Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.     

Unique Features of the Insulin Receptor in Rat Brain

We examined the structure of the affinity-labeled insulin receptors in rat brain, rat liver, and human IM-9 lymphocytes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In gels run under reducing conditions, the alpha-subunit of the insulin receptor in brain had an apparent Mr of 127,000 distinctly lower than that seen in both rat liver and human lymphocytes (apparent Mr = 136,000). Exposure to neuraminidase increased the electrophoretic mobility of the liver receptor, but had no effect on the insulin receptor in brain. The carbohydrate moieties of the insulin receptors in rat brain and liver were further examined by chromatography on wheat-germ agglutinin agarose. The receptors in both tissues adsorbed to the wheat-germ agglutinin; elution with 0.3 M N-acetyl glucosamine resulted in slightly better recovery of the brain than of the liver receptor. Exposure to neuraminidase virtually abolished the interaction of the liver receptor with the lectin, whereas adsorption of the brain receptor was unaffected by neuraminidase. These results indicate that the insulin receptor in brain is distinguished from those in peripheral tissues by structural alterations, including changes in the carbohydrate moiety of the receptor. Such alterations contrast sharply with the previously observed similarities in insulin binding properties between insulin receptors in brain and other tissues. The implications of such structural alterations for the program of insulin action expressed by the receptors in brain remain to be explored.     

Changes of insulin binding in rat tissues after exposure to stress.

The effects of various stressors on insulin receptors in adipose, liver and skeletal muscle tissues were studied in rats exposed to acute or repeated stress. Adult male rats were exposed to immobilization (IMO) for 2.5 hours daily for 1, 7 and 42 days, or to hypokinesia (HK) for 1, 7 and 21 days. We determined the values of specific insulin binding (SIB) and insulin receptor binding capacity (IR) of plasma cell membranes from adipose, liver and muscle tissue (IMO groups), or insulin binding to isolated adipocytes and hepatocytes (HK groups). A significant decrease of SIB and IR was observed in rats exposed to acute stress (1x IMO) in muscle, adipose and liver tissues. However, in animals exposed to repeated stress (7x and 42x IMO), SIB and IR were diminished in the muscle tissue, whereas no significant changes were noted in the liver and adipose tissue. When tissue samples were collected 3-24 hours after exposure to IMO stress, no changes of SIB and IR were found in liver and adipose tissue, but insulin binding was lowered in skeletal muscles. In animals exposed to HK for one day, a decrease of SIB and IR was found in isolated adipocytes, but no changes in insulin binding were noted in the liver tissue. In rats exposed to HK for 7 and 21 days, values of IR were similar as in control group. Our results indicate a) the different changes of IR in the liver, fat and muscle tissues after exposure to stress situations, b) a long-term decrease of insulin binding in muscles of rats exposed to repeated IMO stress, and c) the return of reduced SIB and IR (induced by acute stress) to control values in the liver and adipose tissue after a short recovery period.     

Structural differences between liver- and muscle-derived insulin receptors in rats

The structure of insulin receptors, solubilized from rat skeletal muscle and liver, was studied. The alpha subunit was identified by specific cross-linking to A14 125I-insulin with disuccinimidyl suberate. Muscle- and liver-derived alpha subunits migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a Mr of 131,000 and 135,000, respectively. There was no significant difference in insulin binding affinity. Treatment of cross-linked, immunoprecipitated receptors with either neuraminidase or endoglycosidase H decreased the Mr of muscle- and liver-derived alpha subunits but did not affect the difference in Mr. Autophosphorylated beta subunits migrated with a Mr of 98,000 for muscle and 101,000 for liver. After partial V8 digestion of autophosphorylated, immunoprecipitated receptors the major phosphopeptide fragment migrated on SDS-PAGE at Mr 57,000 from muscle and 60,000 from liver. Glycosidase digestion of autophosphorylated receptors suggested that Mr heterogeneity was due in part to differences in the sialic acid content of beta subunits. Muscle and liver are the major target organs of insulin; the apparent heterogeneity of insulin receptor structure may be relevant to tissue-specific differences in insulin action.     

Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids.

Cytosolic, detergent-solubilized and membrane-bound growth hormone (GH) receptors from rabbit adipose tissue and liver were tested for reactivity with a panel of monoclonal antibodies (MAbs). The cytosolic and detergent-solubilized forms of adipose tissue and liver GH receptors were identically reactive with four precipitating and two hormone-binding-site-directed MAbs. However, the membrane-bound form of the adipose receptor was 1000-fold less reactive with one binding-site-directed MAb (MAb 7) than the membrane-bound liver GH receptor. Reactivity with another inhibitory MAb (MAb 263) was identical for adipose tissue and liver membrane GH receptors. The relative potency of 22,000-Mr and 20,000-Mr forms of human GH was identical in assays with liver and adipose tissue membrane receptors. Thus, contrary to earlier suggestions, the discrepancy between the growth-promoting and insulin-like activities of 20,000-Mr human GH cannot be rationalized by a difference in the affinity of this hormone for 'somatogenic' and 'metabolic' receptors when the comparison is made in the same species. Cross-linking studies showed that the major GH-binding subunit of liver and adipose tissue GH receptors had the same Mr (54,000 +/- 5000, reduced). The ligand-binding subunits of liver and adipose tissue receptors are identical by several criteria, but one epitope on the adipose tissue receptor appears to be masked upon membrane insertion, possibly by close association with a tissue-specific component. Tissue specificity may be determined by association of a ubiquitous GH-binding subunit with tissue-specific membrane components, rather than by differences in amino acid sequence.     

Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis,triglyceride clearance,and regulation of body fat mass

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor that mediates the antidiabetic effects of thiazolidinediones. PPAR gamma is present in adipose tissue and becomes elevated in fatty livers, but the roles of specific tissues in thiazolidinedione actions are unclear. We studied the function of liver PPAR gamma in both lipoatrophic A-ZIP/F-1 (AZIP) and wild type mice. In AZIP mice, ablation of liver PPAR gamma reduced the hepatic steatosis but worsened the hyperlipidemia, triglyceride clearance, and muscle insulin resistance. Inactivation of AZIP liver PPAR gamma also abolished the hypoglycemic and hypolipidemic effects of rosiglitazone, demonstrating that, in the absence of adipose tissue, the liver is a primary and major site of thiazolidinedione action. In contrast, rosiglitazone remained effective in non-lipoatrophic mice lacking liver PPAR gamma, suggesting that adipose tissue is the major site of thiazolidinedione action in typical mice with adipose tissue. Interestingly, mice without liver PPAR gamma, but with adipose tissue, developed relative fat intolerance, increased adiposity, hyperlipidemia, and insulin resistance. Thus, liver PPAR gamma regulates triglyceride homeostasis, contributing to hepatic steatosis, but protecting other tissues from triglyceride accumulation and insulin resistance.     

Effect of treatment with different doses of 17-beta-estradiol on the insulin receptor

The mechanism for the development of insulin resistance in normal pregnancy is complex and is associated with serum levels of sex hormones. However, the influence of these hormones on the early steps of insulin action has not been extensively studied, although the potentially beneficial effect of estradiol on glucose homeostasis has been reported. In this paper, we attempted to determine the effect of 17-beta-estradiol on the insulin receptor of ovariectomized rats treated with different doses of hormones. Our results showed a tissue-dependent response to estradiol. We found that low doses of estradiol increased the amount of insulin receptors in liver and muscle on days 6 and 11 of treatment but not in adipose tissue, and after 16 days only the muscle responsed in this way. On the other hand, high doses of estradiol significantly decreased the amount of insulin receptors, at least in muscle and adipose tissue. We believe that the low concentrations of 17-beta-estradiol (similar to early pregnancy) could be responsible for the increase in insulin sensitivity by increasing the amount of insulin receptors in peripheral tissues. When the hormone levels were high (similar to late pregnancy) the amount of insulin receptors decreased in peripheral tissues, and insulin sensitivity is diminished just as in late pregnancy. The specific molecular mechanism for this action is as yet unknown.     

Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit

Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.     

Differential binding of dexamethasone to ammonium sulfate precipitates of human adipose tissue cytosols

Saturation analysis of the binding of [3H]dexamethasone [( 3H]DEX) to ammonium sulfate precipitates (ASPs) confirmed the presence of a limited-capacity, high-affinity binder in human adipose tissue cytosols. Various non-radioactive steroids competed with [3H]DEX for binding to the ASPs in the following sequence: dexamethasone (DEX) approximately equal to triamcinolone acetonide (TA) greater than progesterone (P) much greater than estradiol (E2). The steroid specificity of the binder precipitated by AS was consistent with the specificities reported for glucocorticoid receptors in a number of systems. In order to investigate possible regional differences, glucocorticoid binding to ASPs derived from adipose tissues removed from two different sites in the same subject was quantitated. ASPs of human omental adipose tissue bound significantly more [3H]DEX than did similar preparations of subcutaneous adipose tissue from the abdominal wall (116 +/- 32 vs. 50 +/- 22 fmol/mg protein; mean +/- SD; p less than 0.02). The findings are consistent with reports from other laboratories suggesting that intra-abdominal fat is more responsive to glucocorticoids than is subcutaneous adipose tissue.     

A new member of the insulin receptor family, insulin receptor-related receptor, is expressed preferentially in the kidney.

Shier and Watt isolated human and guinea pig genomic DNA encoding a putative protein, insulin receptor-related receptor (IRR), the primary structure of which is similar to that of other members of the insulin receptor family, the insulin receptor and type-I IGF receptor (J. Biol. Chem. 264, 14605-14608 (1989)). However, the expression of the IRR gene remained unknown. In this paper, we isolated the IRR cDNA from the rat brain and examined the expression of the IRR mRNA in a variety of rat tissues, including the brain, heart, lung, liver, small intestine, kidney, thymus, spleen, muscle, adipose tissue and cartilage by polymerase chain reaction. In contrast to the wide distribution of the insulin receptor and type-I IGF receptor mRNAs, the IRR mRNA is expressed preferentially in the kidney, which indicates that IRR has unique functions as a member of the insulin receptor family.     

Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation

Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

Vanadate restores glucose 6-phosphate in diabetic rats: a mechanism to enhance glucose metabolism

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of streptozotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 +/- 1 microM vanadate). Lipogenic capacity was elevated when a threshold level of approximately 7.5 +/- 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.     

Assessment of transcapillary glucose exchange in human skeletal muscle and adipose tissue

We studied the kinetics of glucose exchange between plasma and interstitial fluid (ISF) in human skeletal muscle and adipose tissue under fasting conditions. Five normal human subjects received an intravenous [6,6-2H2]glucose infusion in a prime-continuous fashion. During the tracer infusion, the open-flow microperfusion technique was employed to frequently sample ISF from quadriceps muscle and subcutaneous adipose tissue. The tracer glucose kinetics observed in muscle and adipose tissue ISF were found to be well described by a capillary-tissue exchange model. As a measure of transcapillary glucose exchange efficiency, the 95% equilibrium time was calculated from the identified model parameters. This time constant was similar for skeletal muscle and adipose tissue (28.6 +/- 3.2 vs. 26.8 +/- 3.6 min; P = 0.60). Furthermore, we found that the (total) interstitial glucose concentration was significantly lower (P < 0.01) in muscle (3.32 +/- 0.46 mmol/l) and adipose tissue (3.51 +/- 0.17 mmol/l) compared with arterialized plasma levels (5.56 +/- 0.13 mmol/l). Thus the observed gradients and dynamic relationships between plasma and ISF glucose in muscle and adipose tissue provide evidence that transcapillary exchange of glucose is limited in these two tissues under fasting conditions.     

