Mutations of the human insulin receptor gene

BRIEF REVIEWS Mutations of the Human Insulin Receptor Gene

tion (Ellis et a1.1986; Ebina et al. 1987).

Simeon I. Taylor, Alessandro Cama, Hiroko Kadowaki, Takashi Kadowaki, and Domenico Accili

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Mutations in the insulin receptor gene have been identified in patients with genetic forms of insulin resistance. These mutations provide insight into structure-function relationships of the insulin receptor, and also into the causes of insulin resistance in human disease.

Genetic diseases have the potential to provide insight into physiology and biochemistry. The study of inborn errors causing human disease has elucidated both disease mechanisms and normal physiology. For example, investigations of sickle cell disease and thalassemia have shed light upon structure-function relationships of the hemoglobin molecule and the globin genes. In addition, investigations of mutations in low-density lipoprotein (LDL) receptor genes of patients with familial hypercholesterolemia have given enormous insight into the biochemistry and cell biology of LDL transport into cells. In this brief review, we summarize recent studies into mutations in the insulin receptor gene in patients with genetic forms of insulin resistance .

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Structure

of the Insulin

Receptor

The insulin receptor gene encodes a single polypeptide which undergoes Nlinked glycosylation to yield a 190-kDa precursor of the insulin receptor (Ebina et al. 1985; Ullrich et al. 1985). This undergoes additional post-translational processing to yield the mature receptor. Simeon I. Taylor, Alessandro Cama, Hiroko Kadowaki, Takashi Kadowaki, and Domenice Accili are in the Biochemistry and Molecular Pathophysiology Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

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First, the precursor undergoes proteolytic cleavage into two separate subunits (Hedoet al. 1983). Second, the high mannose form of N-linked carbohydrate undergoes maturation with removal of mannose and glucose residues and addition of other sugars, including sialic acid (Hedo et al. 1981). Other posttranslational modifications occur, including fatty acylation (Hedo et al. 1987) and O-linked glycosylation (Herzberg et al. 1985; Collier and Gorden 1989). Thus, the mature insulin receptor consists of two different types of subunits. The a! subunit is entirely extracellular, and provides the binding site for insulin (Ullrich et al. 1985). The fi subunit contains the transmembrane domain that anchors the receptor in the plasma membrane, and possesses enzymatic activity as a tyrosine-specific protein kinase (Kasuga et al. 1982; Ullrich et al. 1985). (Figure 1). a-P dimers further dimerize to produce a heterotetrameric Q& species. When insulin binds to the extracellular domain of the receptor, this activates the receptor tyrosine kinase activity. A growing body of evidence supports the hypothesis that activation of the tyrosine kinase plays a necessary role in mediating insulin action upon the target cell (Rosen 1987). For example, by the technique of sitedirected mutagenesis, mutant insulin receptors have been constructed that lack tyrosine kinase activity and also lack the ability to mediate insulin ac-

Chou et al. 1987;

Genetic Syndromes Associated Extreme Insulin Resistance

with

In insulin-resistant patients, there is a diminished response to insulin despite the delivery of normal or supranormal levels of bioactive insulin to the target cell. Insulin-resistant patients usually have increased levels of insulin in their plasma. Often, the increase in insulin levels is sufficient to maintain relatively normal glucose levels in plasma, although some patients develop hyperglycemia and diabetes. Interestingly, extreme insulin resistance is usually associated with acanthosis nigricans-a hyperpigmented, hyperkeratotic skin rash. In addition, premenopausal women with extreme insulin resistance are often masculinized as a result of increased ovarian production of testosterone. It has been suggested that the acanthosis nigricans and masculinization are caused by toxic effects of the elevated levels of insulin upon the skin and ovary, respectively. In addition to these two clinical features that are common to all of the syndromes of extreme insulin resistance, other features are specific for particular syndromes-e.g., intrauterine growth retardation in leprechaunism or absence of subcutaneous fat in lipoatrophic diabetes (Taylor 1985 and 1987). We have investigated this group of insulin-resistant patients and identified individuals in whom insulin resistance is caused by mutations in the insulin receptor gene. ??

