BADERC- Harvey Lodish

Harvey Lodish, Ph.D.

Molecular Regulation of Glucose and Fatty Acid Uptake

1. Acrp30/Adiponectin Structure and Signaling. Adipocyte complement related protein of 30 kDa (Acrp30), or adiponectin, is an adipocyte-secreted hormone we cloned several years ago that is found abundantly in serum. Its expression and serum concentration are decreased in obese or diabetic humans and animals and it exerts multiple metabolic actions at several tissue sites. The isolated trimeric globular domain of Acrp30 (gAcrp30) simulates fatty acid oxidation in skeletal muscle while full length Acrp30, containing an N- terminal collagen tail and a trimeric C- terminal globular domain, was reported by others to synergize with insulin to inhibit hepatic glucose production. Disruption of the Acrp30 locus in mice resulted in impaired fatty acid clearance, increased TNF-a levels, and aggravated insulin resistance in animals fed a high fat diet.

We showed that Acrp30 purified from transfected human embryonic kidney (HEK) 293T cells or E. coli exists as trimers and hexamers. Transfected HEK cells also secrete an even higher molecular weight (HMW) isoform of Acrp30. All three isoforms of Acrp30 are present in mouse serum and conditioned media of differentiated 3T3-L1 adipocytes, albeit with different relative abundances. The HMW and hexameric Acrp30 activates transcription factor NF-kB in undifferentiated or differentiated C2C12 cells, but trimeric Acrp30 or gAcrp30 cannot. Rather, trimeric Acrp30 and gAcrp30, but not full-length hexamer, enhances muscle fatty acid oxidation by inactivating acetyl-CoA carboxylase following stimulation of AMP-activated protein kinase. Although not necessary for stable trimeric structure, two of the three monomers in Acrp30 trimer are covalently linked by disulfide bonds between the two cysteine residues at position 22 (C22) of each monomer. In contrast, assembly of hexameric and HMW Acrp30 depends upon formation of C22-mediated disulfide bonds since their reduction with dithiothreitol or substitution of C22 with alanine led exclusively to trimers. Accordingly, HMW and hexamer isoforms of Acrp30 activated NF-kB, but not reduced Acrp30 or C22A Acrp30, demonstrating the dependence of NF-kB activation by Acrp30 on its oligomerization state. Using a functional expression strategy we are cloning the multiple receptors for these Acrp30 isoforms and have already characterized one specific for hexameric and HMW isoforms. Understanding how these receptors are coupled to activation of NF-kB and AMP-activated protein kinase is another focus of our current work.

2. Induction of insulin resistance by Tumor Necrosis Factor-a (TNF-a). TNF-a, an autocrine/paracrine factor highly induced in adipose tissues of obese animals and human subjects, has been implicated as a contributing cause of insulin resistance seen in obesity and obesity-linked type 2 diabetes. The mechanisms for TNF-a induction of insulin resistance involve inhibition of insulin signal transduction and down-regulation of many important adipocyte proteins that are essential for insulin action. We recently demonstrated that TNF-a-induced and NF-kB-mediated transcriptional regulation constitutes a principal component of the mechanism by which TNF-a induces insulin resistance in adipocytes. Subsequently, we substantiated the critical role of TNF-a-regulated gene expression in adipocytes in the development of systemic insulin resistance by association of gene expression profiles in major insulin-target tissues with overall in vivo insulin sensitivity in rats infused with TNF-a. Currently we are investigating in depth the mechanism(s) by which NF-kB activation by TNF-a represses adipocyte gene expression, as well as the functional consequences of constitutive activation of NF-kB in adipocytes and the impact of loss of function of individual members of NF-kB family on adipocyte biology. Specifically, we are determining the molecular mechanisms for the functional antagonism between p65 (RelA, a major NF-kB family member) and PPAR-g in 3T3-L1 adipocytes, and whether activation of NF-kB suffices to recapitulate the effects of TNF-a on adipocyte gene expression, endocrine activity, and metabolic function.

