Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study.

125I-human growth hormone (125I-hGH) binds specifically to receptors on cultures human lymphocytes (IM-9). When this process is studied by use of quantitative EM radioautography, under conditions of incubation at 15 degrees C for 5 min, the ligand is localized to the plasma membrane of the cell. At 30 degrees and 37 degrees C, however, 125I-hGH is progressively internalized by the cell as a function of time. The internalized ligand is found predominantly in the Golgi region of the cells, with a five-fold preferential localization to membrane-bounded structures with the morphological and cytochemical characteristics of lysosomes. Up to 59% of these lysosome-like structures are positive for the acid phosphatase reaction under the conditions of incubation at 37 degrees C for 120 min. When the cell associated radioactivity after 15-120 min of incubation at 37 degrees C is extracted in 1 M acetic acid and filtered on a Sephadex G-100 column, 58-73% of the material elutes as intact hGH. When cells are incubated with 125I-hGH at 37 degrees C for 15-120 min, separated from the incubation medium, and washed and diluted 100-fold, the percent 125I-hGH dissociable decreases as a function of increasing time of incubation. When cells are incubated with 125I-hGH for 15 min at 37 degrees C and the radioactivity that dissociates from the cells during 15-90 min is studied, the labeled material appearing in the incubation medium is progressively degraded as a function of time of incubation. When the dissociation process is studied radioautographically, grains are found both in plasma membrane and intracelluar compartments after 30 min of association, but after 30 and 120 min of dissociation a higher proportion of grains are in the intracellular compartment. After 120 min of association, there is less dissociation from either compartment and a preferential increase of grains in the intracellular compartment. These data suggest that receptor-linked internalization of a polypeptide hormone provides a mechanism that couples degradation of the ligand with loss of the cell surface receptor.

"Big" growth hormone components from human plasma: decreased reactivity demonstrated by radioreceptor assay.

Plasma as well as pituitary immunoreactive human growth hormone (HGH) comprises at least two discrete components which have been designated as "big" HGH and "little" HGH. Using a newly developed radioreceptor assay, which depends on the ability of a substance to compete with labeled HGH for binding sites on cultured human lymphocytes, we find that the big HGH component from both normal and acromegalic subjects has much less activity in the radioreceptor assay than in the radioimmunoassay, whereas the little HGH component has simnilar activity in both assays.

Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes.

Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.

Insulin in insects and annelids.

The fruitfly, Drosophila melanogaster, and the earthworm, Annelida oligocheta, were extracted with acid-ethanol by a classic method for recovering insulin from the pancreas. When each extract was filtered on a Sephadex G-50 column, a distinct peak of insulin immunoreactivity. The material in this peak had reactivity insulin (equivalent to 0.1 to 2 ng of insulin/g wet weight) was recovered in the region typical of insulin bioassay, measuring stimulation of glucose oxidation or lipogenesis by isolated rat adipocytes. The bioactivity was partially or largely neutralized by anti-insulin antibodies. In concordance with previous work showing the presence of material very similar to insulin in the blowfly and molluscs, we have confirmed the presence of insulin in insects and extended the observation to the earthworms. These findings suggest that insulin is more widespread in invertebrates than was previously thought. In a companion study (Proc. Natl. Acad. Sci. USA 77:6184-88, 1980), we have demonstrated material similar to insulin in unicellular organisms.

Developmental regulation of insulin and type I insulin-like growth factor receptors and absence of type II receptors in chicken embryo tissues.

Chicken embryos are a suitable model for studying the role of insulin, insulin-like growth factors I and II (IGF-I and IGF-II), and their receptors in embryogenesis. We show that plasma membranes from heart, liver, and limb buds, as reported earlier for brain, each have a distinct developmental profile for insulin receptors and type I IGF receptors. In heart and limb buds, IGF binding is higher than insulin binding, but in liver, insulin receptors dominate. Expression of these receptors is, therefore, developmentally regulated and tissue specific. The wide distribution of high-affinity receptors capable of mediating insulin and IGF actions in early organogenesis further supports the possible importance of this family of peptides for differentiation and growth in vertebrates. In all chicken embryo tissues studied, both IGF-I and IGF-II appeared to bind to a type I IGF receptor. We have not detected a receptor with the peptide binding and structural characteristics of the mammalian type II IGF receptor. The type II receptor was absent in embryos, liver from newly hatched chicks, and adipocytes from older chicks, which suggests that the chicken may lack this subtype of IGF receptor.