Mutations Gene

in the Insulin

Receptor

Mutations can occur in either the structural gene encoding the insulin receptor or in the regulatory domains that determine the level of gene expression. Thus far, all of the reported mutations have been identified in the structural gene (Figure 1). These mutations impair insulin action by several mechanisms: 1)

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Decreased number of insulin receptors on the surface of target cells

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1. Mutations in the insulin receptor gene in insulin resistant patients. A structural map of the insulin receptor. Key structural landmarks are identified in the left half of the

Figure

receptor. The locations of mutations the drawing of the receptor.

causing

a) Decreased

levels of insulin receptor mRNA b) Impaired transport of receptors to the cell surface c) Nonsense mutation d) Accelerated receptor degradation (impaired receptor recycling) Decreased affinity of receptor to bind insulin Decreased receptor-associated tyrosine kinase activity

Decreased Number of Insulin Receptors on the Suvface of the Target Cells In some patients, insulin resistance results from a decrease in the number of insulin receptors on the cell surface (Taylor 1985 and 1987). Because these patients have elevated levels of insulin in plasma, it is possible that the hyperinsulinemia could cause a secondary decrease in insulin binding through a known as down-regulation. process However, the binding defect is preserved in lymphoblastoid cells that have been immortalized by transformation with Epstein-Barr virus (EBV). Because EBV-transformed lymphoblasts are cultivated in vitro under standard conditions in the absence of insulin, this suggests that the decrease in the number of

TEM JanuurvlFrbruary

insulin

resistance

are noted in the right half of

insulin receptors is caused by a primary defect intrinsic to the cell. Despite the common biochemical phenotype observed in this group of patients, at least four distinct molecular defects have been identified in patients with a decreased number of receptors on the surface of patients’ cells (see the preceding list). Decreased Levels of Insulin Receptor mRNA In leprechaun/Minn-1, the number of insulin receptors on the surface of EBV-transformed lymphoblasts is decreased to < 10% of normal (Taylor 1985 and 1987). Furthermore, the rate of receptor biosynthesis is decreased. Thus, the decrease in the number of insulin receptors is caused by a decrease in the rate of receptor biosynthesis. Because insulin receptor mRNA is necessary to provide a template for receptor biosynthesis, we measured the level of insulin receptor mRNA (Figure 2). As originally shown by Ullrich et al. (1985), there are at least five species of human insulin receptor mRNA that vary in size from -5.2 to 10.5 Kb in length. All five species of mRNA appear to encode the same protein sequence, and differ primarily with respect to the length of 3’untranslated RNA. In cells from lepre-

0 1990,

Elsevier

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de-

of insulin

receptor mRNA (Figure 2C) (Ojamaa et al. 1988). Studies are under way to identify the mutation(s) responsible for the decrease in insulin receptor mRNA. Several possible explanations need to be considered: decreased rate of transcription, impaired splicing of RNA, nonsense mutation, or accelerated degradation of mRNA.

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Impaired Receptor Transport to the Cell Surface We have studied two sisters with type A extreme insulin resistance (patients A-5 and A-8) in whom there is an 80-90% decrease in the number of insulin receptors on the cell surface (Taylor 1987). However, unlike what was observed in leprechaun/Minn-1 (see above), there are normal levels of insulin receptor mRNA in EBV-transformed lymphoblasts from patients A-5 and A-8 (Figure 2B). Moreover, the rate of receptor biosynthesis is normal. Why, then, is there a decrease in the number of receptors on the cell surface? In principle, this might result from an accelerated rate of receptor degradation. However, when cell surface receptors were labeled by lactoperoxidase-catalyzed radioiodination, the half-life of the insulin receptors on the cell surface was within normal limits (t,,, = 4.8 h; normal range = 6.5 * 2.4 h, mean -t 2 SD). Thus, we hypothesized that there is a defect in the transport of the receptor to the plasma membrane. A similar defect in the transport of receptors to the cell surface has been described with LDL receptors in the Watanabe heritable hyperlipidemic (WHHL) rabbit and some patients with familial hypercholesterolemia (Yamamoto et al. 1986; Esser and Russell 1988). In those cases, mutations were detected in the structural gene encoding the LDL receptor. The mutant LDL receptors were impaired in their ability to be transported to the cell surface. Inspired by this analogy, we cloned insulin receptor cDNA from patient A-8, and identified a mutation encoding substitution of valine for phenylalanine at position 382 in the a-subunit (Figure 1) (Accili et al. 1989). Two lines of evidence support the conclusion that this mutation is the cause of the patients’ disease (Accili et al. 1989). First, the Va13x2 mutation segregates together with insulin resistant diabetes, suggesting that the mutation causing