3. Expression cloning of TUG, a novel protein mediating insulin stimulation of GLUT4 exocytosis. The rate of glucose utilization in muscle and fat is limited by the number of GLUT4 glucose transporters at the cell surface. GLUT4 is sequestered intracellularly in the absence of insulin, and is redistributed to the plasma membrane within minutes of insulin stimulation. We previously described a GLUT4 reporter protein containing epitope tags in its first extracellular domain and green fluorescent protein (GFP) fused to the carboxyl terminus³. Using this reporter, the relative proportion of GLUT4 at the surface of individual, living cells can be assayed by using flow cytometry to measure fluorescence intensities corresponding to cell-surface and total amounts of GLUT4. Based on this reporter we developed a functional screen to identify proteins that modulate GLUT4 distribution, and identified TUG as a putative tether, containing a UBX domain, for GLUT4. In truncated form, TUG acts in a dominant negative manner to inhibit insulin-stimulated redistribution of GLUT4 in CHO cells and 3T3-L1 adipocytes. Full-length TUG complexes specifically with GLUT4 in transfected cells; in 3T3-L1 adipocytes this complex is present in unstimulated cells and is largely disassembled after brief insulin stimulation. Endogenous TUG colocalizes with the insulin-mobilizable pool of GLUT4 in unstimulated 3T3-L1 adipocytes, and is not mobilized to the plasma membrane by insulin. In transfected, non-adipose cells, TUG confers intracellular retention of GLUT4; distinct regions of TUG are involved in GLUT4 binding and retention. Our data suggest a model, which we currently are testing, in which TUG traps endocytosed GLUT4 and tethers it intracellularly, and insulin mobilizes this pool of retained GLUT4 by releasing this tether.

References

1. Bogan, J. S., and H. F. Lodish. Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous GLUT4 and ACRP30 J. Cell Biol., 146: 609 - 620 (1999)

2. Liu, X., S. Constantinescu,, Y. Sun, J. S. Bogan, D. Hirsch, R. A. Weinberg, and H. F. Lodish. Rapid generation of mammalian cells stably expressing multiple genes at predetermined levels Anal. Biochem. 280: 20 - 28 (2000).

3. Chi, N-W and H. F. Lodish Tankyrase is a Golgi-associated MAP Kinase substrate that interacts with IRAP in GLUT4 vesicles J. Biol. Chem. 275: 38437 - 38444 (2000)

4. Fruebis, J., Tsao, T-S., Erickson, M. R., Lee, L., Yen, F., Bihain, B. E., and Lodish, H. F. A Proteolytic Cleavage Product of ACRP30 Increases Fatty Acid Oxidation in Muscle and Causes Weight Loss in Mice. Proc. Natl. Acad. Sci. USA 98: 2005 - 2010; (2001)

5. Bogan, J. B., A. E. McKee, and H. F. Lodish. Insulin-responsive compartments containing GLUT4 in 3T3-L1 and CHO cells: regulation by amino acid concentrations. Mol. Cell Biol. 21: 4785 – 4806 (2001)

6. Sbodio, J., H. Lodish, and N-W Chi. Tankyrase-2 oligomerizes with tankyrase-1 and binds to both TRF1 and IRAP. Biochem. J 361: 451 - 459 (2002)

7. Tsao, T-S, H. Lodish, and J. Fruebis. ACRP30, a new hormone controlling fat and glucose metabolism. E. Jour. Pharm 440: 213 – 221. (2002)

8. Ruan, H., N. Hacohen, T. R. Golub, L. Van Parijs and H. F. Lodish Tumor necrosis factor-a suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: Nuclear Factor-kB activation by TNF-a is obligatory. Diabetes. 51: 1319 - 1336 (2002)

9. Tsao, T-s, H. E. Murrey, C. Hug, D. H Lee, and H. F. Lodish Oligomerization State-Dependent Activation of NF-kB Signaling Pathway by Acrp30. J. Biol. Chem. 277: 29359 – 29362 (2002)

10. Ruan, H., P. D. G. Miles, C. M. Ladd, K. Ross, T, R. Golub, J. M. Olefsky and H. F. Lodish Profiling Gene Transcription in vivo Reveals Adipose Tissue as an Immediate Target of TNF-a: Implications for Insulin Resistance. Diabetes. 51: 3176-3188 (2002)

