Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

Adipose tissue is required for the antidiabetic, but not for the hypolipidemic, effect of thiazolidinediones.

There is uncertainty about the site(s) of action of the antidiabetic thiazolidinediones (TZDs). These drugs are agonist ligands of the transcription factor PPAR gamma, which is abundant in adipose tissue but is normally present at very low levels in liver and muscle. We have studied the effects of TZDs in A-ZIP/F-1 mice, which lack white adipose tissue. The A-ZIP/F-1 phenotype strikingly resembles that of humans with severe lipoatrophic diabetes, including the lack of fat, marked insulin resistance and hyperglycemia, hyperlipidemia, and fatty liver. Rosiglitazone or troglitazone treatment did not reduce glucose or insulin levels, suggesting that white adipose tissue is required for the antidiabetic effects of TZDs. However, TZD treatment was effective in lowering circulating triglycerides and increasing whole body fatty acid oxidation in the A-ZIP/F-1 mice, indicating that this effect occurs via targets other than white adipose tissue. A-ZIP/F-1 mice have markedly increased liver PPAR gamma mRNA levels, which may be a general property of fatty livers. Rosiglitazone treatment increased the triglyceride content of the steatotic livers of A-ZIP/F-1 and ob/ob mice, but not the "lean" livers of fat-transplanted A-ZIP/F-1 mice. In light of this evidence that rosiglitazone acts differently in steatotic livers, the effects of rosiglitazone, particularly on hepatic triglyceride levels, should be examined in humans with hepatic steatosis.

Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice.

In lipoatrophic diabetes, a lack of fat is associated with insulin resistance and hyperglycemia. This is in striking contrast to the usual association of diabetes with obesity. To understand the underlying mechanisms, we transplanted adipose tissue into A-ZIP/F-1 mice, which have a severe form of lipoatrophic diabetes. Transplantation of wild-type fat reversed the hyperglycemia, dramatically lowered insulin levels, and improved muscle insulin sensitivity, demonstrating that the diabetes in A-ZIP/F-1 mice is caused by the lack of adipose tissue. All aspects of the A-ZIP/F-1 phenotype including hyperphagia, hepatic steatosis, and somatomegaly were either partially or completely reversed. However, the improvement in triglyceride and FFA levels was modest. Donor fat taken from parametrial and subcutaneous sites was equally effective in reversing the phenotype. The beneficial effects of transplantation were dose dependent and required near-physiological amounts of transplanted fat. Transplantation of genetically modified fat into A-ZIP/F-1 mice is a new and powerful technique for studying adipose physiology and the metabolic and endocrine communication between adipose tissue and the rest of the body.

Lack of responses to a beta3-adrenergic agonist in lipoatrophic A-ZIP/F-1 mice.

Stimulation of beta3-adrenergic receptors increases metabolic rate via lipolysis in white adipose tissue (WAT) and thermogenesis in brown adipose tissue (BAT). Other acute effects include decreased gastrointestinal motility and food intake and increased insulin secretion. Chronic treatment with a beta3 agonist ameliorates diabetes and obesity in rodents. We studied the effects of beta3 stimulation in A-ZIP/F-1 mice, which have virtually no WAT, a reduced amount of BAT, severe insulin resistance, and diabetes. In contrast with wild-type mice, treatment of A-ZIP/F-1 mice with CL316243, a beta3-adrenergic agonist, did not increase O2 consumption. A single dose of CL316243 produced a 2-fold increase in serum free fatty acids, a 53-fold increase in insulin, and a 2.4-fold decrease in glucose levels in wild-type mice but no change in A-ZIP/F-1 animals. The A-ZIP/F-1 mice also did not show reduced gastrointestinal motility or 24-h food intake during beta3 stimulation. Chronic administration of CL316243 to the A-ZIP/F-1 mice did not improve their thermogenesis, hyperglycemia, or hyperinsulinemia. Thus, all of the beta3 effects studied were absent in the lipoatrophic A-ZIP/F-1 mice, including the effects on nonadipose tissues. From these results, we suggest that all of the effects of beta3 agonists are initiated at the adipocyte with the nonadipose effects being secondary events presumably mediated by signals from adipose tissue.

Immunological abnormalities in insulin receptors on cultured EBV-transformed lymphocytes from insulin-resistant patient with leprechaunism.

