Gerald J. Taborsky

Evidence of Cosecretion of Islet Amyloid Polypeptide and Insulin by β-Cells

Islet amyloid polypeptide (IAPP) has been identified as the major constituent of the pancreatic amyloid of non-insulin-dependent diabetes mellitus (NIDDM) and is also present in normal β-cell secretory granules. To determine whether IAPP is a pancreatic secretory product, we measured the quantity of lAPP-like immunoreactivity (IAPP-LI), insulin, and glucagon released into 5 ml of incubation medium during a 2-h incubation of monolayer cultures (n = 5) of neonatal (3- to 5-day-old) Sprague-Dawley rat pancreases under three conditions: 1.67 mM glucose, 16.7 mM glucose, and 16.7 mM glucose plus 10 mM arginine and 0.1 mM isobutylmethylxanthine (IBMX). The quantity of IAPP-LI, insulin, and glucagon in the cell extract was also determined. Mean ± SE IAPP-LI in the incubation medium increased from 0.041 ± 0.003 pmol in 1.67 mM glucose to 0.168 ± 0.029 pmol in 16.7 mM glucose (P < 0.05) and 1.02 ± 0.06 pmol in 16.7 mM glucose plus arginine and IBMX (P < 0.05 vs. 1.67 or 16.7 mM glucose). Insulin secretion increased similarly from 4.34 ± 0.27 to 20.2 ± 0.6 pmol (P < 0.05) and then to 135 ± 5 pmol (P < 0.05 vs. 1.67 or 16.7 mM glucose). Glucagon release tended to decrease with the increase in glucose concentration (0.39 ± 0.01 vs. 0.33 ± 0.02 pmol, P < 0.1), whereas with the addition of arginine and IBMX to high glucose, glucagon release increased to 1.32 ± 0.03 pmol (P < 0.05 vs. 1.67 or 16.7 mM glucose). Thus, the molar proportion of IAPP-LI to insulin secreted in low glucose was ∼1% and did not differ significantly with stimulation (0.95 ± 0.08 vs. 0.84 ± 0.15 vs. 0.76 ± 0.05%). In contrast, there was no constant proportional relationship between the release of IAPP-LI and glucagon (10.6 ± 0.8 vs. 51.3 ± 8.7 vs. 77.5 ± 5.2%). After incubation in 1.67 mM glucose, the extracted cells contained 3.7 ± 0.2 pmol IAPP-LI, 944 ± 25 pmol insulin, and 28.2 ±1.5 pmol glucagon. After maximal stimulation, the fractional release of IAPP-LI was 26.7 ± 0.7% vs. 14.7 ± 0.6% of insulin and 4.4 ± 0.2% of glucagon. These data indicate that nondiabetic neonatal rat islet cultures contain IAPP-LI and release it after stimulation by glucose and nonglucose secretagogues. Furthermore, the data suggest that IAPP-LI is a product of the β-cell, which coreleases it with insulin in a molar ratio of ∼1.100.

Increased β-Cell Secretory Capacity as Mechanism for Islet Adaptation to Nicotinic Acid-Induced Insulin Resistance

To determine whether prolonged nicotinic acid (NA) administration produces insulin resistance and, if so, how the normal pancreatic islet adapts to prolonged insulin resistance, we administered incremental doses of NA to 11 normal men for 2 wk, ending at 2 g/day. Insulin sensitivity was measured with Bergmans minimal model. Islet function was evaluated by measurement of acute insulin (AIR) and glucagon (AGR) responses to arginine at three glucose levels. Insulin resistance was demonstrated and quantified by a marked drop in the insulin sensitivity index (S1) from 6.72 ± 0.77 to 2.47 ± 0.36 × 10−5 min−1/pM (P < .0001) and resulted in a doubling of basal immunoreactive insulin levels (from 75 ± 7 to 157 ± 21 pM, P < .001) with no change in fasting glucose (5.5 ± 0.1 vs. 5.7 ± 0.1 mM). Proinsulin levels also increased (from 9 ± 1 to 15 ± 2 pM, P < .005), but the ratio of proinsulin to immunoreactive insulin did not change (12.7 ± 1.9 vs. 10.3 ± 1.9%). β-Cell changes were characterized by increases in the AIR to glucose (from 548 ± 157 to 829 ± 157 pM, P < .005) and in the AIR to arginine at the fasting glucose level (from 431 ± 54 to 788 ± 164 pM, P < .05). At the maximal hyperglycemia level the AIR to arginine represents β-cell secretory capacity, and this increased with administration of NA (from 2062 ± 267 to 2630 ± 363 pM, P < .05). From the AIRs to arginine an estimate of the glucose level giving half-maximal AIR to arginine can be calculated. This measure did not increase (10.0 ± 0.5 vs. 9.6 ± 0.9 mM). The AGRs to argininewere reduced at all glucose levels during NA administration. Thus, the pancreatic islet adapts to the prolonged insulin resistance induced by NA. This adaptation comprises a combination of increased insulin secretion and reduced glucagon secretion. The changes in insulin secretion can be entirely explained by an increase in the secretory capacity of the β-cell.

