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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 6 2241-2244
Copyright © 1999 by The Endocrine Society

Original Studies

Discovery of a Met300Val Variant in Shc and Studies of Its Relationship to Birth Weight and Length, Impaired Insulin Secretion, Insulin Resistance, and Type 2 Diabetes Mellitus1

K. Almind, M. G. Ahlgren, T. Hansen, S. A. Urhammer, J. O. Clausen and O. Pedersen

Steno Diabetes Center and Hagedorn Research Institute (K.A., M.G.A., T.H., S.A.U., O.P.), and the Center of Preventive Medicine, Glostrup University Hospital (J.O.C.), DK-2820 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Katrine Almind, Ms.Sci., Steno Diabetes Center, Niels Steensensvej 2, Gentofte, DK-2820 Copenhagen, Denmark.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results and Discussion
 References
 
The Shc adaptor proteins corresponding to the 46-, 52-, and 66-kDa isoforms are key transducers of growth promotion and gene expression, which are being phosphorylated by all known receptor tyrosine kinases after stimulation by growth factors such as insulin and insulin-like growth factor I. Several studies have demonstrated a relationship between intrauterine growth retardation and impaired glucose tolerance or type 2 diabetes later in life. It is unclear whether this finding is partially explained by genetic factors. In this context, abnormalities in Shc proteins are considered to be a plausible candidate. Therefore, the aim of this study was to analyze whether genetic variability of the Shc isoforms causes a decrease in cell growth and cell differentiation that could be manifested by a decrease in birth weight and length, impaired acute insulin secretion after iv glucose, insulin resistance, and eventually a higher prevalence of type 2 diabetes. By single strand conformation polymorphism-heteroduplex analysis of 70 patients with diabetes mellitus and subsequent nucleotide sequencing of identified single strand conformation polymorphism variant, we discovered a Met300Val substitution of the 52-kDa isoform. The amino acid variant was predicted to be present in all 3 isoforms of Shc. In a genotype-phenotype study of 360 young healthy subjects, the allelic frequency of the codon 300 polymorphism was 4.2%. In this cohort, no significant differences could be shown between carriers and noncarriers in birth weight and length, the acute insulin response to iv glucose, or the insulin sensitivity index, as estimated from an iv glucose tolerance test. In an association study of 313 type 2 diabetic patients and 226 matched glucose-tolerant subjects, there was no significant difference in allelic frequency of the Shc variant (5.1% in diabetic patients vs. 3.1% in control subjects; P = 0.11). In conclusion, by itself the Met300Val polymorphism of Shc has no major impact on birth weight and length, insulin sensitivity index, acute glucose-induced insulin secretion, or prevalence of random type 2 diabetes mellitus.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results and Discussion
 References
 
THE INSULIN-LIKE growth factor I (IGF-I) receptor and the insulin receptor activate intracellular signaling pathways by tyrosine phosphorylation of several substrates, including the adaptor proteins insulin receptor substrate-1 and Shc (termed Shc for the relation to Src homology 2 and {alpha}-collagen). Phosphorylation of Shc leads to an interaction with the SH2 domain of the adaptor protein growth factor receptor-bound protein (Grb2), followed by formation of the Grb2-son of sevenless complex with a consequent activation of the GTP-binding protein Ras (1). The activation of Ras with the subsequent coupling to the mitogen-activated protein kinase pathway leads to many cellular effects, including growth promotion and gene expression. Three isoforms of Shc are identified of 66, 52, and 46 kDa (Fig. 1Go), encoded by the same transcript but using alternative in-frame ATG translation initiation sites (2). The 52- and 46-kDa isoforms are expressed in all tissues, whereas the 66-kDa Shc protein is lacking in some cell types. The roles of the various Shc proteins have been investigated to some extent, and it is suggested that the 66-kDa isoform is a negative regulator of the epidermal growth factor-induced mitogen-activated protein kinase activity, the opposite of the effect demonstrated by the 52- and 46-kDa isoforms (2, 3). During insulin stimulation of Chinese hamster ovary cells it has been demonstrated that the insulin receptor primarily uses the 52-kDa Shc isoform, whereas the 66-kDa isoform does not undergo notable tyrosine phosphorylation. However, the insulin-stimulated serine phosphorylation of the 66-kDa Shc is significant.



