James N. Livingston

Distribution of insulin receptor-like immunoreactivity in the rat forebrain

Previous studies have suggested that insulin may play a role in the hormonal regulation of neurotransmitter metabolisms within the central nervous system. In order to provide additional information to support this hypothesis, we examined the distribution of insulin receptors within the forebrain of adult male rats. Insulin receptors were localized by immunocytochemistry, using an antibody directed against the carboxy-terminus of the beta-subunit of the insulin receptor. The antibody specificity was tested by immunoprecipitation of brain insulin receptors with antiserum and the purity of the receptor-antibody preparation was determined using hormone binding-assays with radiolabeled insulin and insulin-like growth factor-l. Insulin receptor-like immunoreactivity was found in a widespread, but selective, distribution on neurons throughout the rat forebrain. Double-labeling with glial fibrillary acidic protein did not demonstrate any detectable insulin receptor-like immunoreactivity on glial cells. Areas with the highest density of insulin receptor-like immunoreactivity were found in the olfactory bulbs, hypothalamus and median eminence, medial habenula, subthalamic nucleus, subfornical organ, CA 1/2 pyramidal cell layer of the hippocampus and piriform cortex. Double-staining of hypothalamic sections with somatostatin and vasopressin antisera revealed insulin receptor-like immunoreactivity on a subpopulation of somatostatin neurons in the periventricular region and on vasopressin neurons in the supraoptic nucleus. A moderately dense insulin receptor-like immunoreactivity was observed in layers II-IV of cerebral cortex, medial amygdala, reticular thalamic nucleus, zona incerta, and preoptic and septal regions, whereas a low density of insulin receptor-like immunoreactive neurons was found in basolateral amygdala and most thalamic regions. The basal ganglia and most parts of the thalamus were almost devoid of insulin receptor-like immunoreactivity. Our findings provide morphological support for a direct action of insulin on selected regions of the rat forebrain and suggest that the insulin receptor may modulate synaptic transmission or the release of neurotransmitters and peptide hormones in the CNS.

Quinazolinone derivatives as orally available ghrelin receptor antagonists for the treatment of diabetes and obesity

The peptide hormone ghrelin is the endogenous ligand for the type 1a growth hormone secretagogue receptor (GHS-R1a) and the only currently known circulating appetite stimulant. GHS-R1a antagonism has therefore been proposed as a potential approach for obesity treatment. More recently, ghrelin has been recognized to also play a role in controlling glucose-induced insulin secretion, which suggests another possible benefit for a GHS-R1a antagonist, namely, the role as an insulin secretagogue with potential value for diabetes treatment. In our laboratories, piperidine-substituted quinazolinone derivatives were identified as a new class of small-molecule GHS-R1a antagonists. Starting from an agonist with poor oral bioavailability, optimization led to potent, selective, and orally bioavailable antagonists. In vivo efficacy evaluation of selected compounds revealed suppression of food intake and body weight reduction as well as glucose-lowering effects mediated by glucose-dependent insulin secretion.

Immunohistochemical localization of insulin receptors and phosphotyrosine in the brainstem of the adult rat

Previous studies have demonstrated that insulin receptors are widely distributed throughout areas of the forebrain in the adult rat that are involved in modulating neuroendocrine functions and feeding behaviour. In addition, a recent investigation showed that there is a good correlation between the presence of the insulin receptor and phosphotyrosine-containing proteins in these regions, indicating a possible functional activity of insulin receptors in vivo. It is unknown whether neural connections between specific brainstem nuclei to forebrain regions may also be under direct regulation of insulin or related factors. In order to test this possibility, the distribution of insulin receptors and phosphotyrosine was mapped throughout the hindbrain of the adult rat by immunocytochemistry, using specific antibodies against the beta-subunit of the insulin receptor as well as against phosphotyrosine. Both markers showed a high degree of overlap throughout numerous distinct anatomical regions of the hindbrain. In the mesencephalon, insulin receptor and phosphotyrosine-positive neurons were found in the precommissural nucleus, the lateral and dorsal part of the central gray, the mammillary bodies and the interpeduncular nucleus. In addition, immunoreactivity was found in the subependymal layer around the aqueduct along fibres and nerve cells possibly contacting the cerebrospinal fluid. In the pons and medulla, dense immunoreactivity was seen in the lateral superior olive, nucleus of the solitary tract, spinal trigeminal nucleus and nucleus ambiguous. Scattered cells were found in the pontine and vestibular nuclei, as well as in the reticular formation. The cerebellum contained moderately dense immunoreactivity in the granule cell and molecular cell layer of the cortex, as well as in the deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

