Role of SREBPs in Muscle Metabolism

This goal of this work is to understand the regulation of skeletal muscle metabolism. The current focus is on the regulation and function of sterol response element binding proteins (SREBPs) in muscle; how they regulate muscle metabolism; and how they might be involved in the development and/or maintenance of insulin resistance, obesity, and diabetes.

Program Directors: (Click name for biographical information)
Paul S. MacLean, Ph.D.
Paul.MacLean@uchsc.edu
(303)315-1508

Staff and/or Affiliated Personnel: (Click name for biographical information)
Janine Higgins, Ph.D.
Rosie Wei , M.S.
Ginger Johnson, M.S.
Neal Beeman, M.S.
Brooke Fleming, B.S.

Background:
The IRS is a compilation of associated disorders that include resistance to insulin-stimulated glucose uptake, obesity, dyslipidemia, atherosclerosis, and diabetes. The occurrence of these disorders diagnosed alone or in conjunction with one another is increasing at an alarming rate, with staggering consequences on annual health care costs. Several tissues, including skeletal muscle, display an adverse shift in lipid metabolism that is thought to contribute to the development of these associated disorders. While this shift favoring the synthesis and accumulation of lipids and cholesterol is common to many tissues, the underlying mechanisms behind it are poorly understood. Recent studies in some of these tissues implicate SREBPs in the development of perturbed lipid partitioning and the IRS. These studies report that SREBPs: 1) mediate insulin action on gene expression^1-10; 2) have dramatic adverse effects on lipid partitioning/accumulation^11-14; and 3) are elevated in rodent models of insulin resistance and diabetes¹², ¹³. Given these observations, SREBPs present attractive targets for the study of the tissue-specific mechanisms that cause this debilitating syndrome.

General characteristics of SREBP proteins

SREBPs are transcription factors that are members of the basic helix-loop-helix leucine zipper family of DNA binding proteins¹⁵. Three isoforms have been identified in mammalian tissues that vary in structure, regulation, and function. SREBP-1a and SREBP-1c (originally cloned as ADD1¹⁶) are protein products of alternative promoter usage of the SREBP-1 gene¹⁷, ¹⁸. The third isoform is transcribed from a different gene, SREBP-2¹⁹. The relative expression of these isoforms can vary dramatically between tissues and is thought to be linked to the respective regulatory regions that are under the independent control of tissue-specific and metabolic-specific factors. In liver and adipose tissues, SREBPs have a significant influence on lipid and cholesterol accumulation by inducing the transcription of genes involved in these processes. While SREBP-1 is thought to be more important in regulating the expression of genes involved in triglyceride synthesis and accumulation, SREBP-2 has been more closely linked to those involved in cholesterol synthesis and accumulation. Even so, it is important to note that this delineation of function is not distinct, as several studies indicate redundancy between the 1 and 2 isoforms in their effects on transcription. Furthermore, it has been suggested that the ratio of the isoforms is important in altering gene expression, because they have dramatically different efficacies on transcriptional activation while competing for the same response elements ¹⁴,²⁰,²¹.

SREBP expression in skeletal muscle

Studies in human skeletal muscle and cultured muscle cells have indicated that SREBP proteins are also expressed in skeletal muscle¹⁹, ²², ²³. As in other tissues, several studies are suggesting that skeletal muscle SREBPs mediate insulin action on gene expression ^22-25. The function of SREBP proteins (gene targets, physiological effects, etc.) has yet to be elucidated. Adenoviral delivery of the SREBP gene to cultured muscle cells resulted a gene expression profile that would suppress fat oxidation, promote lipid accumulation, and promote a preference for carbohydrate as a fuel source²³. These observations suggest that the function of SREBPs in skeletal muscle may be similar to that found in other tissues and may suggest a role for SREBPs in the development and/or maintenance of skeletal muscle insulin resistance. In our efforts to pursue a better understand skeletal muscle metabolism, we are attempting to characterize the function and regulation of SREBPs in this tissue.

