Diabetes Genome Anatomy Project |
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Projects > Project 6
Project 6:
Transcriptional Profiling of PGC-1 Family in Liver
Primary Investigator: Bruce Spiegelman, Ph.D. (Dana-Farber Cancer Institute)
Specific Aims
- Characterization of transcriptional profile induced by PGC-1α or PGC-1β in cultured hepatocytes.
- Characterization of PGC-1 induced gene profile from livers of adenoviral-infected livers of mice.
- Comparisons of PGC-1-induced patterns of gene expression with those of diabetic and fasted livers.
- Fuctional analysis of novel PGC-1 target genes.
PGC-1α is a transcriptional coactivator of nuclear receptors and other transcription factors. It is a dominant regulator of mitochondrial biogenesis in many cell types and regulates a program of thermogenesis in brown fat, where it is inducible in the cold. PGC-1α is also involved in several other important tissue-specific metabolic programs. This coactivator is preferentially expressed in type 1 muscle fibers; when expressed transgenically in muscle beds that contain predominantly type 2 fibers, it induces a program of fiber-type switching including expression of type 1 myofibrillar proteins.
PGC-1α is expressed in liver, where it plays a crucial role in hepatic gluconeogenesis. It is induced in the liver upon fasting in mice and it is induced in multiple models of type 1 and type 2 diabetes. Importantly, PGC-1α can induce an entire program of hepatic gluconeogenesis when expressed in primary hepatocytes or hepatoma cells, including expression of PEPCK and glucose-6-phosphatase. When expressed in live rats via adenoviral infusion, PGC-1α induces the genes of hepatic gluconeogenesis and causes a rise in circulating glucose and insulin levels. Recently, PGC-1α has been shown to induce other aspects of the fasted liver response, including certain genes of the β-oxidation of fatty acids including MCAD and CPT-1. The full range of genes activated by PGC-1α in the liver has not been explored.
PGC-1α has two homologs: PGC-1β, which is more closely related, and PRC. PGC-1β is mildly induced in the liver in fasting but its biological functions have not been explored. We propose here to examine he genetic program induced by PGC-1α and β in the rodent liver. In particular, we will compare the program induced by PGC-1α,β to the program induced in the liver in fasting, and in diabetes. In addition, we will explore the ability of PGC-1β to induce a genetic program in liver cells and liver in vivo, and compare this to the program induced by PGC-1α.
1. Transcriptional Program Induced by PGC-1α and β in Heptaocytes
Primary hepatocytes will be infected with adenoviral vectors expressing PGC-1α, β or control GFP protein as described previously. Titers will be used that yield levels of protein approximately equal to those observed in fasting liver. RNA from these cells will be used for transcriptional profiling using the Affymetrix MG-U74Av2 chip. This oligonucleotide array profiles the expression of over 12,000 mouse Unigene clusters and is a platform on which other large-scale biology experiments have been previously performed. Using the GFP as a control, we propose to identify genes that are significantly up-regulated or down-regulated in response to PGC-1α, β infection.
This program of gene expression will be compared to primary hepatocytes tested with hormones that mimic the fasted or diabetic state: forskolin (an inducer of cyclic AMP, and a mimic of glucagen) and the glucocorticoid dexamethasone. The extent to which PGC-1α or PGC-1β induces a similar program will be rigorously examined by using a recently developed technique called gene set enrichment analysis (GSEA). GSEA detects changes in groups of a priori defined genes and provides a rigorous statistical framework for microarray analysis. We will order the genes in the hormonally modulated cells on the basis of expression change, and ask if the ordering is enriched by targets of PGC-1α, β. In essence, we will attempt to intersect the two expression datasets to parse the hormone targets that are indeed regulated by PGC-1α, β.
2. PGC-1α and PGC-1β in Liver
Live mice will be infused with adenoviruses expressing PGC-1α, PGC-1β or GFP. mRNA extracted from livers from these mice will be compared with another set of mice that have been starved for 24 hours. We will also render a set of control (GFP) mice type 1 diabetic through the use of streptozotocin injections. By performing pairwise comparisons, we will identify genes induced or suppressed in diabetes or in fasting, and again, these lists can be compared systematically to the targets of PGC-1α, β using GSEA, with the goal of detecting genes in fasting and diabetes regulated by the PGC-1 family members.
3. Integration of in vitro and in vivo Microarray Datasets
The hepatocyte and in vivo liver microarray datasets will provide valuable information about the transcriptional targets of PGC-1α, β. However, a frequently faced problem in microarray analysis is limited sensitivity and imperfect specificity. By integrating the in vitro and in vivo expression sets, we aim to refine the results of the above experiments. The extent to which forskolin or dexamethasone mimic the in vivo fed or fasted state, for example, can be assessed by comparing the microarray results.