A Novel Long-Acting Selective Neuropeptide Y2 Receptor Polyethylene Glycol-Conjugated Peptide Agonist Reduces Food Intake and Body Weight and Improves Glucose Metabolism in Rodents

Selective activation of the neuropeptide Y (NPY)2 receptor to suppress appetite provides a promising approach to obesity management. A selective NPY2 polyethylene glycol-conjugated (PEGylated) peptide agonist is described that consists of a peptide core corresponding to residues 13 to 36 of human peptide YY (PYY) and a nonpeptidic moiety (2-mercaptonicotinic acid) at the peptide N terminus that is derivatized with 20-kDa monomethoxypolyethylene glycol. The PEGylated peptide elicits a dose-dependent reduction in food intake in lean C57BL/6 mice and Wistar rats that persists for 72 and 48 h, respectively. The effect on food intake in lean C57BL/6 mice is blocked by the selective NPY2 antagonist BIIE0246 (N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide formate). A dose-dependent reduction in body weight in diet-induced obese (DIO) mice is seen following daily dosing for 14 days. The reduction in body weight is sustained following dosing for 40 days, and it is accompanied by an increase in plasma adiponectin. Improvements in glucose disposal and in plasma insulin and glucose levels that are risk factors for type II diabetes are observed following once-daily subcutaneous dosing in DIO mice. The results provide evidence from two animal species that the long-acting selective NPY2 peptide agonist has potential for obesity management.

Insulin resistance: Receptor and post-binding defects in human obesity and non-insulin-dependent diabetes mellitus

Insulin resistance is a prominent feature of three clinical conditions: obesity, impaired glucose tolerance, and non-insulin-dependent (type II) diabetes mellitus. Numerous studies over the past 15 years have provided a better understanding, from both a clinical and cellular standpoint, of the pathophysiology of these insulin-resistant states as well as of insulin action. In addition, it has recently been recognized that correction of glucose intolerance leads to an improvement in insulin secretion and a reduction in insulin resistance. Examination of the most recent data suggests that the basis for insulin resistance in these common clinical disorders is often multifactorial. In uncomplicated obesity, the cellular alterations responsible for insulin resistance appear to be at the level of the hepatic insulin receptor and in post-binding processes in peripheral target tissues. In type II diabetes, a post-binding defect(s) in peripheral tissues appears to be the primary lesion. In humans, many of the factors that mediate the changes leading to insulin resistance are still unknown and are the object of current investigations.

Insulin degradation by insulin target cells

Recent findings illustrate the complexities associated with the interaction between insulin and its target cells. These results suggest that the processes involved in insulin action and those involved in insulin degradation may have certain steps in common. Both apparently begin when insulin binds to the insulin receptor. The next step is unknown but it ultimately leads to the internalization of the hormone before insulin dissociates from the cell surface. Furthermore, internalization appears to be a requirement for efficient degradation of insulin since the vast majority (perhaps all in certain cells) of the degrading activity is intracellular. Internalization may not be required to produce certain actions of the hormone, however, and the two processes may diverge at the point. It is not clear how insulin enters the target cell other than the process appears to be receptor-mediated. Also, further work is needed to more fully characterize the vesicles that contain internalized insulin. Finally, the actual location of insulin degradation and the enzyme(s) involved need further study, especially to clarify the relative contributions of lysosomes, cytosolic protease, and GIT to physiological insulin destruction. An understanding of the overall process of insulin degradation is required for a complete description of the physiologic disposition of the hormone at the target cell. Moreover, this system has subtle control mechanisms that may have important implications for the management of diabetes and other endocrine and metabolic disorders.