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes.

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.

Ontogeny of receptors for insulin-like peptides in chick embryo tissues: early dominance of insulin-like growth factor over insulin receptors in brain.

Recently, we confirmed early data showing deleterious effects of exogenous insulin on chick embryos at 2 days of development, although insulin receptors were not clearly demonstrable until days 3-4. Now we report that insulin-like growth factor (IGF) receptors are present in whole embryos on day 2. The developmental patterns of [125I]IGF-I and [125I]IGF-II binding to brain were similar, and IGF-I showed approximately a 2-fold higher binding than IGF-II; there was a sharp increase from days 3 to 6, and a subsequent gradual fall during the second and third weeks of ontogeny. Competitive binding experiments with unlabeled analogs suggested that both labeled IGFs were binding to type I IGF receptors, and insulin interacted with them. The temperature and pH dependence were relatively higher than those for some other known IGF receptors. We have previously reported that [125I]insulin binding to brain is barely detectable on day 3 and shows a progressive rise throughout the rest of embryonic life. The pattern of IGF and insulin receptors appears to be organ specific, since nonneural tissues such as heart, liver, and limb buds showed different binding profiles in ontogeny. We conclude from these data that IGF receptors develop in chick embryo brain before insulin receptors and probably can mediate effects of IGF and insulin at early stages of embryogenesis.

The insulin receptors of chick embryo show tissue-specific structural differences which parallel those of the insulin-like growth factor I receptors.

Since specific binding to receptors and biological effects of insulin and insulin-like growth factors (IGFs) are demonstrable soon after the neural tube closes and organogenesis begins in the chick embryo, in the present study we have analyzed the structural characteristics and specificity of the receptors for insulin and IGFs at this early stage of development. We show that membranes from newly differentiated chick embryo tissues (day 6 brain, day 6 heart, day 8 liver, day 12 skeletal muscle) as well as whole embryos postneurulation (day 2, stage of 27-30 somites) have two populations of receptors with distinct specificity: insulin and type I IGF (IGF-I) receptors. Both insulin and IGF-I alpha-subunits, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had tissue-dependent heterogeneity in Mr (liver greater than heart = skeletal muscle greater than brain) ranging from 138 kilodaltons (kDa) to 129 kDa. Desialylation of the receptors by treatment with neuraminidase produced a significant change in the Mr of the alpha-subunits in liver and heart but not in brain or the whole day 2 embryo. In each tissue the pattern for insulin receptors and IGF-I receptors was strikingly similar. Our studies raise the possibility that postranslational modifications of the insulin and IGF-I receptors, characteristic of terminally differentiated tissues, are already present in early organogenesis. Further, structural heterogeneity of the binding subunit of these receptors among tissues appears to be widespread and not exclusive to the brain receptor. An insulin receptor with features similar to the neural type is the only one detected in embryos at the beginning of organogenesis (day 2). The functional implication of this developmental tissue-specific regulation of insulin and IGF-I receptors, is still speculative. Its possible importance is suggested by the fact that it occurs embryologically early and affects both insulin and IGF-I receptors in parallel.

Insulin and insulin-like growth factor I both stimulate metabolism, growth, and differentiation in the postneurula chick embryo.