Glucose Disposal is Not Proportional to Plasma Glucose Level in Man

Metabolic clearance rate (MCR) of glucose has been defined as the rate of glucose utilization divided by the glucose concentration. This model of glucose transport has been widely used as a measure of hormonally regulated glucose disposal, on the assumption that glucose disposal rate is proportional to glucose concentration. To test this assumption, the relationship between glucose concentration and disposal rate was studied in man during infusion of somatostatin ± exogenous insulin to achieve fixed plasma insulin levels of 1,18, and 46 μU/ml on separate days. When glucose concentration was increased to more than twice basal fasting levels, the glucose disposal rate increased significantly at all three insulin levels. However, the increase was not proportional to the rise in glucose concentration, and MCR fell by 38%, 16%, and 11% at the low, medium, and high insulin levels, respectively. These results are explained by an alternative model of glucose transport in which insulin-independent tissues such as brain have a relatively fixed glucose uptake, while other tissues have glucose transport systems which take up glucose at a rate proportional to its plasma concentration. We conclude that MCR of glucose is not a good measure of hormonally regulated glucose disposal because it is partially dependent on the glucose concentration, particularly at low insulin levels.

An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.

Leptin Activates a Novel CNS Mechanism for Insulin-Independent Normalization of Severe Diabetic Hyperglycemia

The brain has emerged as a target for the insulin-sensitizing effects of several hormonal and nutrient-related signals. The current studies were undertaken to investigate mechanisms whereby leptin lowers circulating blood glucose levels independently of insulin. After extending previous evidence that leptin infusion directly into the lateral cerebral ventricle ameliorates hyperglycemia in rats with streptozotocin-induced uncontrolled diabetes mellitus, we showed that the underlying mechanism is independent of changes of food intake, urinary glucose excretion, or recovery of pancreatic β-cells. Instead, leptin action in the brain potently suppresses hepatic glucose production while increasing tissue glucose uptake despite persistent, severe insulin deficiency. This leptin action is distinct from its previously reported effect to increase insulin sensitivity in the liver and offers compelling evidence that the brain has the capacity to normalize diabetic hyperglycemia in the presence of sufficient amounts of central nervous system leptin.

Minireview: The Role of the Autonomic Nervous System in Mediating the Glucagon Response to Hypoglycemia

In type 1 diabetes, the impairment of the glucagon response to hypoglycemia increases both its severity and duration. In nondiabetic individuals, hypoglycemia activates the autonomic nervous system, which in turn mediates the majority of the glucagon response to moderate and marked hypoglycemia. The first goal of this minireview is therefore to illustrate and document these autonomic mechanisms. Specifically we describe the hypoglycemic thresholds for activating the three autonomic inputs to the islet (parasympathetic nerves, sympathetic nerves, and adrenal medullary epinephrine) and their magnitudes of activation as glucose falls from euglycemia to near fatal levels. The implication is that their relative contributions to this glucagon response depend on the severity of hypoglycemia. The second goal of this minireview is to discuss known and suspected down-regulation or damage to these mechanisms in diabetes. We address defects in the central nervous system, the peripheral nervous system, and in the islet itself. They are categorized as either functional defects caused by glucose dysregulation or structural defects caused by the autoimmune attack of the islet. In the last section of the minireview, we outline approaches for reversing these defects. Such reversal has both scientific and clinical benefit. Scientifically, one could determine the contribution of these defects to the impairment of glucagon response seen early in type 1 diabetes. Clinically, restoring this glucagon response would allow more aggressive treatment of the chronic hyperglycemia that is linked to the debilitating long-term complications of this disease.

Intra-islet insulin permits glucose to directly suppress pancreatic A cell function.

Inhibition of pancreatic glucagon secretion during hyperglycemia could be mediated by (a) glucose, (b) insulin, (c) somatostatin, or (d) glucose in conjunction with insulin. To determine the role of these factors in the mediation of glucagon suppression, we injected alloxan while clamping the arterial supply of the pancreatic splenic lobe of dogs, thus inducing insulin deficiency localized to the ventral lobe and avoiding hyperglycemia. Ventral lobe insulin, glucagon, and somatostatin outputs were then measured in response to a stepped IV glucose infusion. In control dogs glucagon suppression occurred at a glucose level of 150 mg/dl and somatostatin output increased at glucose greater than 250 mg/dl. In alloxan-treated dogs glucagon output was not suppressed nor did somatostatin output increase. We concluded that insulin was required in the mediation of glucagon suppression and somatostatin stimulation. Subsequently, we infused insulin at high rates directly into the artery that supplied the beta cell-deficient lobe in six alloxan-treated dogs. Insulin infusion alone did not cause suppression of glucagon or stimulation of somatostatin; however, insulin repletion during glucose infusions did restore the ability of hyperglycemia to suppress glucagon and stimulate somatostatin. We conclude that intra-islet insulin permits glucose to suppress glucagon secretion and stimulate somatostatin during hyperglycemia.

