J. Endocrinol. Invest. 18: 398-405,1995
REVIEW ARTICLE
Insulin receptor gene expression and insulin resistance A. Brunetti *, and I.D. Goldfine** *Cattedra di Endocrinologia, Universita di Reggio Calabria, Catanzaro, Italy, **Division of Diabetes and Endocrine Research, Mount Zion Medical Center of the University of California and Departments of Medicine and Physiology, University of California San Francisco, San Francisco, California, USA INTRODUCTION
sulin receptor gene that may have major importance in regulating the expression of the insulin receptor in target tissues.
Non-insulin-dependent diabetes mellitus (NIDDM) is one of the more common endocrine diseases, affecting about 5-7% of the population (1, 2). Its prevalence rate is even higher in certain populations such as Hispanic-Americans, and Native Americans (3). One group, Pima Indians, has a prevalence of NIDDM approaching 50% (4, 5). In NIDDM patients there is a decreased B-cell insulin secretory response to glucose stimulation (1,6). In addition, there is resistance to insulin in key target tissues including muscle (1,4,6). Based on a variety of experimental approaches, it is clear that insulin resistance represents a prominent feature in virtually all patients with NIDDM. Studies have suggested that the insulin resistance is genetically determined, and in most instances this resistance precedes the abnormalities in insulin secretion (4). Insulin exerts its biological effects by interacting with its specific cell surface receptors (7-10). Recently, it has been estimated that a small but significant fraction of patients with NIDDM have defects in insulin receptor function and/or expression (11, 12). Insulin resistant patients with reduced or absent insulin receptor expression in target cells have been reported and many of these patients have either defects or deletions in the coding sequence of the insulin receptor gene (11,12). In this review we summarize data concerning the regulation of insulin receptor gene expression during muscle cell differentiation. In addition, we analyze the insulin receptor promoter and show the existence of two nuclear binding proteins for the in-
INSULIN RECEPTOR
Insulin regulates the general metabolism of most differentiated cells (1,8, 10, 13). In myocytes, hepatocytes, and adipocytes, the major target cells for the hormone, insulin produces a wide range of effects, including stimulation of the uptake of glucose and aminoacids, activation of a number of intracellular enzymes, and regulation of RNA and DNA synthesis (13). How insulin carries out its various cellular actions is still not well defined. The initial interaction of insulin with target cells is with the insulin receptor protein that is located on the plasma membrane. After insulin binds, the insulin receptor initiates biological responses (Fig. 1). The human insulin receptor gene has been cloned and characterized. It is located on the short arm of chromosome 19 (14). The gene is greater than 120 kilobases in length, and is comprised of 22 exons ranging from 36 to >2500 base pairs (14). The mature insulin receptor is a transmembrane glycoprotein composed of two a and two B subunits (7). The a subunits lie outside of the cell membrane and contain the binding site for insulin, whereas the B subunits contain a transmembrane domain and a cytoplasmic tyrosine kinase domain. One a and one B subunit of the insulin receptor are derived from a single precursor molecule of 1382 amino acids. After translation and N-glycosylation, the receptor precursor is transferred to the Golgi where it is split into separate subunits, the sugar residues modified, and transported to the plasma membrane as a mature heterotetrameric protein a2 B2 (7). Insulin binds to the insulin receptor a subunit, and then activates B subunit tyrosine kinase activity (15). This enzyme activity then phosphorylates tyrosine
Key-words. Differentiation. insulin resistance, transcription. gene expression, trans-acting factors. Correspondence. Antonio Brunetti. MD.. Ph.D. Cattedra di Endocrillologia, Policlinico Mater Domini, Via T. Campanella 88100, Catanzaro. Italy
398
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teins with IRS-1, leads to enhanced Ras activity by stimulating the formation of GTP-Ras complexes (16). Activation of Ras then leads to activation of a cascade of serine kinases which regulate multiple
residues on the docking protein, insulin receptor substrate 1 (I RS-1) (16). Several classes of intracellular adapter molecules then bind to phosphotyrosines on IRS-1 (16). The interaction of these pro-
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Fig. 2 - Effect of differentiation on insulin binding to insulin-receptor in (a) BC3H-1 and (b) C2 muscle cells. Cells were plated in medium containing 20% fetal bovine serum (FBS) and cultured for two days. Cells were then shifted to differentiation medium containing 1% FBS and cultured for up to 8-10 days. Specific 1251-insulin binding was expressed as the percentage of total 1251-insulm bound/10 fl9 DNA
399
Insulin resistance and NIOOM
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In addition to morphologic changes, there are changes in the content of muscle specific proteins (21, 22). BC3H-1 cultured mouse muscle cells have been studied extensively and show a 5 to 10-fold increase in insulin receptor content when myoblasts differentiate into nonfusing myocytes (Fig. 2a). BC3H-1 cells form nonfusing myocytes, and then disply certain morphological properties of both smooth muscle cells and skeletal muscle cells (20). Mouse C2 and rat L6 muscle cells form fusing myocytes. Like BC3H-1 cells, differentiation in these cells is associated with an increase in insulin receptors (Fig. 2b). Insulin receptor mRNA consists of several species of different molecular sizes (24). In BC3H-1 muscle cells, two major bands can be detected with Northern blot analysis representing the two major species of insulin receptor mRNA at 7.0 and 9.5 kilobases (Fig. 3). Differentiation into myocytes is associated with a significant increase in both species of mRNA and this increase reflects an increase in insulin receptor gene transcription (Fig. 4).