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the decrease in the number 01 insulin receptors on the surface 01 his cells. Corrsistent with this conclusion, [ ‘2iI]insulin binding to the father’s circulating monocytes is decreased by 60-70%.

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Figure 2. Northern blot analysis of insulin receptor mRNA levels in cells from patients with decreased numbers of insulin receptors on the cell surface. Polyadenylated RNA from EBVtransformed lymphoblasts was analyzed by Northern blotting probed with either human insulin receptor (upper panels) or chicken p-actin (lower panels) probes (Ojamaa et al. 1988). Panel B demonstrates that cells from patients A-5 and A-8 (lanes 3 and 4) have normal levels of lanes 1 and 2 with the of insulin receptor mRNA. (Panel A shows the autoradiograph exposure time reduced from 3 days to 1 day in order to allow for better demonstration of the insulin receptor mRNA bands.) Panel C shows a marked reduction in insulin receptor mRNA in IeprechauniMinn-1 (lane 7) as compared with the normal subjects (lanes 8-10).

the disease is genetically linked to the insulin receptor locus. Second, when cDNA encoding the Va1382 mutant receptor was transfected into NIH-3T3 cells, this showed that the Va1382 mutation impairs transport of the insulin receptor to the cell surface. Interestingly, the impairment in intracellular transport is associated with a defect in posttranslational processing of the carbohydrate moiety of the insulin receptor. Normally, the high-mannose form of Nlinked carbohydrate undergoes rapid processing to complex carbohydrate within the Golgi apparatus. However, the receptor with the Va1382 mutation appears not to be transported normally from the endoplasmic reticulum, so that there is a defect in the processing steps that require enzymes present in the Golgi. A mutation in the CSF-1 (colonystimulating factor-l) receptor gene has been reported to cause a similar type of defect in posttranslational processing of N-linked carbohydrate and transport of receptors to the cell surface. Interestingly, this mutation in the CSF-1 receptor is thought to play a key role in activating the protooncogene encoding the CSF-1 receptor and converting it to the v-fms oncogene (Roussel et al. 1988).

Nonsense another

136

Mutation

patient

with

We have studied leprechaunism

(leprechaun/Ark-l) in whom we have identified two mutant alleles of the insulin receptor gene (Kadowaki et al. 1988). In the allele inherited from the father, there is a nonsense mutation substituting a chain termination codon (UAG) for the CAG codon normally encoding Gln672. This premature chain termination leads to deletion of the C-terminus of the o-subunit and the entire P-subunit, including the transmembrane anchor and the tyrosine kinase domain. Lacking a transmembrane anchor, the truncated receptor is not located at the cell surface. In leprechaun/Ark- 1, the allele of the insulin receptor gene with the nonsense mutation is paired with an allele containing a different mutation (see below) (Kadowaki et al. 1988). However, the father is also heterozygous for the allele with the nonsense mutation. The father is also insulin resistant, although not as severely insulin resistant as his daughter. Thus, the nonsense mutation causes insulin resistance in a codominant fashion. This suggests that the father’s second allele does not increase the level of expression to compensate for the nonsense mutation. In fact, because the father’s insulin levels are elevated five- to tenfold above the normal range, it is likely that his receptors become downregulated in vivo, thereby exacerbating