11. Hug, C. and H. F. Lodish. . Diabetes, Obesity, and Acrp30/ Adiponectin. Biotechniques 33: 654 - 658 (2002)

12. Thomas, E., T-s Tsao, A, Saha, H. E. Murrey, C.- c. Zhang, S. I. Itani, H. F. Lodish, and N. Ruderman. Enhancement of muscle fatty acid oxidation by globular domain of ACRP30: inactivation of Acetyl-CoA carboxylase and stimulation of AMP-activated protein kinase. Proc. Natl. Acad. Sci. USA 99: 16309-16313 (2002)

13. Bogan, J., N. Hendon, A. McKee, T-s Tsao, and H.F. Lodish Functional cloning of an insulin-regulated tether that controls GLUT4 glucose transporter distribution. Nature Submitted (2003)

14. Ruan, H., H. J. Pownall, and H. F. Lodish. Troglitazone antagonizes TNF-a-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kB J. Biol. Chem. In the press (2003)

15. Ruan, H. and H. F. Lodish. Insulin Resistance in Adipose Tissue: Direct and Indirect Effects of Tumor Necrosis Factor-a. Cytokine and Growth Factor Reviews In the press (2003).

16. Tsao, T-s., H. E. Murrey, C. Hug, D. H. Lee, J. E. Heuser, and H. F. Lodish. Role of Disulfide Bonds in Acrp30/Adiponectin Structure and Signaling. J Biol Chem. 2003 Dec 12;278(50):50810-7.

17. Bogan JS, Hendon N, McKee AE, Tsao TS, Lodish HF. Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature. 2003;425:727-33.

18. Kim JK, Gimeno RE, Higashimori T, Kim HJ, Choi H, Punreddy S, Mozell RL, Tan G, Stricker-Krongrad A, Hirsch DJ, Fillmore JJ, Liu ZX, Dong J, Cline G, Stahl A, Lodish HF, Shulman GI. Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle. J Clin Invest. 2004 Mar;113(5):756-63.

19. Marszalek JR, Kitidis C, Dararutana A, Lodish HF. Acyl-CoA synthetase 2 overexpression enhances fatty acid internalization and neurite outgrowth. J Biol Chem. 2004;279:23882-91