Fig. 3 - Northern blot analysis of insulin receptor mRNA Poly(A)+ RNA was purified from myoblasts and BC3H-1 myocytes and analyzed. RNA ladder was used for molecular weight standards. 1= myoblast, 2= myocyte. Kb, kilobase.
actions of insulin (16). While the elucidation of insulin action is making progress, it is not well known what regulates insulin receptor. In fact, although the insulin receptor is ubiquitously expressed, the regulatory mechanisms controlling insulin receptor gene expression are poorly understood. Data suggest that the insulin receptor can be regulated in a variety of conditions (6-10). For example, glucocorticoids enhance transcription of the insulin receptor gene, while insulin down-regulates its own receptor through internalization (17, 18). Also, the insulin receptor mRNA levels vary with differentiation, being much higher in differentiated mouse myocytes and adipocytes as compared to undifferentiated mouse myoblasts and preadipocytes (19-22). Recently, we and others have carried out a series of investigations to understand what factors regulate the insulin receptor expression during muscle cell differentiation.
Insulin receptor promoter To further understand the regulation of the insulin receptor, we and others have identified and analyzed the promoter of the human insulin receptor gene (14, 24-32) (Fig. 5). This region extends over 1800 bases 5' upstream from the insulin receptor gene ATG codon. It is extremely GC-rich and contains several GC boxes which are putative binding sites for the mammalian transcription factor Sp1 (33). The insulin receptor promoter has no readily identi-
Insulin receptor expression in muscle Muscle tissue is the predominant peripheral site of insulin action and insulin resistance in muscle is a feature of several pathological states including NIDDM and obesity (23). Therefore, studies of insulin receptor expression during muscle cell differentiation are important for providing insights into the development of insulin resistance in these and other pathological states. In muscle, insulin regulates the transport of glucose which leads to major changes in blood glucose concentrations (23), as well as a variety of other functions. Cultured mouse and rat cell lines have been employed to study the in vitro regulation of the muscle insulin receptor (7-10). These cells grow as undifferentiated myoblasts when kept at low densities in media with a high serum concentration (20). However, when confluent cells are grown in media with a low serum concentration, the cells differentiate into myocytes (20).
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Fig. 4 - Effect of muscle differentiation on insulin receptor (fR) gene transcription. Nuclei were isolated from myoblasts and BC3H-1 myocytes and nuclear run-on assays were performed. As control, the transcription of the mouse glyceraldehyde-3phosphate dehydrogenase (GAPDH) gene was also measured. 1= myoblast, 2= myocyte.
400
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control point in gene expression. Gene expression in eukaryotic cells is controlled by nuclear regulatory proteins (transacting factors) that modulate RNA polymerase II transcription of genes and gene networks (33-35). During the last decade, unique DNA sequences that are involved in gene regulation (cis elements) have been identified, and this identification process has led to the detection and characterization of DNA regulatory proteins (33). The insulin receptor is expressed at low levels in most cells and this expression is most likely due to the interaction of GGGCGGG sequences with the nuclear binding protein Sp1 (33). The insulin receptor is expressed at higher levels in differentiated target tissues such as muscle and fat, but the factors regulating its expression are unknown. In BC3H-1 muscle cells we have recently identified two DNA nuclear binding proteins that appear concomitant with differentiation, and bound to two unique AT-rich sequences of the insulin receptor promoter (-674/-874 and -1662/-1823 upstream of the insulin receptor ATG codon) (Figs. 5 and 6) (36). In vivo studies of transcription demonstrate that these two sequences of the insulin receptor gene are both functionally active (Fig. 7). These two insulin receptor DNA nuclear binding proteins are also associated with cells that readily express insulin receptors, whereas they are almost unde-
fiable TATA or CAAT boxes, reflecting the common features for the promoters of constitutively expressed genes (so-called housekeeping genes). In mammalian cells, the binding of proteins to specific DNA sequences is critical for the regulation of many nuclear events such as replication and transcription. Initiation of mRNA synthesis is a major
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Fig. 5 - (Top) Sequence of the 5' flanking region of the insulin receptor gene and exon 1. Nucleotide numbering is relatIVe to the first nucleotide of the A TG codon. Regions of the gene which are putative binding sites for the transcription factor Sp 1 are underlined. (Bottom) Restriction map of the insulin receptor gene 5' region showing fragments used In gel retardation assays. Regions of the gene which were used as probe in this study are indicate by A, B, C, 0, E Regions that demonstrated binding actIVity are indicated by solid bars. Sites of endonuclease cleavage are shown. The smallest probes that interact with DNA binding proteins are indicated by C2 and E3.