Accelerated Receptor Deggmdution In the same patient with leprechaunism (leprechaun/Ark-l) in whom we identified the nonsense mutation (see above), there is a second mutant allele containing a missense mutation that encodes the substitution of glutamic acid for lysine at position 460 in the a-subunit of the insulin receptor. This mutation, inherited from the patient’s mother, is recessive in that the mother has normal glucose tolerance and does not appear to be insulin resistant. Prior to the cloning, we had identified multiple abnormalities in insulin binding to receptors on the surface of the patient’s EBV-transformed lymphoblasts, including decreased sensitivity to changes in temperature and pH (Taylor et al. 1981). The patient’s receptor has a fivefold increase in binding affinity at physiological temperature (37” C) and pH (pH 7.4). Furthermore, insulin stimulates the receptor-associated tyrosine kinase normally in receptors from the patient’s EBVtransformed lymphoblasts. How does the GIu~~’mutation impair receptor function to cause insulin resistance in vivo? An analogy to a site-directed mutant of the LDL receptor suggests an answer (Davis et al. 1987). When the portion of the extracellular domain of the LDL receptor homologous to the epidermal growth factor (EGF) precursor molecule is deleted, this causes LDL binding to become insensitive to changes in pH. After LDL receptors bind LDL, the ligand-receptor complex is internalized into endocytotic vesicles (Figure 3). These endocytotic vesicles develop an acidic pH within their lumens. This acidic pH plays a crucial role in dissociating the ligand from its receptor. Subsequent to internalization, at least two distinct pathways are available to the receptor: recycling to the cell surface for reutilization or degradation within the lysosomes. In the case of the deletion mutant of the LDL receptor, desensitization to changes in pH was associated with inhibition of the recycling pathway. When ligand bound to the receptor, it was not dissociated from the receptor in the endocytotic vesicle. The receptor was targeted preferentially for degradation within the lysosome.

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Figure 3. Pathway of receptor-mediated endocytosis and receptor recycling. After insulin binds to its receptor, the hormone-receptor complex undergoes endocytosis. Because of the presence of proton pumps in their membranes, the endosomes acquire an acidic pH in their lumens. Once within the cell, two alternative fates are available to the ligand and its receptor. They can be targeted to the lysosome where they will be degraded. Alternatively, the receptor can be recycled back to the plasma membrane for reutilization. If the ligand escapes degradation, it can be retroendocytosed intact into the extracellular fluid.

With normal insulin receptors (LYs~~‘), decreasing the pH from 7.8 to 6.0 causes a tenfold acceleration in the rate at which [‘251]insulin dissociates from its receptor. The effect of acid pH to accelerate [‘251]insulin dissociation is markedly blunted with the G1u460 mutant receptor. In analogy to the deletion mutant of the LDL receptor (see above), it seems likely that the G1u460 mutation causes insulin resistance by accelerating the rate of receptor degradation. According to this hypothesis, the cause of insulin resistance is a decrease in the number of insulin receptors on the surface of target cells which results from an accelerated rate of receptor degradation.

affinity causes insulin resistance because, at any concentration of insulin in the physiological range, fewer insulin molecules will bind to the receptor. Recently, another mutation has been identified in the insulin receptor of leprechaun/Geldermalsen: substitution of proline for leucine at position 233 in the (Ysubunit (Klinkhamer et al. 1989). The patient, who is part of a consanguineous pedigree, is homozygous for the Pro233 mutation. Although this mutation is associated with a marked decrease in insulin binding to the surface of the patient’s cells, the mechanism of the decrease in binding has not been elucidated. 3. Decreased Receptor-Associated

2. Decreased Afinity

of the Receptor to

Bind Insulin Two sisters with type A extreme

insulin

resistance have been described who are homozygous for a mutation that prevents proteolytic cleavage of the receptor precursor into two separate subunits (Yoshimasa et al. 1988; Kobayashi et al. 1988). This mutation substitutes serine for arginine at position 735, the fourth amino acid in the tetrabasic amino acid sequence (Arg-Lys-Arg-Arg) in the proteolytic processing site. In the patients’ cultured cells, the uncleaved receptor has a decreased affinity to bind insulin (Kakehi et al. 1988; Kobayashi et al. 1988). The reduction of the binding