Insulin induced changes in insulin binding and insulin-sensitivity of adipocytes

The ability of insulin to regulate the insulin sensitivity of adipose tissue was directly studied by maintaining fat in Medium 199 for 17 hr in the presence or absence of the hormone (10.5 X 10 −9 M ). Following this “chronic” treatment with insulin, the tissue was washed and the adipocytes isolated by collagenase treatment that removes essentially all of the insulin from the cells. The insulin responsiveness of the cells was then tested in “acute” 1–2 hr experiments and the results correlated with their ability to specifically bind 125 I-labeled insulin. “Chronic” insulin treatment did not change the basal glucose transport rate as measured by 2-deoxyglucose uptake. Also, the maximum insulin-stimulated rate of 2-deoxyglucose uptake in the “acute” experiments was similar for both chronically insulin-treated and untreated cells. In these experiments, the only difference noted between the two groups of adipocytes was in their response to submaximally stimulating concentrations of insulin. Insulin-treated cells required approximately three times the amount of insulin to produce half-maximal stimulation of 2-deoxyglucose uptake than the amount needed by the untreated cells. “Chronic” insulin treatment also produced an alteration in the high affinity interaction between the fat cells and 151-labeled insulin. This alteration was characterized by a decrease in insulin binding at low concentrations of the labeled hormone, while at higher concentrations the binding was similar to that of untreated cells. These results show that a high concentration of insulin can produce insulin resistance in fat cells by causing a rightward shift in the insulin dose-response curve. Furthermore, this change in hormonal sensitivity may be a consequence of the insulin-induced decrease in the insulin binding ability of the fat cells.

Role of the Glucagon Receptor COOH-Terminal Domain in Glucagon-Mediated Signaling and Receptor Internalization

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Insulin receptors and insulin effects on type II alveolar epithelial cells

Type II alveolar epithelial cells (pneumocytes) were isolated to purity from adult rabbits and analyzed for the presence of cell surface insulin receptors and for effects of insulin on cells. Assays were performed on cells cultured for 24 h in Eagles minimum essential medium. Insulin binding to cells in culture approached a steady-state level by 180 min at 15 degrees C and remained constant for at least 1 h. Competition experiments using native insulin, proinsulin and desoctapeptide supported specificity of binding. Scatchard analysis of binding revealed a class of high-affinity receptors with Kd = 1.5 X 10(-10) M and a low-affinity component with Kd = 4 X 10(-9) M. The number of receptors was estimated at 2000-4000/cell. Insulin added to cell cultures of type II pneumocytes in concentrations from 5 X 10(-11) to 5 X 10(-8) M resulted in a dose-related increase in uptake of 2-deoxyglucose by cells. Insulin also stimulated the incorporation of choline and glucose into phosphatidylcholine and disaturated phosphatidylcholine.

Insulin degradation by adipose tissue. Studies at several levels of cellular organization

A systematic study of the degradation of physiological concentrations of 125I-labelled insulin was performed in intact fat-pads, isolated adipocytes and subcellular fractions of isolated adipocytes. The findings indicate that insulin is rapidly degraded to low-molecular-weight peptides and/or amino acids by the intact tissue and isolated cells. Of the total insulin-degradation products present after incubation with an intact fat-pad, 94% is recovered in the medium, indicating that these products are not retained by the cells or tissue. The plasma membranes do not degrade insulin significantly in the absence of reduced glutathione, and over 99% of the cellular degradative capacity is found in the postmicrosomal supernatant (cytosol). The cytosol degrades insulin to several labelled fragments that are intermediate in size between insulin and insulin A chain, as well as to the low-molecular-weight tissue degradation products. Inclusion of plasma membranes with cytosol accelerates the cleavage of the intermediate fragments to the size of the small products seen with the intact tissue. However, plasma membranes do not increase the initial step in the degradation of insulin when incubated with cytosol, suggesting that the insulin receptor is not involved with the direct cleavage of insulin. This study supports the hypothesis that the bulk of insulin degradation occurs in the adipocyte cytosol, where intermediate-sized fragments are generated and rapidly cleaved to smaller products by the plasma membrane and quickly released into the surrounding medium.