			Harvey Lodish, Ph.D. Molecular Regulation of Glucose and Fatty Acid Uptake 1. Acrp30/Adiponectin Structure and Signaling. Adipocyte complement related protein of 30 kDa (Acrp30), or adiponectin, is an adipocyte-secreted hormone we cloned several years ago that is found abundantly in serum. Its expression and serum concentration are decreased in obese or diabetic humans and animals and it exerts multiple metabolic actions at several tissue sites. The isolated trimeric globular domain of Acrp30 (gAcrp30) simulates fatty acid oxidation in skeletal muscle while full length Acrp30, containing an N- terminal collagen tail and a trimeric C- terminal globular domain, was reported by others to synergize with insulin to inhibit hepatic glucose production. Disruption of the Acrp30 locus in mice resulted in impaired fatty acid clearance, increased TNF-a levels, and aggravated insulin resistance in animals fed a high fat diet. We showed that Acrp30 purified from transfected human embryonic kidney (HEK) 293T cells or E. coli exists as trimers and hexamers. Transfected HEK cells also secrete an even higher molecular weight (HMW) isoform of Acrp30. All three isoforms of Acrp30 are present in mouse serum and conditioned media of differentiated 3T3-L1 adipocytes, albeit with different relative abundances. The HMW and hexameric Acrp30 activates transcription factor NF-kB in undifferentiated or differentiated C2C12 cells, but trimeric Acrp30 or gAcrp30 cannot. Rather, trimeric Acrp30 and gAcrp30, but not full-length hexamer, enhances muscle fatty acid oxidation by inactivating acetyl-CoA carboxylase following stimulation of AMP-activated protein kinase. Although not necessary for stable trimeric structure, two of the three monomers in Acrp30 trimer are covalently linked by disulfide bonds between the two cysteine residues at position 22 (C22) of each monomer. In contrast, assembly of hexameric and HMW Acrp30 depends upon formation of C22-mediated disulfide bonds since their reduction with dithiothreitol or substitution of C22 with alanine led exclusively to trimers. Accordingly, HMW and hexamer isoforms of Acrp30 activated NF-kB, but not reduced Acrp30 or C22A Acrp30, demonstrating the dependence of NF-kB activation by Acrp30 on its oligomerization state. Using a functional expression strategy we are cloning the multiple receptors for these Acrp30 isoforms and have already characterized one specific for hexameric and HMW isoforms. Understanding how these receptors are coupled to activation of NF-kB and AMP-activated protein kinase is another focus of our current work. 2. Induction of insulin resistance by Tumor Necrosis Factor-a (TNF-a). TNF-a, an autocrine/paracrine factor highly induced in adipose tissues of obese animals and human subjects, has been implicated as a contributing cause of insulin resistance seen in obesity and obesity-linked type 2 diabetes. The mechanisms for TNF-a induction of insulin resistance involve inhibition of insulin signal transduction and down-regulation of many important adipocyte proteins that are essential for insulin action. We recently demonstrated that TNF-a-induced and NF-kB-mediated transcriptional regulation constitutes a principal component of the mechanism by which TNF-a induces insulin resistance in adipocytes. Subsequently, we substantiated the critical role of TNF-a-regulated gene expression in adipocytes in the development of systemic insulin resistance by association of gene expression profiles in major insulin-target tissues with overall in vivo insulin sensitivity in rats infused with TNF-a. Currently we are investigating in depth the mechanism(s) by which NF-kB activation by TNF-a represses adipocyte gene expression, as well as the functional consequences of constitutive activation of NF-kB in adipocytes and the impact of loss of function of individual members of NF-kB family on adipocyte biology. Specifically, we are determining the molecular mechanisms for the functional antagonism between p65 (RelA, a major NF-kB family member) and PPAR-g in 3T3-L1 adipocytes, and whether activation of NF-kB suffices to recapitulate the effects of TNF-a on adipocyte gene expression, endocrine activity, and metabolic function. 