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Tyrosine Kinase Activity Several patients have been described in whom insulin resistance is associated with defects in the insulin receptor tyrosine kinase activity (Grunberger et al. 1984; Grigorescu et al. 1984). The fact that several different naturally occurring mutations have been identified that impair tyrosine kinase activity provides additional strong support for the central role of tyrosine kinase activity in mediating the physiological actions of insulin in vivo. We have cloned insulin receptor cDNA from one such patient (Odawara et al. 1989). In that patient, we identified a missense mutation which encodes sub-

stitution of valine for G~Y’~“, the third glycine in the Gly-X-Gly-X-X-Gly motif that is part of the putative ATP binding site (Hanks et al. 1988). By transfecting mutant insulin receptor cDNA into CHO cells, we have shown that the Va11m8 mutation essentially abolishes tyrosine kinase activity. The patient’s other allele of the insulin receptor gene has the normal sequence in this region. Because the patient’s other allele has not been fully sequenced, however, we do not yet know whether it is normal or has a second mutation. Thus, we do not know whether insulin resistance caused by the Val*““s mutation has a dominant or recessive pattern of inheritance. Moller and Flier (1988) reported a variant sequence that substitutes serine for Trp “O”in the P-subunit. This variant sequence is found in only one allele of the patient’s insulin receptor gene; the other allele is normal in this region. Because tryptophan is a highly conserved amino acid in the homologous region of other tyrosine kinases (Hanks et al. 1988), it seems likely that the Serizoo substitution inhibits tyrosine kinase activity. Finally, two patients (a mother and daughter) have been described who have chromosomal deletions that disrupt the portion of the insulin receptor gene encoding the p-subunit (Taira et al. 1989). Because the patients are heterozygous for this deletion, the mutation appears to cause insulin resistance in a dominant fashion. (This implicitly assumes that the nucleotide sequence of the second allele is normal in these patients although this has not been directly demonstrated.) Of course, in evaluating the significance of deletions of large portions of the receptor, multiple receptor functions may be compromised in addition to the defect in tyrosine kinase activity. ??

Is Insulin Resistance in Patients with Non-Insulin-Dependent Diabetes Mellitus Caused by Mutations in the Insulin Receptor Gene?

In light of the central role of insulin resistance in predisposing to development of non-insulin-dependent diabetes mellitus (NIDDM) (Reaven 1988), it is reasonable to inquire whether patients with NIDDM may have mutations in the insulin receptor gene. In favor of this hypothesis, some patients with extreme

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137

insulin resistance

have had relatives

in

whom heterozygosity for mutations in the insulin receptor gene is associated with a moderate degree of insulin resistance. For example, the father of leprechaun/Ark-l, who is heterozygous for a nonsense mutation in the insulin receptor gene, has a moderate degree of insulin resistance comparable to what is observed in patients with NIDDM (Kadowaki et al. 1988). Furthermore, the mutations detected thus far in the insulin receptor gene have all caused major defects in the function of the insulin receptor. Patients with a milder degree of insulin resistance might have mutations that cause less severe disruption of the function of the insulin receptor. Analyses of restriction fragment length polymorphisms (RFLPs) have yielded equivocal results (Elbein et al. 1986; McClain et al. 1988). However, there are many reasons that this approach might fail to detect association of the disease with the insulin receptor gene. For example, there may be more than one mutation causing insulin resistance in the diabetic population, and these mutations may not all be in linkage disequilibrium with the same RFLP. If this were true, population studies would not detect strong association of NIDDM with a particular RFLP. Furthermore, it may be difficult to demonstrate linkage of NIDDM to a particular gene even in studies of the inheritance of RFLPs in families. For example, if development of NIDDM requires simultaneous mutations at more than one locus, linkage may be difficult to detect unless all of the relevant loci are analyzed simultaneously. Accordingly, we have embarked upon a more direct approach-to determine the nucleotide sequence of the insulin receptor gene in diabetic patients. Thus far, we have studied one insulin-resistant Pima Indian in a family with a high prevalence of NIDDM (Cama et al. 1989). Both alleles of his insulin receptor gene encode

