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Structural and biochemical studies relevant to Diabetes

Our work falls into two areas: the first uses structural and biochemical approaches to define potential targets for therapeutic intervention in Type 1 and 2 diabetes, and a second approach involves biochemical and translational studies to elucidate the role of the IkB kinase, IKKb, as a mediator of insulin resistance.

We are using X-ray crystallography to determine the structure of polypeptides relevant to insulin signaling or its antagonism. Major project areas for structural analysis include: 1) insulin receptor substrates and associated SH2 domain proteins; 2) genetic causes of diabetes, lipodystophy and obesity; 3) potential drug targets for insulin sensitization and reversing diabetic complications, and 4) potential mediators of autoimmunity in type 1 diabetes. Specific ongoing projects include: a) HNF1a (MODY3) bound to DNA; this is the most common monogenic causes of diabetes (solved). b) HNF4a (MODY1) an orphan nuclear receptor involved in insulin secretion. c) Complexes between HNF4a and the coactivator, PGC-1, which regulate glucogenesis and hepatic glucose production. d) A complex of CREB (or CREM), DNA and the KIX domain of CBP,. e) Lamin A/C is mutated in diabetes and hyperlipidemia (solved) f) The kinase domain in IKKb, a major mediator of insulin resistance in type 2 diabetes and obesity, (for inhibitor/drug discovery efforts). g) The kinase domain of PKCb, a protein implicated in diabetic retinopathy (for inhibitor/drug discovery efforts. h) The kinase domain of AMPK, a mediator of exercise induced glucose uptake (for inhibitor/drug discovery efforts). i) The AIRE protein, which controls central tolerance (by driving the transcription of many potential autoantigens in the thymus,. j) The major type 1 diabetes autoantigens I1-2, phogrin and GAD.

A second area of effort derives from recent studies in our laboratory that identified the IkB kinase, IKKb, as a mediator of insulin resistance in obese rodents and patients with type 2 diabetes. IKK can be inhibited by high-dose salicylates, which explains their well-known but poorly understood hypoglycemic effects. Salicylate-inhibition of IKKb enhances signaling through insulin receptor cascades. Expression of IKKb , or activators of it, attentuated insulin signaling in cultured cells, whereas expression of dominant inhibitory forms of IKKb or IKKa reversed insulin resistance. Reductions in IKKb activity brought about by crossing IKKb ^+/- to obese Lep ob/ob mice prevented the development of insulin resistance. These findings implicate IKKb in the pathogenesis of insulin resistance in type 2 diabetes mellitus and obesity, and identify IKKb as a new target for the reversal of insulin resistance in these conditions. We’re now pursuing broad avenues of study to validate IKKb as a target for treating insulin resistance in patients with type 2 diabetes or subject predisposed to developing it. We have already conducted studies with high dose asiprin; parallel studies with salicylate are underway. Plans are being drawn up to expand these preliminary trials into large, multi-center trials of greater duration – to evaluate salicylate and IKKb in terms of both treatment and prevention. Additional studies are looking at alternative inhibitors of IKKb as treatments for insulin resistance in rodents. A variety of genetically altered mice (tissue-specific transgenes and knockouts) are being used to identify primary tissue of action and up- and down-stream activators and targets.

References:

1. Duda K, Chi YI, Shoelson SE. Structural basis for HNF-4alpha activation by ligand and coactivator binding. J Biol Chem. 2004;279:23311-6.

2. Cai D, Dhe-Paganon S, Melendez PA, Lee J, Shoelson SE. Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. J Biol Chem. 2003;278:25323-30.

3. Chi YI, Frantz JD, Oh BC, Hansen L, Dhe-Paganon S, Shoelson SE. Diabetes mutations delineate an atypical POU domain in HNF-1alpha. Mol Cell. 2002;10:1129-37.

4. Dhe-Paganon S, Duda K, Iwamoto M, Chi YI, Shoelson SE. Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand. J Biol Chem. 2002;277:37973-6.

5. Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest. 2002;109:1321-6.

6. Dhe-Paganon S, Werner ED, Chi YI, Shoelson SE. Structure of the globular tail of nuclear lamin. J Biol Chem. 2002;277:17381-4.

7. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science. 2001;293:1673-7.

8. Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI. Prevention of fat induced insulin resistance by salicylate. J Clin Invest.2001;108:437-46.

9. Dhe-Paganon S, Shigeta R, Chi YI, Ristow M, Shoelson SE. Crystal structure of human frataxin. J Biol Chem. 2000;275:30753-6.

10. Dhe-Paganon S, Ottinger EA, Nolte RT, Eck MJ, Shoelson SE. Crystal structure of the pleckstrin homology-phosphotyrosine binding (PH-PTB) targeting region of insulin receptor substrate 1. Proc Natl Acad Sci U S A. 1999;96:8378-83.


