The Molecular and Cellular Basis of Obesity (Obesity and Energy Metabolism) Core has been assisting with the development of several basic research programs.
In one set of studies, the Core as provided further support to the Mariash laboratory in determining the function of the S14 gene in lipid regulation. The core facilities were used to develop a mouse in which the mouse S14 gene is knocked out. The knockout model shows that this gene plays a significant role in lipogenesis. Since the original publication, we have shown that the S14 gene is a member of a larger gene family that compensates for the lack of the S14 gene in the liver but not the mammary gland. We have now shown the S14 gene regulates lipogenesis by a unique pathway involved in the allosteric regulation of fatty acid synthase.
The manuscript describing these aspects of the knockout mouse has been recently published in Endocrinology. We also have a manuscript under review showing that deletion of the S14 gene improves glucose tolerance and protects against the development of obesity. An additional manuscript is under review describing the carbohydrate responsiveness of the S14 related protein in the liver. The mouse model will be extremely helpful in understanding intracellular factors that regulate lipid synthesis and the accumulation of fat.
The Obesity and Energy Metabolism (Basic Mechanisms) Core has helped with the Bernlohr laboratory studies of Fatty Acid Transport Proteins. The Fatty Acid Transport Protein (FATP) has been implicated by a combination of classical and molecular genetic studies to facilitate FA uptake in adipose cells and is hypothesized to be bifunctional, catalyzing both FA flip-flop across planar biological membranes and esterification with CoA. To test this hypothesis, FATP1-his and FATP4-flag were purified and enzymatically characterized as long/very long chain acyl CoA synthetases carrying out ATP-dependent FA esterification with broad specificity for a variety of lipids (C12 to C24). FATP4 is 10 to 40-fold more active than FATP1 and is approximately 3-times more abundant in 3T3-L1 cells than is FATP1. In response to insulin (100 nM) FATP1, but not FATP4, translocates from intracellular sites to the plasma membrane similar to that for GLUT4. 293 cell lines stably expressing FATP1 on the cell surface stimulate FA uptake and storage as triacylglycerol while cell lines expressing FATP4 in intracellular sites do not.
To evaluate the role of FA influx to produce sufficient AMP to regulate the AMP-activated protein kinase, 3T3-L1 adipocytes stabely expressing either shRNA directed to either scrambled or FATP1 sequences were developed. FATP1 knockdown adipocytes exhibit markedly reduced FATP1 (~90% reduction) protein expression and differentiate normally but have diminished FA uptake, acyl CoA synthetase activity and triacylglycerol accumulation. FA influx led to the rapid phosphorylation of AMPK in scrambled adipocytes but markedly reduced phosphorylation in FATP1 knock down cells. Consistent with reduced phospho-AMPK, FATP1 cells exhibited reduced phosphorylation of acetyl CoA carboxylase relative to scrambled adipocytes. These results suggest that in fat cells FATP1 facilitates FA influx and that the product of the acyl CoA synthetase reaction, AMP, regulates AMP-activated protein kinase leading to the phosphorylation and regulation of downstream targets.
Another project in part supported by the Obesity and Energy Metabolism (Basic Mechanisms) Core is the development of FABP null and transgenic animal models which exhibit increased or decreased levels of adiposity, respectively, compared to wild type C57Bl/6J mice. Stable isotope infusions (collaborations between D. Bernlohr, E. Parks and A. Lange) into FABP null mice demonstrated reduced lipolysis from fat cells with no effect on FA influx. Consistent with a role for FABPs in mediating lipolysis, FABPs from fat cells physically associate with the Hormone Sensitive Lipase and mediate the removal of FFA from the surface of the lipid droplet during lipolysis. In contrast, FABP null mice exhibit increased de novo FA biosynthesis. Real time PCR analysis of target genes in adipose tissue indicates that the expression of the adipose triglyceride lipase (ATGL) and HSL are markedly down regulated in the FABP null animals while being increased in the FABP transgenic mice. Concomitant with changes in lipolysis/lipogenesis, the expression of droplet associated proteins perilipin and S3-12 were also altered leading to an overall decrease in lipid flux in the adipose tissue from null mice and increased in the transgenic.
The Obesity and Energy Metabolism (Basic Mechanisms) Core is participating with H. Towle in his studies of excess dietary carbohydrate converion to triglycerides through the process of de novo lipogenesis, which occurs predominantly in the liver in mammals. In part, this process is mediated by the transcriptional induction of key rate-limiting enzymes involved in lipogenesis. The transcriptional induction requires both insulin and glucose signaling pathways. The studies have been exploring the glucose-mediated pathway that is responsible for transcriptional induction. A candidate transcription factor that mediates this action was recently reported to be ChREBP (ChoRE Binding Protein). ChREBP binds to DNA as an obligate heterodimer with the protein Mlx. To evaluate the role of ChREBP and Mlx in glucose regulation, we prepared dominant negative forms of Mlx and introduced these into adenoviral expression constructs. When transduced into primary hepatocytes, dominant negative Mlx was capable of blocking the normal glucose-mediated induction of mRNAs for many lipogenic enzyme genes, including acetyl-CoA carboxylase and fatty acid synthase. No effect on insulin induction was observed. These studies indicate the ChREBP/Mlx is directly involved in glucose regulation and that this pathway works independently from the insulin-mediated induction by SREBP-1c. The Basic Mechanisms core was instrumental in providing instrumentation and support for performing the RT-PCR measurements used in these studies. A portion of these studies was recently published: Ma, L., Tsatsos, N. G. and Towle, H. C., "Direct Role of ChREBP/Mlx in Regulating Hepatic Glucose-responsive Genes," J. Biol. Chem. 280: 12019-12027 (2005).
C. Kotz studies central nervous system gene expression changes before, during and after the development of obesity, and how the gene changes relate to feeding and activity measures. The Obesity and Energy Metabolism (Basic Mechanisms) Core has helped Dr. Kotz incorporate rtPCR and microarray technology into her laboratory procedures. Dr. Kotz uses the Affymetrix gene rat brain gene chip (~33,000 genes) available at academic rates through the University of Minnesota. Dr. Kotz uses real-time rtPCR to test direct hypotheses regarding gene changes associated with physical activity and obesity, and to follow-up on results from the microarray work. Several sets of primers of interest have been successfully tried. Some of the experimental results have recently been published and others are under review.