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The mechanism by which insulin elicits its multiple biological responses in target cells is extremely complex. Insulin has multiple effects upon multiple different target cells. For target cells to respond to insulin, this requires the function of many proteins encoded by many genes. At least in theory, each of these genes is a candidate to be the locus of a mutation causing insulin resistance in NIDDM. As progress is made in the identification and cloning of the many genes that allow for the normal response to insulin, it seems likely that it will be possible to identify the loci of the mutations that cause insulin resistance in NIDDM.

References Accili D, Frapier C, Mosthaf L, et al.: A mutation in the insulin receptor gene which impairs transport of the receptor to the plasma membrane and causes insulin resistant diabetes. EMBO J 1989; 8:2509. Cama A, Patterson A, Kadowaki T, Lillioja S, Roth J, Taylor SI: Cloning of insulin receptor cDNA from an insulin resistant patient. In Proceedings of the 75th Annual Meeting of the Endocrine Society, Seattle, WA, 1989, p 211 (Abstract 756). Chou CK, Dull TJ, Russell DS, Gherzi R, Lebwohl D, Ullrich A, Rosen OM: Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem 1987; 262:1842. Collier E, Gorden P: The insulin receptor contains O-linked oligosaccharide (Abstract). Diabetes 1989; 38 (suppl. 2): 178A. Davis CG, Goldstein JL, Sudhof TC, Anderson RG, Russell DW, Brown MS: Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 1987; 326:760. Ebina Y, Ellis L, Jarnagin K, et al.: The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell 1985; 40:747. Ebina Y, Araki E, Taira M, et al.: Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity. Proc Nat1 Acad Sci USA 1987; 84:704. Elbein SC, Corsetti L, Ullrich A, Permutt MA: Multiple restriction fragment length polymorphisms at the insulin receptor locus: a

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highly informative

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of the gene have not yet been clearly delineated, so that it is more difficult to address this question at the present time.

marker tar linkage anal-

ysis. Proc Nat1 Acad Sci USA 1986: 83:5223. Ellis L, Clauser E, Morgan DO, Edery M, Roth RA, Rutter WJ: Replacement of insulin n-. ceptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinasr activity and uptake of 2-deoxyglucosc. Cell 1986; 45:72 1. Esser V, Russell DW: Mutational analysis of the ligand binding domain of the low density lipoprotein receptor. J Biol Chem 1988; 263~13276. Grigorescu F, Flier JS, Kahn CR: Defect in insulin receptor phosphorylation in erythrocytes and fibroblasts associated with severe insulin resistance. J Biol Chem 1984; 259: 15003. Grunberger G, Zick Y, Gorden P: Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding. Science 1984; 223:932. Hanks SK, Quinn AM, Hunter T: The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988; 241:42. Hedo JA, Kahn CR, Hayashi M, Yamada KM, Kasuga M: Biosynthesis and glycosylation of the insulin receptor: evidence for a single polypeptide precursor of the two major subunits. J Biol Chem 1983; 258:10020. Hedo JA, Kasuga M, VanObberghen E, Roth J, Kahn CR: Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity. Proc Nat1 Acad Sci USA 1981; 78:4791. Hedo JA, Collier E, Watkinson A: Myristyl and palmityl acylation of the insulin receptor. J Biol Chem 1987; 262:954. Herzberg VL, Grigorescu F, Edge ASB, Spiro RG, Kahn CR: Characterization of insulin receptor carbohydrate by comparison of chemical and enzymatic deglycosylation. Biochem Biophys Res Commun 1985; 129:789. Kadowaki T, Bevins CL, Cama A: Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science 1988; 2401787. Kakehi T, Hisatomi A, Kuzuya H, et al.: Defective processing of insulin-receptor precursor in cultured lymphocytes from a patient with extreme insulin resistance.J Clin Invest 1988; 81:2020. Kasuga M, Karlsson FA, Kahn CR: Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science 1982; 215:185. Klinkhamer M, Groen NA, Van der Zon GCM, et al: A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance. EMBO J 1989; 8:2053. Kobayashi M, Sasaoka T, Takata Y, Hisatomi A, Shigeta Y: Insulin resistance by uncleaved insulin proreceptor. Emergence of