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  		Figure 1. Schematic structure of the three human Shc isoforms. The methionine to valine variant at codon 300 (numbered according to 52-kDa Shc) is shown. The variant domains (CH, collagen homology; PTB, phosphotyrosine binding; SH2, Src homology2) and tyrosine residues (Y) of Shc are displayed. The three sites initiating translation of the 66-, 52-, and 46-kDa Shc isoforms are indicated by arrows.

 
Several studies have demonstrated a relationship between intrauterine growth retardation and impaired glucose tolerance (IGT) or type 2 diabetes later in life (4, 5). Exactly how low birth weight may be related to adult glucose intolerance is not clear. Furthermore, it is not yet known how much potential fetal malnutrition could be involved in these mechanisms or how big an impact an underlying genetic defect might have on alterations in insulin secretion and insulin sensitivity. A study of a positive association between low birth weight and IGT in discordant monozygotic and dizygotic twin pairs could not exclude the possibility that the association could be due to a coincidence with a certain genotype causing both low birth weight and IGT in some subjects (6).

The molecular mechanisms behind abnormal human fetal growth might occur as a result of alterations in the insulin or IGF-I molecules, in the receptors for insulin or IGF-I, or, alternatively, in postreceptor events (7). The importance of IGF-I during fetal life has been clearly demonstrated in mice, in which a targeted disruption of the IGF-I gene resulted in growth deficiencies of 60% the normal birth weight, and disruption of the IGF-I receptor conferred an even more severe growth retardation (45% of normal size), and the mice invariably died at birth (8). The role of insulin in the regulation of fetal growth was illustrated in mice lacking insulin receptor substrate-1 (9, 10). These mice had a 50% reduction in intrauterine growth compared to their wild-type littermates, which is due to IGF-I resistance. However, the insulin receptor-deficient mice exhibited normal prenatal growth but died from ketoacidosis within 3–7 days after birth (11, 12), suggesting, together with the above data, that the IGF-I receptor in the mouse embryo is the predominant signal transducer of the growth-promoting effects of IGF-I and insulin. A recent observation in humans of a mutation in the glucokinase gene (13) and polymorphisms of the variable number of tandem repeat locus that influence the transcription of the insulin gene (14) were both shown to have an influence on birth weight. These data support the hypothesis of an association between inherited alterations in genes affecting insulin secretion or insulin action and variations in fetal growth.

The purpose of the present study was to analyze whether genetic variability in Shc is associated with a decrease in birth weight and length, impaired acute insulin secretion or insulin resistance in young adulthood and with a higher prevalence of type 2 diabetes.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results and Discussion
 References
 
Subjects

The analysis was performed on complementary DNA (cDNA) from 70 Danish type 2 diabetic patients (female, n = 28; male, n = 42) who tested negative for glutamic acid decarboxylase antibodies. Their mean age was 52 ± 1 yr, and their mean body mass index was 29.9 ± 0.5 kg/m2 (values are the mean ± SD). The diabetes diagnosis was made in accordance to 1985 WHO criteria. The association study was performed in a cohort of middle-aged Danish Caucasians comprising 226 glucose-tolerant subjects (mean age, 52 yr) and 313 type 2 diabetic subjects (mean age, 62 yr); the glucose-tolerant subjects had been examined by a 1985 WHO standardized oral glucose tolerance test (15). The examination of birth weight, birth length, acute insulin response, and insulin sensitivity index was made in a sample of 360 young (18- to 32-yr-old) healthy Danish Caucasian subjects (16) who had all been examined by an iv glucose tolerance test in combination with iv injection of tolbutamide (16), and their birth weights and lengths were obtained from the midwives’ records. The studies were approved by the ethical committee of Copenhagen, and all participants gave informed consent to the experiments, which were carried out in accordance with the principles of the Declaration of Helsinki.

Single strand conformation polymorphism (SSCP) and heteroduplex analysis of Shc

The cDNA (purified from biopsies of vastus lateralus muscle as described in Ref. 17) encoding all three isoforms of Shc was amplified by PCR in eight segments (Table 1Go), followed by a SSCP-heteroduplex analysis using two different experimental conditions (15). The sensitivity of the combined SSCP-heteroduplex method in our laboratory to detect known mutations is 90%. Primers were made from the available information on the cDNA sequence of the three Shc isoforms (Table 1Go) (2). The segments that showed aberrant mobility were sequenced and analyzed on an ABI 377 automated sequencer (PE Applied Biosystems, Foster City, CA).


