Insulin exerts direct, IGF-1 independent actions in growth plate chondrocytes

Abstract

Insufficient insulin production or action in diabetic states is associated with growth retardation and impaired bone healing, while the underling mechanisms are unknown. In this study, we sought to define the role of insulin signaling in the growth plate. Insulin treatment of embryonic metatarsal bones from wild-type mice increased chondrocyte proliferation. Mice lacking insulin receptor (IR) selectively in chondrocytes (CartIR ^−/−) had no discernable differences in total femoral length compared to control littermates. However, CartIR ^−/− mice exhibited an increase in chondrocyte numbers in the growth plate than that of the controls. Chondrocytes lacking IR had elevated insulin-like growth factor (IGF)-1R mRNA and protein levels. Subsequently, IGF-1 induced phosphorylation of Akt and ERK was enhanced, while this action was eliminated when the cells were treated with IGF-1R inhibitor Picropodophyllin. Deletion of the IR impaired chondrogenic differentiation, and the effect could not be restored by treatment of insulin, but partially rescued by IGF-1 treatment. Intriguingly, the size of hypertrophic chondrocytes was smaller in CartIR ^−/− mice when compared with that of the control littermates, which was associated with upregulation of tuberous sclerosis complex 2 (TSC2). These results suggest that deletion of the IR in chondrocytes sensitizes IGF-1R signaling and action, IR and IGF-1R coordinate to regulate the proliferation, differentiation and hypertrophy of growth plate chondrocytes.

Bone growth: The role of insulin

Insulin and insulin-like growth factors (IGFs) have independent, overlapping effects on bone growth. Insulin deficiency or insensitivity, caused by diseases such as diabetes, is known to impair bone growth but the mechanism remains unknown. An international team led by Chao Wan at the Chinese University of Hong Kong investigated how insulin affects chondrocytes, collagen-producing cells essential for building new bone, using genetically modified mice whose chondrocytes lacked insulin receptors (IRs). The mice had normal-length bones, and the IR-deficient chondrocytes showed increased production of and sensitivity to IGF-1, indicating that IGFs can compensate for reduced insulin. However, chondrocytes in the zone where new bone has yet to harden were small, indicating that insulin plays an important role in regulating chondrocyte size. These results will help clarify how diseases like diabetes impair bone growth and healing.

References

[1] Fulzele K, Clemens TL. Novel functions for insulin in bone. Bone, 2012, 50: 452-456

[2] Nef S, Parada LF. Hormones in male sexual development. Genes Dev, 2000, 14: 3075-3086

[3] Ebina Y, Ellis L, Jarnagin K et al The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signaling. Cell, 1985, 40: 747-758

[4] Kim JJ AcciliD. Signalling through IGF-1 and insulin receptors: where is the specificity? Growth Horm IGF Res, 2002, 12: 84-90

[5] Woods KA, Camacho-Hubner C, Savage MO, Clark AJ. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med, 1996, 335: 1363-1367

[6] Wang YM, Nishida S, Sakata T et al Insulin-like growth factor-I is essential for embryonic bone development. Endocrinology, 2006, 147: 4753-4761

[7] Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell, 1993, 75: 73-82

[8] Govoni KE, Lee SK, Chung YS et al Disruption of insulin-like growth factor-I expression in type IIalphaI collagen-expressing cells reduces bone length and width in mice. Physiol Genomics, 2007, 30: 354-362

[9] Wertheimer E, Lu SP, Backeljauw PF, Davenport ML, Taylor SI. Homozygous deletion of the human insulin receptor gene results in leprechaunism. Nat Genet, 1993, 5: 71-73

[10] Accili D, Drago J, Lee EJ et al Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat Genet, 1996, 12: 106-109

[11] Bailyes EM, Nave BT, Soos MA, Orr SR, Hayward AC, Siddle K. Insulin receptor/IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting. Biochem J, 1997, 327 Pt 1 209-215

[12] Fulzele K, Digirolamo DJ, Liu Z, Xu J, Messina JL, Clemens TL. Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action. J Biol Chem, 2007, 282: 25649-25658

[13] Long F, Joeng KS, Xuan S, Efstratiadis A, McMahon AP. Independent regulation of skeletal growth by Ihh and IGF signaling. Dev Biol, 2006, 298: 327-333

[14] Chan SJ, Plisetskaya EM, Urbinati E, Jin Y, Steiner DF. Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage. Proc Natl Acad Sci USA, 1997, 94: 12446-12451

[15] Hill DJ, De SD. Insulin is a mitogen for isolated epiphyseal growth plate chondrocytes from the fetal lamb. Endocrinology, 1990, 126: 2661-2670

[16] Serra R, Chang C. TGF-beta signaling in human skeletal and patterning disorders. Birth Defects Res C Embryo Today, 2003, 69: 333-351

[17] Efstratiadis A. Genetics of mouse growth. Int J Dev Biol, 1998, 42: 955-976

[18] Ornitz DM, Marie PJ. FGF signaling pathways inendochondral and intramembranous bone development and human genetic disease. Genes Dev, 2002, 16: 1446-1465

[19] Treadway JL, Frattali AL, Pessin JE. Intramolecular subunit interactions between insulin and insulin-like growth factor 1 alpha beta half-receptors induced by ligand and Mn/MgATP binding. Biochemistry, 1992, 31: 11801-11805

[20] Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri R. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocr Rev, 2009, 30: 586-623

[21] Brüning JC, Michael MD, Winnay JN et al A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell, 1998, 2: 559-569

[22] Baffi MO, Slattery E, Sohn P, Moses HL, Chytil A, Serra R. Conditional deletion of the TGF-beta type II receptor in Col2a expressing cells results in defects in the axial skeleton without alterations in chondrocyte differentiation or embryonic development of long bones. Dev Biol, 2004, 276: 124-142

[23] Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR. Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell, 1999, 96: 329-339

[24] Chen M, Lichtler AC, Sheu TJ et al Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase. Genesis, 2007, 45: 44-50

[25] Wang Y, Wan C, Deng L et al The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development. J Clin Invest, 2007, 117: 1616-1626

[26] Inoki K, Zhu TQ, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell, 2003, 115: 577-590

[27] Phornphutkul C, Wu KY, Gruppuso PA. The role of insulin inchondrogenesis. Mol Cell Endocrinol, 2006, 249: 107-115

[28] Phorphutkul C, Wu KY, Yang X, Chen Q, Gruppuso PA. Insulin-like growth factor-1 signaling is modified during chondrocyte differentiation. J Endocrinol, 2004, 183: 477-486

[29] Böhni R, Riesgo-Escovar J, Oldham S et al Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell, 1999, 97: 865-875

[30] Verdu J, Buratovich MA, Wilder EL, Birnbaum MJ. Cell-autonomous regulation of cell and organ growth in Drosophila by AKT/PKB. Nat Cell Biol, 1999, 1: 500-506

[31] Johnston LA, Gallant P. Control of growth and organ size in Drosophila. Bioessays, 2002, 24: 54-64

[32] Jewell JL, Guan KL. Nutrient signaling to mTOR and cell growth. Trends Biochem, 2013, 38: 233-242

[33] Sasaki AT, Firtel RA. Regulation of chemotaxis by the orchestrated activationof Ras, PI3K, and TOR. Eur J Cell Biol, 2006, 85: 873-895

[34] Howell JJ, Manning BD. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends Endocrinol Metab, 2011, 22: 94-102

[35] Inoki K, Li Y, Zhu TQ, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by AKT and suppresses mTOR signaling. Nat Cell Biol, 2002, 4: 648-657