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site

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Clinical Applications of Somatostatin Analogs Steven W. J. Lamberts, Eric P. Krenning, Jan G. M. Klijn, and Jean-Claude Reubi The somatostatin analog Sandostatin is successfully used in the treatment of metastatic endocrine pancreatic tumors, carcinoids, and acromegaly. In addition, somatostatin receptors are also present on other tumors in man, therefore making it possible to demonstrate these tumors by the administration of ‘231-coupled to a somatostatin analog.

Growth hormone (GH) release-inhibiting hormone, also called somatostatin, is a cyclic peptide consisting of 14 amino acids (Brazeau et al. 1973). This peptide Steven W. J. Lamberts is at the Department of Medicine and Eric P. Krenning is at the Departments of Medicine and Nuclear Medicine at Erasmus University, Rotterdam, The Netherlands. Jan G.M. Klijn is at the Dr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands. Jean-Claude Reubi is at the Sandoz Research Institute, Berne, Switzerland.

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secretion,

Science

Publishing

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like insulin,

the secretion glucagon,

cretin, gastrin, and vasoactive

se-

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polypeptide. In these different activities, somatostatin acts as a neurohormone, a neurotransmitter, as a local factor acting via autocrine or paracrine mechanisms, or even as a lumone (a hormone secreted into the lumen of the intestines). Reichlin (1983a and b) described the phylogeny, the anatomic distribution, the mechanism of action and the functional significance of somatostatin in an excellent review. In view of its ability to inhibit such a variety of physiological processes, somatostatin was expected to be of therapeutic value in clinical conditions involving hyperfunction of the systems mentioned above (Guillemin 1978). However, the multiple simultaneous effects of pharmacological concentrations of somatostatin in different organs, the need for intravenous administration, its short half-life in the circulation, and the postinfusion rebound hypersecretion of hormones considerably hampered its successful clinical use. Many investigators and drug companies therefore have extensively looked for long-acting somatostatin analogs. The Sandoz Company in Basel, Switzerland (Bauer 1982), finally succeeded in preparing an analog, code-named SMS 20 l-995 [Sandostatin; octreotide; H-[D]-Phe-Cys-Phe[D]Trp-Lys-Thr-Cys-Thr(ol)], which has the characteristics required for a clinically usable drug: (a) it inhibits GH preferentially over insulin; (b) it is active after subcutaneous administration; (c) it has a long half-life in the circulation (about 2 h), causing a prolonged inhibitory effect in target organs of somatostatin; and (d) the administration of the drug is not followed by rebound hypersecretion (Lamberts 1985a). In Figure 1 is an example of the effect of a single

is present and plays an inhibitory role in the normal regulation of three organ systems in man and other species: (a) the central nervous system, the hypothalamus, and the pituitary gland; (b) the gastrointestinal tract; and (c) the exocrine and endocrine pancreas. Apart from the inhibition of GH and thyrotropin (TSH) secretion, somatostatin also inhibits a variety of other physiological functions like gastrointestinal motility, gastric acid secretion, pancreatic enzyme secretion, bile, and colonic fluid

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subcutaneous injection of 50 pg Sandostatin on circulating GH levels of an acromegalic patient, in comparison to a placebo: plasma GH concentrations are suppressed for about 8h, after which they return slowly to control values, while the paradoxical GH response to thyrotropin-releasing hormone (TRH) is attenuated. ?? Acromegaly

Large numbers of somatostatin receptors are present in most GH-secreting

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