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  		Table 1. Sequence of oligonucleotide primers used for PCR amplification of Shc

 
Screening for the identified nucleotide substitution in Shc

The nucleotide substitution (atg->gtg) at codon 300 (according to the 52-kDa Shc isoform) was detected by PCR amplification of genomic DNA (extracted as described in Ref. 15) followed by digestion with restriction enzyme BanI (New England Biolabs, Inc., Beverly, MA). The forward primer had a long tail that contained a BanI site to confirm that each digestion had worked properly.


   Results and Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results and Discussion
References
 
SSCP-heteroduplex analysis of the human Shc

The SSCP-heteroduplex analysis of cDNA from 70 patients with type 2 diabetes and the subsequent sequencing revealed 1 nucleotide substitution (atg->gtg) that resulted in a change of methionine to valine at codon 300 (numbered according to the 52-kDa Shc; Fig. 1Go). The remaining parts of the coding sequence of Shc was unaltered. The identified Shc amino acid polymorphism at codon 300 is conserved in murine Shc and is located in the CH1 domain, which is common to all three human Shc isoforms. The variant is placed in the vicinity of the tyrosine residue at codon 317, which upon phosphorylation interacts with Grb2 (ref. 1).

Association study and clinical characteristics of middle-aged glucose-tolerant subjects

Part of the genomic DNA structure surrounding codon 300 of Shc was resolved (results not shown) to make an assay based on genomic DNA for determination of the Shc genotype on larger populations. The allelic frequencies of the Met300Val polymorphism in the middle-aged type 2 diabetic patients (n = 313) were 5.1% (95% confidence interval, 3.5–6.7) and 3.1% (1.5–4.7) in the matched glucose-tolerant subjects (n = 226; P = 0.11), which is in equilibrium according to Hardy-Weinberg. The serum insulin and C peptide responses obtained during the oral glucose tolerance test of the glucose-tolerant subjects showed no significant differences between wild-type and heterozygous carriers (data not shown).

Birth weight, birth length, acute insulin response to iv glucose, and insulin sensitivity index in young healthy carriers

To address the question as to what extent the Shc variant influenced the birth weight and length and the insulin sensitivity index, we determined the Shc genotype in a cohort of 360 young healthy Danish Caucasian subjects (16). These subjects had all been examined by an iv glucose tolerance test in combination with iv injection of tolbutamide, and their birth weights and lengths were obtained from the midwives’ records. The allelic frequency of the Met300Val polymorphism in this group of young individuals was 4.2% (95% confidence interval, 2.7–5.7). Carriers of the variant had a nonsignificant decrease in birth weight of 127 g compared to wild-type carriers (P = 0.14; Table 2Go). When the mean birth weights of female and male subjects were determined separately, a decrease in birth weight of 71 g was found in female carriers of the polymorphism vs. that of noncarriers (P = 0.35), whereas the birth weight of the male carriers was decreased by 179 g (P = 0.47). The power to detect a presumed difference of 10% in birth weight was more than 85% in the present study. Insulin sensitivity, as estimated by Bergman’s minimal model, and the acute insulin response were not different at this young stage in life between heterozygous and wild-type carriers (Table 2Go).


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  		Table 2. Biochemical and clinical characteristics obtained during an IVGTT in combination with iv injection of tolbutamide from 360 young healthy Danish Caucasians carrying either the wild-type or the heterozygous Met300Val polymorphism in Shc

 
In conclusion, given the role of Shc in IGF-I and insulin receptor signaling, it is credible that inherited defects in Shc might cause reduced growth, later impaired insulin sensitivity or secretion, and eventually increased risk of type 2 diabetes. Analysis of the coding region of Shc for mutations revealed a Met300Val polymorphism with an allelic frequency of 4.2% in young healthy subjects. Although, transfection studies are needed to exclude a minor biological impact of this infrequent Shc variant, in phenotype and association studies we were able to exclude a major effect of the polymorphism on birth weight and length, acute glucose-induced insulin response, insulin sensitivity index, and prevalence of type 2 diabetes mellitus. Our approach was not designed to address the presence of variability in noncoding regions of the Shc gene. Obviously, the presence of such variability might affect fetal growth.