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Fig. 6 - Gel retardation assays performed with fragments E3 and C2 in nuclear extracts of BC3H-1 muscle cells. 4 ~g of extracts were incubated with E3 and C2 probes and DNA-protein complexes were resolved on a non-denaturing polyacrylamide gel. Arrows show the position of the DNA-protein complexes. 1= control, 2= myoblast; 3= myocyte.
401
Insulin resistance and NIOOM
Table 1 - Insulin receptor concentration and DNA binding activity to E3 and C2 in other cell types.
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Fig. 7 - Functional analysis of fragments C2 and E3 of the insulin receptor gene. Recombinant plasmids containing C2 or E3 sequences, either in the sense or antisense orientation, were transfected into HEP-G2 cells and chloramphenicol acetyltransferase (CA T) activity was measured. (Top) Autoradiogram illustrating CA T activity associated with each constructs after TLC separation. (Botton) CA T activity was assayed by liquid scintillation counting (LSC) of the two major acetylated forms of 14C-labeled chloramphenicol in the upper xylene phase. Lanes: 1=Basic vector without an insert; 2=C2-containing vector;3= E3-containing vector; 4= C2 antisense-containing vector, 5= E3 antisense-containing vector; 6= a positive control having the simian virus 40 gene promoter upstream of the CA T.
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Decre~lsed
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As mentioned above, the mechanism of insulin action involves multiple steps. Defects in any of these steps may give rise to insulin resistance. In as much as the insulin receptor is responsible for mediating the first step in insulin action, the gene encoding the insulin receptor has been intensively investigated and several types of mutations in the insulin receptor gene have been identified in patients with genetic syndromes of insulin resistance. Figure 8 shows a classification of mutations of the insulin re-
Decreased tyrosine kinase activity
Increased receptor degradation
r-.-X X X X X
402
Fig. 8 - Classification of mutations in the insulin receptor gene Modified from Brown & Goldstein Science, 232, 34, 1986
A. Brunetti and 1.0. Goldfine
sion in patients with insulin resistance, NIDDM, and decreased levels of insulin receptor mRNA, could be caused by a defect in the expression of these nuclear binding proteins. We have preliminary data indicating that in cells from certain insulin resistant patients, the expression of nuclear binding proteins for the regulatory region of the insulin receptor gene is markedly reduced. Using both C2 and E3 sequences of the insulin receptor gene, we found a decreased amount of nuclear proteins for these regulatory regions in EBV-transformed Iymphoblasts from two unrelated insulin resistant patients (Table 2 and Fig. 9). One patient had extreme insulin resistance with acanthosis nigricans (Type A insulin resistance). However, the other patient had features of a typical patient with NIDDM. The decrease in DNA binding activity in patients' cells paralleled the decrease in both insulin receptor mRNA abundance and 1251-insulin binding on intact EBV-transformed Iymphoblasts (Fig. 9).
Table 2 - Clinical data of patients. PATIENT 1 Age (yr) Sex Plasma glucose (mg/dl) fasting 2 h after 75 g glucose Plasma insulin (mU/ml) fasting 2 h after 75 g glucose AcanthosIs nlgricans
PATIENT 2
11 Male
45 Male
100 310
250 NO
300 1845 Yes
34 NO No
NO= not determined.
ceptor gene. According to their biochemical phenotype, five classes of insulin receptor mutations have been described: Class 1 (impaired insulin receptor mRNA synthesis) (37, 38); Class 2 (impaired transport of receptors to the cell surface) (39); Class 3 (decreased insulin binding) (40); Class 4 (defective tyrosine kinase) (41); Class 5 (increased receptor degradation) (42). Patients with apparently normal insulin receptor genes and reduced expression of the insulin receptor have been described (43-12, 43, 44). In these patients, indirect evidence strongly suggest the existence of a molecular defect in the insulin receptor gene that act by decreasing insulin receptor mRNA levels (43-2,43,44). Since we have identified nuclear binding proteins for the regulatory region of the insulin receptor gene, we are investigating whether the defects in insulin receptor expres-
DNA-binding activity
CONCLUSIONS
Insulin receptor is regulated by muscle differentiation. The molecular mechanisms by which insulin receptor gene expression is regulated in muscle involve a factor that binds to AT-rich sequences of the regulatory region of the insulin receptor gene. This factor is induced on differentiation of the musclelike BC3H-1 cell. Interaction of this factor with upstream sequences of the insulin receptor gene activates insulin receptor gene transcription and stimulates insulin receptor protein expression.