Defects in insulin-receptor structure can impair insulin-receptor function. We have previously identified qualitative abnormalities in insulin binding to insulin receptors from an insulin-resistant patient (Lep/Ark-1). The defects in insulin binding are probably caused by a defect in receptor structure. In this study, we used immunological probes to investigate the structural defect(s) responsible for the abnormal function. Several anti-receptor antibodies were impaired in their abilities to bind to the insulin receptor of Lep/Ark-1. For example, monoclonal antibody MoAb-51 was much less effective in inhibiting binding to insulin receptors from Lep/Ark-1 (ID50 70 nM) than to those of normal subjects (ID50 8 nM). In addition, there was a 10-fold reduction of the avidity with which human polyclonal antibody B-d immunoprecipitated the patient's insulin receptors. The avidity of antibody B-10 was also reduced, although to a lesser extent. In contrast, several site-specific antibodies against epitopes on the beta-subunit of the receptor bound to receptors from Lep/Ark-1 with normal avidity. The data with monoclonal and polyclonal antibodies are consistent with the hypothesis that the structural defect resides in the extracellular domain of this patient's insulin receptor. The normal immunoreactivity of two putative phosphorylation sites on the beta-subunit with site-specific antibodies gives further support to the conclusion that this patient's receptors have normal tyrosine kinase activity.

Binding of insulin to its receptor impairs recognition by monoclonal anti-insulin antibodies.

The interaction between insulin and its receptor was investigated using both monoclonal and polyclonal anti-insulin antibodies. After covalent cross-linking of 125I-insulin to the insulin receptor on cultured human lymphocytes (IM-9 cells) using disuccinimidyl suberate, we inquired whether the insulin-receptor complex could be immunoprecipitated with anti-insulin antibodies. While a polyclonal guinea pig anti-insulin antiserum succeeded in immunoprecipitating receptor-bound 125I-insulin, binding to the receptor decreased the avidity of the antiserum for the insulin moiety by a factor of approximately 1000-fold. Sixteen distinct monoclonal murine anti-insulin antibodies were employed to immunoprecipitate receptor-bound 125I-insulin. Of these 16 monoclonal antibodies, only one (antibody 5.9F4) could be shown to recognize receptor-bound 125I-insulin. Moreover, even with antibody 5.9F4, binding of 125I-insulin to its receptor reduced the affinity of the antibody by a factor of 10- to 100-fold. These data strongly suggest that, when insulin binds to its receptor, the majority of the insulin molecule is unavailable for binding by anti-insulin antibodies. It seems likely that the hormone binding site on the receptor may be very large, thereby allowing for sequestration of the majority of the insulin molecule with relatively little of the hormone remaining exposed.

Estrogens increase the number of thyrotropin-releasing hormone receptors on mammotropic cells in culture.

The number of plasma membrane receptors for TRH on tumor-derived mammotropic cells in culture, GH3 and GC cells, but not their affinity for TRH, was increased by estrogens. For GH3 cells, exposure to 10 nM 17 beta-estradiol for 48 h increased the receptor level from 54,000 to 90,000 sites/cell, while for GC cells, the number of receptors increased from 29,000 to 46,000 after 28 h. PRL accumulation in the medium was also increased by 17 beta-estradiol. 17 beta-Estradiol and diethylstilbestrol were equally potent in increasing the TRH receptor level, while estrone was only 1/10th as potent. Diethylstilbestrol bound to the cytoplasmic estrogen receptor with an apparent affinity approximately 2.5 times higher than 17 beta-estradiol in GH3 and GC cells, while the affinity for estrone was only 1/12th to 1/20th that of 17 beta-estradiol. Tamoxifen, an antiestrogenic compound, inhibited the increase in TRH receptor number induced by 0.3 nM 17 beta-estradiol and was capable of binding to the estrogen receptor. Modulation of the TRH receptor level on mammotropic cells by estrogens, which is likely mediated through cytoplasmic estrogen receptors, may be an important mechanism for regulation of TRH action.

Anti-receptor antibodies mimic the effect of insulin to down-regulate insulin receptors in cultured human lymphoblastoid (IM-9) cells.

Autoantibodies directed against the insulin receptor mimicked the effect of insulin to down-regulate insulin receptors in IM-9 cells. This down-regulation occurred at 37 C, but not at 15 or 22 C. As with insulin itself, antireceptor antibodies caused down-regulation by accelerating the rate of receptor degradation. Down-regulation induced by antireceptor antibody may play a role in desensitizing target cells to the effects of insulin.

Atypical antiinsulin receptor antibodies in a patient with type B insulin resistance and scleroderma.

We studied a 23-yr-old woman with scleroderma and type B insulin resistance. The association with autoimmune disease suggested that the insulin resistance resulted from autoantibodies to the insulin receptor. However, in preliminary studies, serum antireceptor antibodies were not detected in an assay that measures the ability of the antibodies to inhibit insulin binding to the insulin receptor. Antireceptor antibodies were subsequently detected by their ability to immunoprecipitate affinity-labeled receptors. After the patient had received immunosuppressive therapy with prednisone and cyclophosphamide for 3 months, her insulin resistance remitted, and she developed hypoglycemia. Simultaneously with the remission of insulin resistance, the titer of serum antireceptor antibody (measured by the immunoprecipitation assay) fell to less than 1% of the previous level. In a series of 21 patients, this is the first patient with antireceptor antibodies that bound to the insulin receptor without inhibiting insulin binding.