Chick embryos after 48 h of development (day 2) maintained in ovo provide an adequate model to study hormonal influences in early organogenesis in vertebrates. In previous studies at this (prepancreatic) stage of chick embryogenesis we demonstrated not only the presence of an insulin-related material but also insulin receptors and insulin-like growth factor I (IGF-I) receptors. Further, when embryos developed in the presence of antiinsulin antibodies, we showed retardation in both morphological and biochemical events which strongly suggested a physiological requirement for insulin in normal embryogenesis. In the present study we have evaluated the effects of insulin, proinsulin, desoctapeptide insulin, and IGF-I when applied to day 2 chick embryos. At day 4 of development biochemical indices were compared in treated vs. control groups. Insulin (10-100 ng/embryo) increased the content of protein, total creatine kinase, creatine kinase MB isozyme, triglycerides, cholesterol, phospholipids, DNA, and RNA, in a dose-dependent fashion. IGF-I had a lower potency than insulin in stimulating both metabolic and growth indices and was nearly equipotent in stimulating the creatine kinase MB content (marker of muscle differentiation). The high relative potency of insulin together with the effects of proinsulin (less than 15%) and desoctapeptide insulin (less than 10%) compared to insulin on the chick embryo, led us to infer that at low doses (nanograms per embryo) insulin stimulates developmental processes mainly through the insulin receptor, with the possible exception of muscle differentiation. The broad range of metabolic, growth, and differentiation indices stimulated by insulin and IGF-I in chick embryos, at a stage when specific receptors for both peptides are present, suggests that insulin and IGF-I may have a regulatory, complementary, or overlapping role in normal chick embryo early development.

Effect of pH and lysosomotropic agents on membrane-associated and internalized 125I-iodinated human growth hormone in cultured human lymphocytes: a quantitative biochemical and electron microscopic study.

When 125I-iodinated human GH ([125I]iodo-hGH) interacts with cultured human lymphocytes at 15 C, the reaction is reversible, but at 37 C the reaction becomes less dissociable as a function of incubation time. Acidification of the incubation medium results in rapid ligand dissociation at 15 C, but at 37 C the acid-dissociable component decreases as a function of incubation time. Under conditions where approximately 50% of the ligand is internalized by the cell, 90% is nondissociable. When the 37 C incubation is carried out in the presence of 25 mM NH4Cl, cell-associated radioactivity is increased. Under these conditions approximately 90% of cell-associated radioactivity also is nondissociable. Using quantitative electron microscopic autoradiography, the proportion of [125I]iodo-hGH associated with the plasma membrane and internalized by the cell is indistinguishable in the presence or absence of NH4Cl. Irreversible [125I]iodo-hGH association with cultured human lymphocytes is due to time- and temperature-dependent effects in the plasma membrane of the cell. These effects cannot be distinguished from internalization by acidification. Furthermore, lysosomotropic agents increase cell-associated radioactivity, but the proportion internalized is not increased.

Development of receptors for insulin and insulin-like growth factor-I in head and brain of chick embryos: autoradiographic localization.

In whole brain of chick embryos insulin receptors are highest at the end of embryonic development, while insulin-like growth factor-I (IGF-I) receptors dominate in the early stages. These studies provided evidence for developmental regulation of both types of receptors, but they did not provide information on possible differences between brain regions at each developmental stage or within one region at different embryonic ages. We have now localized the specific binding of [125I]insulin and [125I]IGF-I in sections of head and brain using autoradiography and computer-assisted densitometric analysis. Embryos have been studied from the latter part of organogenesis (days 6 and 12) through late development (day 18, i.e. 3 days before hatching), and the binding patterns have been compared with those in the adult brain. At all ages the binding of both ligands was to discrete anatomical regions. Interestingly, while in late embryos and adult brain the patterns of [125I]insulin and [125I] IGF-I binding were quite distinct, in young embryos both ligands showed very similar localization of binding. In young embryos the retina and lateral wall of the growing encephalic vesicles had the highest binding of both [125I]insulin and [125I]IGF-I. In older embryos, as in the adult brain, insulin binding was high in the paleostriatum augmentatum and molecular layer of the cerebellum, while IGF-I binding was prominent in the hippocampus and neostriatum. The mapping of receptors in a vertebrate embryo model from early prenatal development until adulthood predicts great overlap in any possible function of insulin and IGF-I in brain development, while it anticipates differential localized actions of the peptides in the mature brain.

Receptors for insulin-like growth factors I and II: autoradiographic localization in rat brain and comparison to receptors for insulin.