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6
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Patient 2
Fig. 9 - Comparison of C2 and E3 DNA binding actlvtty to insulin receptor mRNA levels and 1251-lnsulin binding in human EBV-transformed Iymphoblasts Values of insulin receptor DNA bindtng activity and mRNA levels represent arbttrary Units as measured by laser densitometry. Specific 1251-insulin binding is expressed as percent of total/l0 7 cells.
Insulin resistance and NIOOM
12. Taylor S.I., CamaA, Accili D., Barbetti F., Quon M.J., Sierra M.L, Suzuki Y., Koller E., Levy Toledano R. , Wertheimer E., Moncada V.Y., Kadovahi H., Kadovaki T. Mutations in the insulin receptor gene. Endocr. Rev. 13: 566,1992.
Considerable progress has been made in understanding the role of receptor defects in syndromes of extreme insulin resistance. Nevertheless, the molecular causes of most cases of NIDDM remain to be elucidated. Our observations suggest that defects in the expression of nuclear proteins that regulate insulin receptor gene transcription may induce insulin resistance in affected individuals. Further studies will be necessary to determine the prevalence of these nuclear binding protein abnormalities in a general population of NIDDM patients.
13. Goldfine ID. Effects of insulin on intracellular functions. Litwach In: G.(Ed), Biochemical Actions of Hormones. Academic Press, Inc. New York , 1981 , vol. 8, p. 272. 14. Seino S , Seino M , Nishi S, Bell G.I. Structure of the human insulin receptor gene and characterization of its promoter. Proc. Natl. Acad. Sci. USA 86: 114, 1989.
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35. Yamamoto K.R. Steroid receptor regulated transcription of specific genes and gene networks. Ann. Rev. Genet. 19.' 209, 1985. 36. Brunetti A. , Foti D., Goldfine 1.0. Identification of unique nuclear regulatory proteins for the insulin receptor gene, which appear during myocyte and adypocyte differentiation. J. Clin. Invest. 92' 1288, 1993. 37. Kadovaki T., Kadovaki H., Rechler M.M., SerranoRios M. , Roth J., Gorden P , Taylor S.I. Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. J. Clin. Invest. 86.254, 1990. 38. Shimada F., Taira M, Suzuki Y , Hashimoto N. , Nozaki 0., Taira M., Tatibana M., Ebina Y, Tawata M. , Onaya T. , Makino H., Yoshida S. Insulin-resistant diabetes associated with p::>rtial deletion of insulinreceptor gene. Lancet 335. 1179, 1990. 39. Kadovaki T., Kadovaki H., Accili D., Taylor SJ Subsitution of lysine for asparagine-15 in the human insulin receptor impairs intracellular transport of the receptor to the cell surface and decreases the affinity of insulin binding. J. BioI. Chem. 265: 19143, 1990. 40. Yoshimasa Y., Seino S., Whittaker J., Kakehi T., Kosaki A., Kuzuya H., Imura 1., Bell G.I., Steiner D.F Insulin-resistant diabetes due to a pOint mutation that prevents insulin proreceptor processing. Science 240.' 784, 1988. 41. Odawara M., Kadovaki T. , Yamamoto R. , Shibasaki Y. , Tobe K., Accili D., Bevins C, Mikami Y., Matsuura N., Akanuma Y. , Takaku F. , Taylor S.I., Kasuga M. Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor. Science 245. 66, 1989. 42. Taylor S.I., Kadovaki T., Kadovaki H. , Accili D., Cama A., McKeon C. Mutations in insulin-receptor gene in insulin-resistant patients. Diabetes Care 13.' 257,1990. 43. Imano E. , Kadowaki H. , Kadowaki T. , Iwama N. , Watarai T., Kawamori R., Kamada T. , and Taylor S.I. Two patients with insulin resistance due to decreased levels of insulin-receptor mRNA. Diabetes. 40.548, 1990. 44. Ojamaa K., Hedo J.A., Roberts CT, Gorden P , Ullrich A., Taylor S.I. Defects in human insulin receptor gene expression. Mol. Endocrinol. 2: 242,1 988.
