insulin. Based on these observations, we propose that hyperinsulinemia, as seen in insulin resistant states such as obesity, induces an SREBP-mediated increase in hepatic GLDC gene expression, increased glycine degradation, and ultimately reduced levels of glycine in circulation. We further propose that the increase in hepatic glycine degradation induces a feed-forward cascade of events, including increased oxidative stress, that exacerbates the development of type 2 diabetes and other age-related diseases. The overarching goal of this project is to test this model and determine if there is a causal link between glycine metabolism and diabetes susceptibility. This will be accomplished by characterizing the metabolic effects of experimentally altered hepatic GLDC gene expression in lean and obese mice. These studies represent an initial effort to explore the novel and potentially important possibility that hepatic glycine degradation affects disease susceptibility, which if true, would identify GLDC as a potential target for therapeutic intervention for type 2 diabetes and other age- related diseases.