Receptors for insulin-like growth factor I (IGF-I) in rat brain were visualized using autoradiography with [125I]IGF-I. The binding of the labeled peptide was competed for fully by high concentrations of unlabeled IGF-I. At intermediate concentrations of unlabeled peptide the binding of [125I]IGF-I was competed for by unlabeled IGF-I more effectively than by IGF-II or insulin, which is typical of receptors for IGF-I. Essentially every brain section shows specific binding of IGF-I, and the pattern of binding of IGF-I to its receptors correlated well with the cytoarchitectonic structures. In parallel studies we showed that [125I]IGF-II was bound to tissue sections of rat brain and that the binding was competed for by an excess of unlabeled IGF-II. However, intermediate concentrations of unlabeled peptides gave inconclusive results. To confirm that the binding of [125I]IGF-II was to IGF-II receptors, we showed that antibodies specific for the IGF-II receptor inhibited the binding of labeled IGF-II. Furthermore, the binding of the antibody to regions of the brain section, visualized by the application of [125I]protein-A, gave patterns indistinguishable from those obtained with [125I]IGF-II alone. Again, the binding was very widely distributed throughout the central nervous system, and the patterns of distribution corresponded well to the underlying neural structures. Densitometric analysis of the receptors enabled us to compare the distribution of IGF-I receptors with that of IGF-II receptors as well as retrospectively with that of insulin receptors.

Studies on binding and mitogenic effect of insulin and insulin-like growth factor I in glomerular mesangial cells.

The mesangial cells, as part of their smooth muscle cell function, are actively involved in regulating glomerular hemodynamics. Their overlying endothelium is fenestrated; therefore, these cells are directly exposed to plasma substances, including hormones such as insulin and insulin-like growth factor I (IGF-I). These peptides may contribute to the mesangial sclerosis and cellular hyperplasia that characterize diabetic glomerulopathy. We report herein the characterization of the receptors and the mitogenic effects of IGF-I and insulin on mouse glomerular mesangial cells in culture. The IGF-I receptor was characterized on intact cells. The Kd of the IGF-I receptor was 1.47 X 10(-9) M, and the estimated number of sites was 64,000 receptors/cell. The binding was time, temperature, and pH dependent, and the receptor showed down-regulation after exposure to serum. The expression of the receptor did not change on cells at different densities. The specific binding for insulin was too low to allow characterization of the insulin receptor on intact cells. However, it was possible to identify the insulin receptor in a wheat germ agglutinin-purified preparation of solubilized mesangial cells. This receptor showed the characteristic features of the insulin receptor, including pH dependence of binding and a curvilinear Scatchard plot. The mitogenic effects of insulin and IGF-I on mesangial cells were measured by the incorporation of [3H]thymidine into DNA. IGF-I was more potent than insulin. The half-maximal response to IGF-I stimulation occurred at 1.3 X 10(-10) M, and a similar increase with insulin was observed at concentrations in the range of 10(-7) M, suggesting that this insulin action was mediated through the IGF-I receptor. These data show that the mouse microvascular smooth muscle cells of the glomerulus express a cell surface receptor for IGF-I in vitro and that this peptide is a potent mitogen for these mesangial cells. It may, therefore, play a role in glomerular proliferative lesions. The insulin receptor is present in small numbers and does not mediate mitogenesis in mesangial cells.

Water-soluble specific growth hormone binding sites from cultured human lymphocytes: preparation and partial characterization.

Cultured human lymphocytes (IM-9 cell line) have specific binding sites or receptors for human growth hormone (hGH). Under appropriate conditions, this specific binding material is spontaneously released into the incubation medium and is solubilized without the use of detergents. This water-soluble preparation binds [125I]iodo-hGH with the same specifictiy as receptors on the intact cell. Unlabeled hGH, but not porcine growth hormone or insulin, competes with labeled hGH for binding to the soluble preparation. Growth hormone preparations of varying purity compete for binding to the soluble binding preparation in the same rank order as they compete for binding to the intact IM-9 lymphocyte. [125I]iodo-hGH incubated with, but not bound to, the soluble preparation is partially degraded, while the [125I]iodo-hGH that is bound to the soluble preparation is protected from degradation, and its ability to rebind to fresh cells is enhanced. The [125I]iodo-hGH-soluble binding preparation complex can be dissociated by the addition of large quantities of unlabeled hGH or by lowering the pH, and [125I]iodo-hGH in both instances remains intact and undegraded. The soluble binding preparation did not sediment when centrifuged at 200,000 X g for 4 hours and was not retained on 0.20 micron Millipore filters. The soluble binding preparation was not retarded on Sephadex G-200. Binding activity was abolished by tryptic digestion. These studies demonstrate that hGH-binding sites, like previously reported insulin binding sites, can be spontaneously solubilized from cultured human lymphocytes, without the use of detergents; these soluble binding preparations are of high molecular weight and are, at least in part, protein in nature.