   Acknowledgments
 
The authors thank Annemette Forman, Bente Mottlau, Lene Aabo, Sandra Urioste, Lisbeth Drastrup, Susanne Kjellberg, and Jane Broennum for technical assistance, and Grete Lademann for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the Danish Medical Research Council (the Research Center for Growth and Regeneration), the University of Copenhagen, the Danish Diabetes Association, the Velux Foundation, and the European Economic Community (BMH4-CT-950662). Back

Received January 7, 1999.

Revised February 18, 1999.

Accepted February 23, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results and Discussion
 References
 

  1. Ouwens DM, Vanderzon GCM, Pronk GJ, et al. 1994 A mutant insulin-receptor induces formation of a Shc-growth factor-receptor bound protein-2 (Grb2) complex and p21(Ras)-GTP without detectable interaction of insulin-receptor substrate-1 (IRS1) with Grb2: evidence for IRS1-independent p21(Ras)-GTP formation. J Biol Chem. 269:33116–33122.[Abstract/Free Full Text]
  2. Migliaccio E, Mele S, Salcini AE, et al. 1997 Opposite effects of the p52(Shc)/p46(Shc) and p66(Shc) splicing isoforms on the EGF receptor-Map kinase-Fos signaling pathway. EMBO J. 16:706–716.[CrossRef][Medline]
  3. Okada S, Kao AW, Ceresa BP, Blaikie P, Margolis B, Pessin JE. 1997 The 66-kda Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein-kinase pathway. J Biol Chem. 272:28042–28049.[Abstract/Free Full Text]
  4. Hales CN. 1997 Non-insulin-dependent diabetes-mellitus. Br Med Bull. 53:109–122.[Abstract/Free Full Text]
  5. Phillips DIW. 1996 Insulin resistance as a programmed response to fetal undernutrition. Diabetologia. 39:1119–1122.[Medline]
  6. Poulsen, P, Vaag AA, Kyvik KO, Jensen DM, Beck-Nielsen H. 1997 Low-birth-weight is associated with NIDDM in discordant monozygotic and dizygotic twin pairs. Diabetologia. 40:439–446.[CrossRef][Medline]
  7. Gluckman PD, Harding JE. 1997 Fetal growth-retardation: underlying endocrine mechanisms and postnatal consequences. Acta Paediatr. 86:69–72.
  8. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor-I (IGF-I) and type-1 IGF receptor (IGF1R). Cell. 75:59–72.[Medline]
  9. Araki E, Lipes MA, Patti ME, et al. 1994 Alternative pathway of insulin signaling in mice with targeted disruption of the IRS-1 gene. Nature. 372:186–190.[CrossRef][Medline]
  10. Tamemoto H, Kadowaki T, Tobe K. 1994 Insulin-resistance and growth-retardation in mice lacking insulin-receptor substrate-1. Nature. 372:182–186.[CrossRef][Medline]
  11. Accili D, Drago J, Lee EJ. 1996 Early neonatal death in mice homozygous for a null allele of the insulin-receptor gene. Nat Genet. 12:106–109.[CrossRef][Medline]
  12. Joshi RL, Lamothe B, Cordonnier N. 1996 Targeted disruption of the insulin-receptor gene in the mouse results in neonatal lethality. EMBO J. 15:1542–1547.[Medline]
  13. Hattersley AT, Beards F, Ballantyne E, Appleton M, Harvey R, Ellard R. 1998 Mutations in the glucokinase gene of the fetus result in reduced birth weight. Nat Genet. 19:268–270.[CrossRef][Medline]
  14. Dunger DB, Ong KKL, Huxtable SJ, et al. 1998 Association of the INS VNTR with size at birth. Nat Genet. 19:98–100.[Medline]
  15. Almind K, Frederiksen SK, Ahlgren MG, et al. 1998 Common amino acid substitutions in insulin receptor substrate-4 are not associated with type II diabetes mellitus or insulin resistance. Diabetologia. 41:969–974.[CrossRef][Medline]
  16. Clausen JO, Borch Johnsen K, Ibsen H, et al. 1996 Insulin sensitivity index, acute insulin-response, and glucose effectiveness in a population-based sample of 380 young healthy Caucasians: analysis of the impact of gender, body-fat, physical-fitness, and life-style factors. J Clin Invest. 98:1195–1209.[Medline]
  17. Vestergaard H, Bjørbæk C, Hansen T, Larsen FS, Granner DK, Pedersen O. 1996 Impaired activity and gene-expression of hexokinase-II in muscle from non-insulin-dependent diabetes-mellitus patients. J Clin Invest. 96:2639–2645.



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