Genetics of the insulin receptor defect in a patient with extreme insulin resistance.

A patient with extreme insulin resistance (leprechaun/Ark-1) had an 80-90% decrease in the number of insulin receptors on her circulating monocytes. In contrast, while a normal number of insulin receptors was expressed on the surface of Epstein-Barr (EB) virus-transformed lymphocytes from the patient, the receptors had decreased sensitivity to changes in temperature and pH. The father, who had a moderate degree of insulin resistance, resembled the patient in that his monocytes had a 60-80% decrease in the number of insulin receptors. Binding to the father's EB virus-transformed lymphocytes was normal. The mother was normally sensitive to insulin and had a normal number of insulin receptors on her circulating monocytes. In contrast, insulin receptors on the mother's EB virus-transformed lymphocytes were qualitatively abnormal, closely resembling the daughter's cultured cells. These observations suggest that each parent has transmitted a different genetic defect to the patient. When both mutations coexist in the same individual, they fail to complement, but, rather, result in extreme insulin resistance.

Transgenic mice lacking white fat: models for understanding human lipoatrophic diabetes.

The human disease lipoatrophic (or lipodystrophic) diabetes is a rare syndrome in which a deficiency of adipose tissue is associated with Type 2 diabetes. This disease is an interesting contrast to the usual situation in which diabetes is associated with obesity, an excess of fat. Aside from obesity, patients with lipodystrophic diabetes have the other features associated with Metabolic Syndrome X, including hypertension and dyslipidemia. The contrast between diabetes with a lack of fat and diabetes with an excess of fat provides an opportunity to study the mechanisms causing Type 2 diabetes and its complications. Recently, three laboratories have produced transgenic mice that are deficient in white adipose tissue. These mice have insulin resistance and other features of lipoatrophic diabetes, and are a faithful model for the human disease. Here we review the different murine models of fat ablation and compare the murine and human diseases, addressing the questions: Is the lack of fat causative of the diabetes, and if so by what mechanism? How could the other clinical features be explained mechanistically? And finally, what can be gleaned about insight into treatment options?

Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor.

Leptin is a hormone produced in adipose cells that regulates energy expenditure, food intake, and adiposity. In mice, we observed that circulating leptin levels increase 20-40-fold during pregnancy. Pregnant ob/ob females had no detectable serum leptin, demonstrating that the heterozygous conceptus was not the source of the leptin. However, leptin RNA and protein levels in maternal adipose tissue were not elevated. The circulating leptin was in a high molecular weight complex, suggesting that the rise in leptin was due to expression of a binding protein. Indeed, quantitative assays of serum leptin binding capacity revealed a 40-fold increase, coincident with the rise in serum leptin. Leptin binding activity reached a capacity of 207 +/- 15 nmol/liter of serum at day 18 of gestation, and half-maximal binding was observed with approximately 3 nM leptin. The binding protein was purified and partially sequenced, revealing sequence identity to the extracellular domain of the leptin receptor. We found that the placenta produces large amounts of the OB-Re isoform of leptin receptor mRNA, which encodes a soluble binding protein. Thus, the extreme hyperleptinemia of late pregnancy is attributable to binding of the leptin by a secreted form of the leptin receptor made by the placenta.

Insulin stimulates phosphorylation of a 120-kDa glycoprotein substrate (pp120) for the receptor-associated protein kinase in intact H-35 hepatoma cells.

The insulin receptor possesses protein kinase activity, which may play a role in mediating insulin action. Recently, we have identified a glycoprotein (pp120) in rat liver plasma membranes that is phosphorylated by the solubilized insulin receptor in a cell-free system. We now report that insulin stimulates phosphorylation of pp120 in intact H-35 cells. H-35 cells were preloaded with [32P]orthophosphate to label the intracellular ATP pool. Insulin caused a 10-fold increase in the phosphorylation of its receptor and a 2-fold increase in phosphorylation of pp120 (P less than 0.001). The time course of insulin's stimulation of pp120 closely paralleled that of insulin receptor phosphorylation over the time period investigated (15-45 min). This effect had the specificity corresponding to the insulin receptor. Epidermal growth factor was inactive, and insulin-like growth factor I had approximately equal to 1% the potency of insulin in this regard. Insulin increased 32P incorporation into pp120 in a linkage that was stable to alkaline hydrolysis, as would be expected for tyrosine-specific phosphorylation. Direct phosphoamino acid analysis confirmed that insulin increased 32P incorporation into phosphotyrosine residues in pp120.

Receptor affinity and biological potency of thyroid hormones in thyrotropic cells.