Specialized Techniques and Facilities:
Adenoviral gene delivery to skeletal muscle

In vitro models do not always reflect the physiology, metabolism, and gene expression of intact skeletal muscle fibers²⁶. In addition, disease etiology and metabolic regulation of genes can be multifactorial, involving neural, endocrine, and autocrine inputs that can only be mimicked in vivo, making it difficult to translate findings in tissue culture to the in vivo setting. For these reasons, it is particularly important with skeletal muscle to have in vivo models to examine the functional aspects of gene expression in this tissue. Recently we have been employing a novel approach to delivery genes to intact skeletal muscle with genetically engineered adenoviruses. The muscle-specific expression of the coxsackie/adenoviral receptor (CAR), the docking protein for adenoviruses, improves AdV gene delivery to skeletal muscle ²⁷. With transgenically expressed CAR, the resistance of skeletal muscle to adenoviral infection is dramatically attenuated, as has been found with other resistant tissues²⁸, ²⁹, making this approach to studying the functional aspects of gene expression more practical. We are currently employing these mice in our studies to understang the function of SREBPs in muscle, and we are developing ways to refine and improve the physiological relevance of this model.

Hyperinsulinemic-euglycemic clamp

This technique allows us to determine whole body insulin sensitivity and to estimate the sensitivity of some specific tissues to insulin action30. Catheter extensions are inserted into carotid and jugular cannulae so that animals can move freely in the experimental cage. These extensions will exit the cage through the top of the cage in such a manner that prevents the rat from tangling or chewing on them during the procedure. Following a rest period, a primed, continuous infusion of insulin is begun and followed by a variable rate glucose infusion to clamp plasma glucose at basal levels. Arterial blood samples are drawn at regular intervals in order to monitor blood glucose levels and to properly adjust the glucose infusion rate. Tracers of glucose and 2-deoxyglucose are injected late in the procedure for determining the decay rate and tissue-specific glucose uptake. The amount of glucose infused at this high level of insulin is used to indicate the body's sensitivity to insulin action. Tracer uptake into the tissues allows us to estimate each tissue's sensitivity to insulin action.

Indirect Calorimetry

This program employs the use of the JFK Indirect Calorimetry Satellite Facility (JFK ICSF). This satellite facility provides temporary housing of rats in the vicinity of an open indirect calorimetry system. The use of indirect calorimetry allows us to estimate metabolic rate (energy expenditure) and respiratory exchange ratio (relative measurement of fat and carbohydrate oxidation)31. This is accomplished by utilizing a sophisticated system whereby the amount of oxygen consumed and carbon dioxide produced by an animal is precisely measured over a period of time. The system consists of cages (chambers), air pumps, air flow controllers, valves, and gas analyzers, and is computer controlled to simultaneously measure oxygen and carbon dioxide in an individual chamber, recording data from each of the four chambers in a sequential manner. The system operates as follows. Air taken from a common source is pulled through each chamber via separate intake lines. Rats are placed in four chambers (1 rat/chamber). These cylindrical chambers are designed to collect food spillage, feces, and urine for the determination of energy intake, unabsorbed calories, and nitrogen excretion. The air exiting each chamber is then passed through a cooling unit for dehumidification, a requirement of the gas analyzers. The air then passes through a flow controller/pump system (1 per line) that maintains a constant air flow through each chamber (1 L/min). The air then passes to a manifold containing 4 switch valves. At predefined intervals, the computer sends a signal to close a specified valve corresponding to a particular chamber, resulting in the shunt of air from that chamber to the analyzers. The time of measurement, carbon dioxide concentrations, oxygen concentrations, flow rate, vCO2, vO2, RQ, and MR (Weir equation) are calculated and stored with software designed and written by researchers here at the Center for Human Nutrition. This procedures acquires and records thousands of data points per variable over a 24 hr period. A similar system for mice is available for use at another satellite facility. ((picture of rat IC chamber))

Program Support:
We appreciate the support of the following funding agencies:

National Institutes of Health Colorado Clinical Nutrition Research Unit Parke Davis and Pfizer Inc., Atorvastatin Research Awards Program American Physiological Society

Relevant References:

1. Shimomura I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS, Goldstein JL. Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc Natl Acad Sci U S A 1999; 96:13656-61.
2. Foretz M, Pacot C, Dugail I, et al. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol Cell Biol 1999; 19:3760-8.
3. Foretz M, Guichard C, Ferre P, Foufelle F. Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis- related genes. Proc Natl Acad Sci U S A 1999; 96:12737-42.
4. Kotzka J, Muller-Wieland D, Koponen A, et al. ADD1/SREBP-1c mediates insulin-induced gene expression linked to the MAP kinase pathway. Biochem Biophys Res Commun 1998; 249:375-9.
5. Kotzka J, Muller-Wieland D, Roth G, et al. Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP-kinase cascade. J Lipid Res 2000; 41:99-108.
6. Kim JB, Spiegelman BM. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev 1996; 10:1096-107.
7. Horton JD, Bashmakov Y, Shimomura I, Shimano H. Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc Natl Acad Sci U S A 1998; 95:5987-92.
8. Kim JB, Sarraf P, Wright M, et al. Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest 1998; 101:1-9.
9. Shimano H, Yahagi N, Amemiya-Kudo M, et al. Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes. J Biol Chem 1999; 274:35832-9.
10. Streicher R, Kotzka J, Muller-Wieland D, et al. SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I. J Biol Chem 1996; 271:7128-33.
11. Shimomura I, Bashmakov Y, Horton JD. Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem 1999; 274:30028-32.
12. Kakuma T, Lee Y, Higa M, et al. Leptin, troglitazone, and the expression of sterol regulatory element binding proteins in liver and pancreatic islets. Proc Natl Acad Sci U S A 2000; 97:8536-41.
13. Shimomura I, Hammer RE, Richardson JA, et al. Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 1998; 12:3182-94.
14. Boizard M, Le Liepvre X, Lemarchand P, Foufelle F, Ferre P, Dugail I. Obesity-related overexpression of fatty-acid synthase gene in adipose tissue involves sterol regulatory element-binding protein transcription factors. J Biol Chem 1998; 273:29164-71.
15. Osborne TF. Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action. J Biol Chem 2000; 275:32379-82.
16. Tontonoz P, Kim JB, Graves RA, Spiegelman BM. ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol Cell Biol 1993; 13:4753-9.
17. Miserez AR, Cao G, Probst LC, Hobbs HH. Structure of the human gene encoding sterol regulatory element binding protein 2 (SREBF2). Genomics 1997; 40:31-40.
18. Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, Goldstein JL. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 1997; 99:846-54.
19. Hua X, Yokoyama C, Wu J, et al. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci U S A 1993; 90:11603-7.
20. Chouinard RA, Jr., Luo Y, Osborne TF, Walsh A, Tall AR. Sterol regulatory element binding protein-1 activates the cholesteryl ester transfer protein gene in vivo but is not required for sterol up- regulation of gene expression. J Biol Chem 1998; 273:22409-14.
21. Gauthier B, Robb M, Gaudet F, Ginsburg GS, McPherson R. Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1. J Lipid Res 1999; 40:1284-93.
22. Guillet-Deniau I, Carre D, Achouri Y, Girard J, Foufelle F, Ferre P. Insulin and glucose regulate SREBP-1c expression in rat skeletal muscle., American Diabetes Association 61st Scientific Sessions, Philadelphia, PA, 2001. Vol. 50(2).
23. Ferre P, Achouri Y, Guillet-Deniau I, Girard J, Foufelle F. Insulin-like action of sterol response element binding protein-1c in liver and muscles. Diabetes Mellitus: Molecular Mechanisms, Genetics, and New Therapies 2002; Keystone Symposia:183.
24. Dohm GL. Skeletal muscle SREBP expression in lean, obese, obese diabetic, and weight-reduced subjects. In: MacLean PS, ed. Greenville, NC, 2000.
25. Ducluzeau PH, Perretti N, Laville M, et al. Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes. Diabetes 2001; 50:1134-42.
26. Michael LF, Wu Z, Cheatham RB, et al. Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci U S A 2001; 98:3820-5.
27. Nalbantoglu J, Larochelle N, Wolf E, Karpati G, Lochmuller H, Holland PC. Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle. J Virol 2001; 75:4276-82.
28. Schmidt MR, Piekos B, Cabatingan MS, Woodland RT. Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes. J Immunol 2000; 165:4112-9.
29. Wan YY, Leon RP, Marks R, et al. Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in naive T cells. Proc Natl Acad Sci U S A 2000; 97:13784-9.
30. Podolin DA, Gayles EC, Wei Y, Thresher JS, Pagliassotti MJ. Menhaden oil prevents but does not reverse sucrose-induced insulin resistance in rats. Am J Physiol 1998; 274:R840-8.
31. Commerford SR, Pagliassotti MJ, Melby CL, Wei Y, Gayles EC, Hill JO. Fat oxidation, lipolysis, and free fatty acid cycling in obesity-prone and obesity-resistant rats. Am J Physiol Endocrinol Metab 2000; 279:E875-85.

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