Reactivity of non-primate growth hormones and prolactins with human growth hormone receptors on cultured human lymphocytes.

Ovine placental lactogen is as reactive as human growth hormone with the human growth hormone receptor of cultured human (IM-9) lymphocytes, which confirms the findings of Carr and Friesen with receptors of human liver. We now also show that bovine and ovine growth hormones and ovine prolactin have reactivity for the human growth hormone receptor on IM-9 lymphocytes that is of the same order of magnitude (0.03%) as that previously reported for human placental lactogen. The binding studies predict that these non-primate hormones will have biological effects on skeletal growth in primates, either as agonists or antagonists. Previous studies have shown that when IM-9 lymphocytes are exposed to human growth hormone for 18 h at 37 C, there is a time and concentration dependent loss of human growth hormone receptors, and the magnitude of the loss of receptors after preincubation for 18 h at 37 C is greater than the average occupancy of receptors under steady state conditions for 90 min at 30 C. In the present study we show that human and ovine placental lactogens, ovine prolactin, and bovine and ovine growth hormones also produce this effect on the human growth hormone receptor. Since the cellular process by which a hormone induces loss of its own receptors appears to require binding of the hormone to its receptor as well as one or more subsequent steps in hormone action, it is likely that all of the preparations that induce receptor loss will be shown to have some agonist activity of human growth hormone in promoting skeletal growth in primates. Further, these studies extend the interrelationships between primate and non-primate pituitary and placental hormones from what has been suggested previously from biological and structural studies.

Regulation of receptor by homologous hormone enhances sensitivity and broadens scope of radioreceptor assay for human growth hormone.

In standard competitive binding assays (including radioreceptor assays) unlabeled ligand (hormone) competes with labeled ligand (hormone) for binding to a fixed number of binding (receptor) sites. Detection of the unlabeled ligand occurs when the occupancy of binding sites by the unlabeled ligand is sufficient to reduce the binding of labeled ligand. A common feature of the hormone-receptor interaction is the ability of the hormone to regulate the affinity and/or the concentration of its homologous receptor. In the present study, by exploiting the ability of human GH to regulate by negative feedback the concentration of its own receptors, we have enhanced the sensitivity of the human GH radioreceptor assay 5-fold. The ability of hormone to regulate receptor concentration and affinity affords wide opportunities to broaden the scope as well as to enhance the sensitivity of radioreceptor assays.

Evidence for higher proportion of "little" growth hormone with increased radioreceptor activity in acromegalic plasma.

Plasma growth hormone (GH) from both normal and acromegalic patients comprises multiple immunoreactive components that can be separated by G-100 Sephadex gel filtration and measured by radioimmunoassay (RIA) and radioreceptor assay (RRA). The higher molecular weight immunoreactive components, from both normal and acromegalic subjects, have a lower RRA/RIA than does the lower molecular weight "little" GH component. The "little" GH component comprises a higher proportion of the total immunoreactive GH in acromegalic plasma than in normal plasma whether the data are expressed only in terms of the "big" and "little" components (89 vs. 71%), or as a function of total immunoreactive GH (76 vs. 55%), or whether the plasma is obtained in the basal or stimulated state for the acromegalic patients. When the RRA/RIA x 100 is determined for the isolated "little" component, the acromegalic has a significantly greater ratio than the normal (110 vs. 75%). We conclude that acromegalic plasma contains a higher proportion of the more radioreceptor active "little" GH component than does the normal and, in addition, that the "little" component from the acromegalic is more radioreceptor-active than the normal.