	Steven Shoelson, M.D., Ph.D. Structural and biochemical studies relevant to Diabetes Our work falls into two areas: the first uses structural and biochemical approaches to define potential targets for therapeutic intervention in Type 1 and 2 diabetes, and a second approach involves biochemical and translational studies to elucidate the role of the IkB kinase, IKKb, as a mediator of insulin resistance. We are using X-ray crystallography to determine the structure of polypeptides relevant to insulin signaling or its antagonism. Major project areas for structural analysis include: 1) insulin receptor substrates and associated SH2 domain proteins; 2) genetic causes of diabetes, lipodystophy and obesity; 3) potential drug targets for insulin sensitization and reversing diabetic complications, and 4) potential mediators of autoimmunity in type 1 diabetes. Specific ongoing projects include: a) HNF1a (MODY3) bound to DNA; this is the most common monogenic causes of diabetes (solved). b) HNF4a (MODY1) an orphan nuclear receptor involved in insulin secretion. c) Complexes between HNF4a and the coactivator, PGC-1, which regulate glucogenesis and hepatic glucose production. d) A complex of CREB (or CREM), DNA and the KIX domain of CBP,. e) Lamin A/C is mutated in diabetes and hyperlipidemia (solved) f) The kinase domain in IKKb, a major mediator of insulin resistance in type 2 diabetes and obesity, (for inhibitor/drug discovery efforts). g) The kinase domain of PKCb, a protein implicated in diabetic retinopathy (for inhibitor/drug discovery efforts. h) The kinase domain of AMPK, a mediator of exercise induced glucose uptake (for inhibitor/drug discovery efforts). i) The AIRE protein, which controls central tolerance (by driving the transcription of many potential autoantigens in the thymus,. j) The major type 1 diabetes autoantigens I1-2, phogrin and GAD. A second area of effort derives from recent studies in our laboratory that identified the IkB kinase, IKKb, as a mediator of insulin resistance in obese rodents and patients with type 2 diabetes. IKK can be inhibited by high-dose salicylates, which explains their well-known but poorly understood hypoglycemic effects. Salicylate-inhibition of IKKb enhances signaling through insulin receptor cascades. Expression of IKKb , or activators of it, attentuated insulin signaling in cultured cells, whereas expression of dominant inhibitory forms of IKKb or IKKa reversed insulin resistance. Reductions in IKKb activity brought about by crossing IKKb ^+/- to obese Lep ob/ob mice prevented the development of insulin resistance. These findings implicate IKKb in the pathogenesis of insulin resistance in type 2 diabetes mellitus and obesity, and identify IKKb as a new target for the reversal of insulin resistance in these conditions. We’re now pursuing broad avenues of study to validate IKKb as a target for treating insulin resistance in patients with type 2 diabetes or subject predisposed to developing it. We have already conducted studies with high dose asiprin; parallel studies with salicylate are underway. Plans are being drawn up to expand these preliminary trials into large, multi-center trials of greater duration – to evaluate salicylate and IKKb in terms of both treatment and prevention. Additional studies are looking at alternative inhibitors of IKKb as treatments for insulin resistance in rodents. A variety of genetically altered mice (tissue-specific transgenes and knockouts) are being used to identify primary tissue of action and up- and down-stream activators and targets. References: 1. Duda K, Chi YI, Shoelson SE. Structural basis for HNF-4alpha activation by ligand and coactivator binding. J Biol Chem. 2004;279:23311-6. 2. Cai D, Dhe-Paganon S, Melendez PA, Lee J, Shoelson SE. Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. J Biol Chem. 2003;278:25323-30. 3. Chi YI, Frantz JD, Oh BC, Hansen L, Dhe-Paganon S, Shoelson SE. Diabetes mutations delineate an atypical POU domain in HNF-1alpha. Mol Cell. 2002;10:1129-37. 4. Dhe-Paganon S, Duda K, Iwamoto M, Chi YI, Shoelson SE. Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand. J Biol Chem. 2002;277:37973-6. 5. Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest. 2002;109:1321-6. 6. Dhe-Paganon S, Werner ED, Chi YI, Shoelson SE. Structure of the globular tail of nuclear lamin. J Biol Chem. 2002;277:17381-4. 7. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science. 2001;293:1673-7. 8. Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI. Prevention of fat induced insulin resistance by salicylate. J Clin Invest.2001;108:437-46. 9. Dhe-Paganon S, Shigeta R, Chi YI, Ristow M, Shoelson SE. Crystal structure of human frataxin. J Biol Chem. 2000;275:30753-6. 10. Dhe-Paganon S, Ottinger EA, Nolte RT, Eck MJ, Shoelson SE. Crystal structure of the pleckstrin homology-phosphotyrosine binding (PH-PTB) targeting region of insulin receptor substrate 1. Proc Natl Acad Sci U S A. 1999;96:8378-83.