Binding and antilipolytic action of insulin in isolated adipocytes from cortisol-treated rats

The effects of an in vivo cortisol-treatment to rats (2 X 2 mg/rat/day, for one week) on insulin plasma levels, insulin binding and antilipolytic activity in rat adipose tissue were investigated. Hyperinsulinemia together with an increase in insulin degradation in the serum of cortisol-treated rats were observed. The adipocytes from cortisol-treated animals showed a statistically significant decrease in insulin binding but no change in receptor numbers [cortisol-treated 103,000 +/- 8,000 (n = 8) receptors/cell and controls 138,000 +/- 15,000 (n = 16) receptors/cell], together with unchanged receptor affinity [ED50: cortisol-treated 3 X 10(-9) M and controls 3.2 X 10(-9) M], and a decreased sensitivity to the antilipolytic effect of insulin. The evidence presented for pre-receptor, receptor and post-receptor insulin defects on the action of cortisol in isolated rat adipocytes could represent a coordinated mechanism by which cortisol exerts "insulin resistance" in this tissue.     

Effect of hypothermia on insulin-receptor interaction in different rat tissues

Experimental hypothermia caused extensive changes in the number of both classes of insulin receptors in different rat tissues. In the liver, the number of high affinity insulin receptors (HAIRs) decreased by 50% (from 25.3 to 12.6 fmol/mg membrane protein), whereas number of low affinity insulin receptors (LAIRs) was almost unchanged in comparison to normothermic animals (5.63 and 4.39 pmol/mg, respectively). In the adipose tissue, number of both classes was reduced--HAIRs by 81% (from 24.0 to 4.50 fmol/mg) and LAIRs by 92% (from 16.0 to 1.29 pmol/mg). In the skeletal muscle, capacity of HAIRs was not changed (16.2 and 19.3 fmol/mg in normo- and hypothermic animals, respectively), whereas number of LAIRs increased by 150% (from 6.65 to 16.6 pmol/mg). Hypothermic rats also showed lower amount (by 85%) of LAIRs in the heart muscle (9.37 and 1.43 pmol/mg in control and experimental animals, respectively). Simultaneously, no significant changes were found in HAIRs (16.3 and 11.9 fmol/mg, respectively) and LAIRs (4.43 and 3.88 pmol/mg, respectively) in the brain. These differences in insulin receptors responses to hypothermia may reflect different physiological role of insulin in the regulation of target cell metabolism and/or the differences in tissue distribution of the insulin receptor isoforms.     

Comparison of insulin and insulin-like growth factor I receptors from rat skeletal muscle and L-6 myocytes

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.     

Beta-adrenergic receptors and adenylate cyclase activity in heart, muscle and adipose tissue of German Landrace pigs selectively bred for differences in backfat deposition

The density of beta-adrenergic receptors was examined in heart, muscle (m. longissimus dorsi) and backfat tissue of German Landrace pigs using (-)-[3H]-dihydroalprenolol. The animals were selected for high (E(+)-line) or low (E(-)-line) activity of lipogenic enzymes in backfat and for low backfat thickness (U(-)-line) based on ultrasonic measurement. An unselected control (K0-line) also exists. The correlated selection response on stress susceptibility (measured by halothane reaction and by meat quality parameters) showed the following sequence of selection lines: U- greater than E- greater than E+ greater than K0. The strains used for examination descended from the 9th generation (heart and muscle tissue) and the 10th generation (heart, muscle and adipose tissue) of this selection experiment. No further selection took place between the 9th and 10th generation. Basal adenylate cyclase activity was determined as well as (-)-Isoproterenol + GTP or sodium fluoride (NaF) stimulated activity in heart and muscle of the 10th generation measuring [3H]-cAMP formation from [3H]-ATP. A good correlation (r greater than or equal to 0.59, n = 9) between beta-adrenergic receptor density and adenylate cyclase activity was found which pointed to an efficient receptor-effector system. The number of beta-adrenergic receptors and adenylate cyclase activity was higher in the U(-)-line in all investigated tissues compared to the other lines in the 9th and 10th generation. Most differences between these lines were also significant.(ABSTRACT TRUNCATED AT 250 WORDS)