Variant sequences of insulin receptor substrate-1 in patients with noninsulin-dependent diabetes mellitus.

The pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM) is characterized by insulin resistance and insulin deficiency. To search for genetic defects causing NIDDM, we have screened for mutations in the gene encoding insulin receptor substrate-1 (IRS-1), an intracellular protein that is phosphorylated by the insulin receptor and is thought to play an important role in mediating insulin action. The coding sequence of the IRS-1 gene (divided into 12 overlapping fragments) was amplified by polymerase chain reaction and screened for the presence of single stranded conformational polymorphisms. This led to the identification of 6 variants in the nucleotide sequence. There were 3 nonconservative amino acids substitutions: Gly819-->Arg, Gly972-->Arg, and Arg1221-->Cys. In addition, there were three silent polymorphisms: GAC vs. GAT encoding Asp90, GGG vs. GGA encoding Gly235, and GCA vs. GCG encoding Ala805. The previously reported Arg972 substitution was identified in 7 of 31 patients with NIDDM, 4 of 32 normal subjects, and 4 of 16 nondiabetic obese individuals. The 2 novel amino acid substitutions (Arg819 and Cys1221) were both detected in 1 patient with NIDDM, but not in either of the other 2 groups of nondiabetic individuals. All 3 amino acid residues are identically conserved in the amino acid sequences of human, mouse, and rat IRS-1, suggesting that Gly819, Gly972, and Arg1221 are important for the normal function of IRS-1. Furthermore, the prevalence of amino acid substitutions in IRS-1 is increased in patients with NIDDM. These observations suggest that mutations in the IRS-1 gene may play a causal role in the pathogenesis of NIDDM.

Autoradiographic localization of insulin receptors in rat brain: prominence in olfactory and limbic areas.

The binding of 125I-labeled insulin in thin sections of frozen fresh rat brain (95% specific binding) was shown using autoradiography. By several criteria including structure-activity relationship analysis, the brain insulin receptors were qualitatively indistinguishable from insulin receptors previously characterized on brain and other more typical target tissues and distinct from receptors for the insulin-like growth factors. The 125I-labeled insulin binding sites in brain formed a distinct pattern with high levels of binding in all olfactory areas and in closely related limbic regions. Binding was also prominent in the neocortex and the accessory motor areas of the basal ganglia and the cerebellum. Among ancillary brain structures only the choroid plexus demonstrated a high density of insulin receptors. When insulin receptors were located on cortical or laminated structures, the highest binding occurred in the superficial ("molecular" or "plexiform") layer, i.e. the layer which receives afferent input and/or is rich in the dendritic branches of principal neurons (e.g. external plexiform layer of the olfactory bulb and the molecular layer of the dentate gyrus). The enrichment of insulin receptors in the olfactory and limbic systems, which is a characteristic feature of other known neuropeptides, in addition to their prevalence in the strata occupied by the dendritic fields of principle neurons, suggests a neuromodulatory function for insulin in the brain.

Insulin-related material extractable from brain and other tissues of rat: Possible biologic and methodologic variables.

Insulin, the amount and its source, in brain and other extrapancreatic tissues remains a subject of disagreement. One major obstacle in understanding the source of the disagreement is the wide range of variations in methods used in the extraction and processing of the tissue insulin. In the present study we showed that glass homogenizers, in our hands, are superior to the Waring blender; that storage of the acid ethanol supernatants (at ?80 degrees C) before processing or evaporation to dryness during early stages may be deleterious; gel filtration of extracts results in significant losses. Losses with hydrophobic (C(18)) chromatography were less severe than we had noted earlier with this method. With paired samples from controlled hypoinsulinemic (fasted), and hyperinsulinemic (fasted-refed) animals, we showed that insulin concentrations in brain, lung and probably liver are unaffected by changes in plasma insulin, whereas kidney insulin is directly related to plasma insulin concentrations. These findings confirm the essential data of previous studies. The unresponsiveness of brain insulin and most other tissue insulin compartments to fluctuations in plasma insulin, whether due to local synthesis or transport from extracellular sources followed by intracellular storage, needs to be incorporated into any overall conceptualization of tissue insulin.