3. Expression cloning of TUG, a novel protein mediating insulin stimulation of GLUT4 exocytosis. The rate of glucose utilization in muscle and fat is limited by the number of GLUT4 glucose transporters at the cell surface. GLUT4 is sequestered intracellularly in the absence of insulin, and is redistributed to the plasma membrane within minutes of insulin stimulation. We previously described a GLUT4 reporter protein containing epitope tags in its first extracellular domain and green fluorescent protein (GFP) fused to the carboxyl terminus³. Using this reporter, the relative proportion of GLUT4 at the surface of individual, living cells can be assayed by using flow cytometry to measure fluorescence intensities corresponding to cell-surface and total amounts of GLUT4. Based on this reporter we developed a functional screen to identify proteins that modulate GLUT4 distribution, and identified TUG as a putative tether, containing a UBX domain, for GLUT4. In truncated form, TUG acts in a dominant negative manner to inhibit insulin-stimulated redistribution of GLUT4 in CHO cells and 3T3-L1 adipocytes. Full-length TUG complexes specifically with GLUT4 in transfected cells; in 3T3-L1 adipocytes this complex is present in unstimulated cells and is largely disassembled after brief insulin stimulation. Endogenous TUG colocalizes with the insulin-mobilizable pool of GLUT4 in unstimulated 3T3-L1 adipocytes, and is not mobilized to the plasma membrane by insulin. In transfected, non-adipose cells, TUG confers intracellular retention of GLUT4; distinct regions of TUG are involved in GLUT4 binding and retention. Our data suggest a model, which we currently are testing, in which TUG traps endocytosed GLUT4 and tethers it intracellularly, and insulin mobilizes this pool of retained GLUT4 by releasing this tether. References 1. Bogan, J. S., and H. F. Lodish. Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous GLUT4 and ACRP30 J. Cell Biol., 146: 609 - 620 (1999) 2. Liu, X., S. Constantinescu,, Y. Sun, J. S. Bogan, D. Hirsch, R. A. Weinberg, and H. F. Lodish. Rapid generation of mammalian cells stably expressing multiple genes at predetermined levels Anal. Biochem. 280: 20 - 28 (2000). 3. Chi, N-W and H. F. Lodish Tankyrase is a Golgi-associated MAP Kinase substrate that interacts with IRAP in GLUT4 vesicles J. Biol. Chem. 275: 38437 - 38444 (2000) 4. Fruebis, J., Tsao, T-S., Erickson, M. R., Lee, L., Yen, F., Bihain, B. E., and Lodish, H. F. A Proteolytic Cleavage Product of ACRP30 Increases Fatty Acid Oxidation in Muscle and Causes Weight Loss in Mice. Proc. Natl. Acad. Sci. USA 98: 2005 - 2010; (2001) 5. Bogan, J. B., A. E. McKee, and H. F. Lodish. Insulin-responsive compartments containing GLUT4 in 3T3-L1 and CHO cells: regulation by amino acid concentrations. Mol. Cell Biol. 21: 4785 – 4806 (2001) 6. Sbodio, J., H. Lodish, and N-W Chi. Tankyrase-2 oligomerizes with tankyrase-1 and binds to both TRF1 and IRAP. Biochem. J 361: 451 - 459 (2002) 7. Tsao, T-S, H. Lodish, and J. Fruebis. ACRP30, a new hormone controlling fat and glucose metabolism. E. Jour. Pharm 440: 213 – 221. (2002) 8. Ruan, H., N. Hacohen, T. R. Golub, L. Van Parijs and H. F. Lodish Tumor necrosis factor-a suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: Nuclear Factor-kB activation by TNF-a is obligatory. Diabetes. 51: 1319 - 1336 (2002) 9. Tsao, T-s, H. E. Murrey, C. Hug, D. H Lee, and H. F. Lodish Oligomerization State-Dependent Activation of NF-kB Signaling Pathway by Acrp30. J. Biol. Chem. 277: 29359 – 29362 (2002) 10. Ruan, H., P. D. G. Miles, C. M. Ladd, K. Ross, T, R. Golub, J. M. Olefsky and H. F. Lodish Profiling Gene Transcription in vivo Reveals Adipose Tissue as an Immediate Target of TNF-a: Implications for Insulin Resistance. Diabetes. 51: 3176-3188 (2002) 11. Hug, C. and H. F. Lodish. . Diabetes, Obesity, and Acrp30/ Adiponectin. Biotechniques 33: 654 - 658 (2002) 12. Thomas, E., T-s Tsao, A, Saha, H. E. Murrey, C.- c. Zhang, S. I. Itani, H. F. Lodish, and N. Ruderman. Enhancement of muscle fatty acid oxidation by globular domain of ACRP30: inactivation of Acetyl-CoA carboxylase and stimulation of AMP-activated protein kinase. Proc. Natl. Acad. Sci. USA 99: 16309-16313 (2002) 13. Bogan, J., N. Hendon, A. McKee, T-s Tsao, and H.F. Lodish Functional cloning of an insulin-regulated tether that controls GLUT4 glucose transporter distribution. Nature Submitted (2003) 14. Ruan, H., H. J. Pownall, and H. F. Lodish. Troglitazone antagonizes TNF-a-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kB J. Biol. Chem. In the press (2003) 15. Ruan, H. and H. F. Lodish. Insulin Resistance in Adipose Tissue: Direct and Indirect Effects of Tumor Necrosis Factor-a. Cytokine and Growth Factor Reviews In the press (2003). 16. Tsao, T-s., H. E. Murrey, C. Hug, D. H. Lee, J. E. Heuser, and H. F. Lodish. Role of Disulfide Bonds in Acrp30/Adiponectin Structure and Signaling. J Biol Chem. 2003 Dec 12;278(50):50810-7. 17. Bogan JS, Hendon N, McKee AE, Tsao TS, Lodish HF. Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature. 2003;425:727-33. 18. Kim JK, Gimeno RE, Higashimori T, Kim HJ, Choi H, Punreddy S, Mozell RL, Tan G, Stricker-Krongrad A, Hirsch DJ, Fillmore JJ, Liu ZX, Dong J, Cline G, Stahl A, Lodish HF, Shulman GI. Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle. J Clin Invest. 2004 Mar;113(5):756-63. 19. Marszalek JR, Kitidis C, Dararutana A, Lodish HF. Acyl-CoA synthetase 2 overexpression enhances fatty acid internalization and neurite outgrowth. J Biol Chem. 2004;279:23882-91