Matthew Brady, Ph.D - Research Summary - Kovler Diabetes Center

Matthew Brady, Ph.D.

Research Summary

The overall focus of the lab is metabolic signaling.  We are particularly interested in the insulin-mediated regulation of energy storage in adipocytes and hepatocytes, the interplay of glucose and lipid metabolism, the regulation of hepatic glucose production, and the molecular mechanisms underlying the development of insulin resistance in animal models and humans.  Research is conducted using cell lines, primary murine hepatocytes and adipocytes, and samples collected from human fat biopsies.

Hormonal Regulation of Energy Metabolism
Insulin is the most potent physiological anabolic agent known, promoting the synthesis and storage of carbohydrates and lipids, and inhibiting their degradation and release into the circulation. These effects are achieved in part by the acute regulation of metabolic enzymes through changes in their phosphorylation state. In fat, liver, and muscle, insulin stimulates the dephosphorylation of a number of enzymes involved in glycogen and lipid metabolism via activation of protein phosphatase-1 (PP1). Although PP1 is a cytosolic protein, the phosphatase is compartmentalized throughout the cell by discrete targeting subunits. These proteins confer substrate specificity to PP1 and mediate the specific regulation of intracellular pools of PP1 by a variety of extracellular signals. A main focus of the laboratory is the hormonal regulation of glycogen metabolism. We have identified a novel PP1 regulatory subunit, Protein Targeting to Glycogen (PTG). This molecule binds to PP1 and glycogen, thus targeting the phosphatase to the glycogen particle. Additionally, PTG specifically binds to several PP1 substrates which are key enzymatic regulators of glycogen metabolism. Overexpression of PTG in cultured cells and intact animals induces the intracellular redistribution of PP1 and glycogen metabolizing enzymes, and a marked increase in glycogen stores. Conversely, reduction of PTG levels using RNA interference causes an 85% reduction in cellular glycogen levels.  These results suggest that PTG acts as a molecular scaffold, assembling PP1 with specific substrate proteins, allowing for the efficient hormonal regulation of glycogen metabolism.

Currently, there are three main projects in the lab in this area.  The first involves studying the impact of altering glycogen metabolism in adipocyte energy sensing and function.  A variety of biochemical and metabolic assays are being used to assess the impact of altering glycogen metabolism in a novel transgenic animal model where PTG is expressed in adipose tissue.  Additionally, we are currently generating a PTGfl/fl animal which will be used to tissue specific PTG knockout lines.  These animals will then be fully characterized using in vitro and in vivo assays, including the use of metabolic cages.  A second project addresses the surprising fact that now seven different proteins have been identified that target PP1 to glycogen particles.  The need for multiple proteins that apparently serve the same function and exhibit overlapping tissue distribution remains poorly understood.  We are using an adenoviral system to overexpress wild type, chimeric and shRNA constructs in order to modulate individual protein expression in cultured primary murine hepatocytes in an effort to delineate the roles of two principle PP1 targeting subunits, PTG and GL.  These studies will be extended in the future through adenoviral administration into mice to modulate these proteins in livers in vivo.  In both sets of studies the regulation of glucose production, storage and metabolism and the expression and activity of critical enzymes in these processes are studied under a variety of hormonal and metabolic conditions. A final project is addressing the molecular mechanisms by which environmental endocrine disrupting chemicals (EDCs) exert their effects.  EDCs are synthetic pollutants and contaminants that inappropriately activate or inactivate endocrine systems.  Their effects on the transcriptional regulation of adipocyte differentiation and induction of insulin resistance and disruption of energy metabolism in primary adipocytes and hepatocytes are currently under study.

Effects of Modulating Sleep Patterns on Insulin Sensitivity in Human Adipose Tissue
Adipose tissue plays a central role in the control of systemic energy metabolism.  Fat is by far the largest energy depot in the body, and additionally secretes a growing number of endocrine factors that influence feeding behavior and insulin sensitivity in other tissues.  Dysregulation of adipocyte function during obesity is a key contributor to the development of insulin resistance and subsequent type 2 diabetes.  Over the past 10 years, there have been an increasing number of studies that report a link between decreased sleep duration and/or quality and an increased risk for obesity and diabetes. In collaboration with Drs. Van Cauter, Ehrmann and Tasali in the Department of Medicine at the University of Chicago, we have initiated a series of studies to examine the effects of sleep disruption on insulin sensitivity in primary human adipocytes using cells obtained by subcutaneous needle biopsy.  In all studies described below, fat biopsies are obtained before and after the experimental intervention.  Insulin sensitivity is assessed by phospho-specific immunoblotting and we also have a series of metabolic assays (lipogenesis, lipolysis, glucose uptake) established for use in human adipocytes.  Finally, in more recent experiments with obese subjects, mRNA has been isolated for use in microarray analysis.

In the first series of experiments, lean, healthy, young subjects were subjected to 4 nights of sleep deprivation (4.5 hr/night).  A second group of subjects were subjected to 4 nights of specific disruption of slow-wave sleep, such that while subjects slept for 7-8 hr/night, restorative slow wave sleep was never achieved.  In both instances, there was a marked induction of insulin resistance detected at the cellular level using the primary human adipocytes.  The molecular mechanisms by which these changes occur are being investigated.

In a second study, obese subject with polycystic ovary syndrome (PCOS) will be screened for degree of obstructive sleep apnea (OSA).  For reasons that are poorly understood, the incidence of OSA in women with PCOS is far higher than in age and weight matched controls.  This disturbance in sleep related breathing results in hypoxia and frequent micro-arousals that disrupt sleep quality and duration.  The hypothesis to be tested in these studies is that restoration of sleep quality though 8 week treatment with continuous positive airway pressure (CPAP) will result in an improvement in insulin resistance and global energy metabolism.  Subjects will be fully characterized in the sleep lab and undergo metabolic profiling in the clinic before and after CPAP intervention.  In parallel, fat biopsies will be performed and insulin action in primary adipocytes will be assessed as described above.  Additionally, mRNA is prepared for gene profiling to elucidate the molecular changes occurring in fat tissue upon improvement in sleep in these subjects.