The nuclear receptor affinity for L-triiodothyronine (L-T3), L-thyroxine (L-T4), L-triiodothyroacetic acid (triac), and D-triiodothyronine (D-T3) was compared to the potency of these thyroid hormone analogues in regulating thyrotropin (TSH) production and the number of membrane receptors for thyrotropin-releasing hormone (TRH) in mouse thyrotropic tumor cells in culture. L-T3 and triac were equally potent and D-T3 was one-sixth to one-fifth as potent in binding to the receptor and in regulating TSH production and TRH receptor number. L-T4 was the least potent analogue in each instance, but its relative receptor-binding affinity, measured after 3 h, was significantly less than its somewhat variable relative biological potency, measured after 48 h. The cells were shown to monodeiodinate L-[125I]T4 to L-[125I]T3 in a time-dependent manner, and the enhanced biological potency of L-T4 was ascribed to its conversion to L-T3. Thyroid hormones appear to regulate TSH production and the number of receptors for TRH in thyrotropic cells in culture through interaction with a nuclear receptor.

Leptin and its role in pregnancy and fetal development--an overview.

Leptin is a hormone that is secreted by adipose cells in proportion to adipose mass, and therefore a low leptin level signifies depletion of energy stores. It has been proposed that leptin is one of the signals controlling sexual maturation. For example, humans and rodents lacking leptin fail to undergo complete puberty, while overexpression of leptin in mice causes early puberty. The placenta also produces leptin in human pregnancy, increasing the amount in the maternal circulation. The effects of the increased leptin levels during pregnancy are not clear. In contrast, the mouse placenta does not produce endocrinologically significant amounts of leptin. The mouse placenta does secrete a leptin-binding protein, the production of which correlates with a large increase in maternal leptin levels. The physiology of leptin during pregnancy and fetal development differs significantly between species, and is not well understood in any.

Mutagenesis of lysine 460 in the human insulin receptor. Effects upon receptor recycling and cooperative interactions among binding sites.

Mutations in the insulin receptor gene can cause insulin resistance. Previously, we have identified a mutation substituting glutamic acid for lysine at position 460 in the alpha-subunit of the insulin receptor in a patient with a genetic form of insulin resistance. In the present work, we have investigated the effect upon receptor function of amino acid substitutions at position 460. Decreasing the pH from 8.0 to 5.5 caused a progressive acceleration of the dissociation of 125I-insulin from the wild-type insulin receptor. Substitution of acidic amino acids (Glu or Asp) for Lys460 decreased the ability of acid pH to accelerate dissociation of 125I-insulin. In contrast, substitution of Arg or neutral amino acids (Val, Met, Thr, or Gln) had no effect upon the sensitivity to acid pH. Correlated with decreased sensitivity to acid pH, substitution of Glu or Asp at position 460 retarded the dissociation of 125I-insulin from intracellular receptors subsequent to receptor-mediated endocytosis. Furthermore, retardation of dissociation of 125I-insulin from the internalized receptor was associated with a decreased half-life of the receptor. In summary, the Glu460 mutation appears to cause insulin resistance by accelerating receptor degradation and, thereby, decreasing the number of insulin receptors on the cell surface. Additional studies suggested that Lys460 may provide the amino groups whereby disuccinimidyl suberate cross-links the two alpha-subunits to each other. Consistent with the hypothesis that Lys460 is located at the interface between adjacent alpha-subunits, substitutions at position 460 impair cooperative interactions among insulin binding sites. The Glu460 mutation decreases positively cooperative binding interactions; the Arg460 mutation impairs negative cooperativity. Mutations at position 460 in the alpha-subunit did not decrease the ability of insulin to stimulate receptor tyrosine kinase.

Hypoglycemia associated with antibodies to the insulin receptor.

Antibodies to the insulin receptor are insulinomimetic in vitro, although they generally induce insulin resistance in vivo. We report the novel case of a patient who presented with fasting hypoglycemia as the sole manifestation of autoantibodies to the insulin receptor. Prednisone therapy (120 mg per day) produced a rise in fasting glucose to more than 100 mg per deciliter (6 mmol per liter) within 48 hours, although there was no detectable change in the titer of antireceptor antibodies. After 10 weeks of therapy, the titer of antireceptor antibodies had fallen approximately 100-fold, and prednisone could be discontinued without recurrence of hypoglycemia. This case demonstrates that antireceptor antibodies must be considered in the differential diagnosis of hypoglycemia, especially in patients with other manifestations of autoimmunity.

Torpor in mice is induced by both leptin-dependent and -independent mechanisms.

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP/F-1 and ob/ob mice. A-ZIP/F-1 mice have virtually no white adipose tissue and low leptin levels, whereas ob/ob mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP/F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP/F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or beta-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP/F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24 degrees C, 2 degrees C above ambient. In ob/ob mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP/F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.