Leptin Deficiency Causes Insulin Resistance Induced by Uncontrolled Diabetes

OBJECTIVE Depletion of body fat stores during uncontrolled, insulin-deficient diabetes (uDM) results in markedly reduced plasma leptin levels. This study investigated the role of leptin deficiency in the genesis of severe insulin resistance and related metabolic and neuroendocrine derangements induced by uDM. RESEARCH DESIGN AND METHODS Adult male Wistar rats remained nondiabetic or were injected with the β-cell toxin, streptozotocin (STZ) to induce uDM and subsequently underwent subcutaneous implantation of an osmotic minipump containing either vehicle or leptin at a dose (150 μg/kg/day) designed to replace leptin at nondiabetic plasma levels. To control for leptin effects on food intake, another group of STZ-injected animals were pair fed to the intake of those receiving leptin. Food intake, body weight, and blood glucose levels were measured daily, with body composition and indirect calorimetry performed on day 11, and an insulin tolerance test to measure insulin sensitivity performed on day 16. Plasma hormone and substrate levels, hepatic gluconeogenic gene expression, and measures of tissue insulin signal transduction were also measured. RESULTS Physiologic leptin replacement prevented insulin resistance in uDM via a mechanism unrelated to changes in food intake or body weight. This effect was associated with reduced total body fat and hepatic triglyceride content, preservation of lean mass, and improved insulin signal transduction via the insulin receptor substrate–phosphatidylinositol-3-hydroxy kinase pathway in the liver, but not in skeletal muscle or adipose tissue. Although physiologic leptin replacement lowered blood glucose levels only slightly, it fully normalized elevated plasma glucagon and corticosterone levels and reversed the increased hepatic expression of gluconeogenic enzymes characteristic of rats with uDM. CONCLUSIONS We conclude that leptin deficiency plays a key role in the pathogenesis of severe insulin resistance and related endocrine disorders in uDM. Treatment of diabetes in humans may benefit from correction of leptin deficiency as well as insulin deficiency.

Galanin—Sympathetic Neurotransmitter in Endocrine Pancreas?

The effects of sympathetic neural activation on basal pancreatic hormone secretion cannot be explained solely by the actions of the classic sympathetic neurotransmitter norepinephrine. The nonadrenergic component may be mediated by the 29-amino acid peptide galanin in that this neuropeptide meets several of the criteria necessary to be considered a sympathetic neurotransmitter in the endocrine pancreas. 1) Galanin administration inhibits basal insulin and somatostatin secretion and stimulates basal glucagon secretion from the pancreas, qualitatively reproducing the effects of sympathetic nerve stimulation. These sympathomimetic effects appear to be mediated by direct actions of galanin on the islet. 2) Galanin-like immunoreactivity exists in fibers that innervate pancreatic islets. 3) Galanin is released during electrical stimulation of pancreatic nerves. The quantity released is sufficient to reproduce sympathetic nerve stimulation-induced effects on insulin secretion and to contribute to the neural effects on somatostatin and glucagon release. 4) Whether interference with galanin action or release reduces the islet response to sympathetic nerve stimulation remains to be determined. We hypothesize that galanin and norepinephrine act together to mediate the islet response to sympathetic neural activation. If galanin is a sympathetic neurotransmitter in the endocrine pancreas, it may contribute to the inhibition of insulin secretion that occurs during stress and thereby to the hyperglycemic response. Moreover, the local presence of this potent β-cell inhibitor in the islet leads to speculation on galanins contribution to the impairment of insulin secretion that occurs in non-insulin-dependent diabetes mellitus and therefore on the potential utility of a galanin antagonist in the treatment of this disease.

Reduction of Glycemic Potentiation: Sensitive Indicator of β-Cell Loss in Partially Pancreatectomized Dogs

To determine which test of islet function is the most sensitive indicator of subclinical β-cell loss, we studied six conscious dogs before and 1 and 6 wk after removal of the splenic and uncinate lobes [64 ± 2% pancreatectomy (PX)]. To assess hyperglycemic potentiation, acute insulin secretory responses (AIR) to 5 g i.v. arginine were measured at the fasting plasma glucose (FPG) level after PG was clamped at ∼250 mg/dl and after PG was clamped at a maximally potentiating level of 550–650 mg/dl. FPG levels were unaffected by PX (112 ± 4 mg/dl pre-PX vs. 115 ± 5 mg/dl 6 wk after PX, P NS). Similarly, basal insulin levels remained constant after PX (11 ± 2 μU/ml pre-PX vs. 11 ± 1 μU/ml 6 wk after PX, P NS). The AIR to 300 mg/kg i.v. glucose decreased slightly from 42 ± 9 μU/ml pre-PX to 32 ± 5 μU/ml 6 wk after PX (P NS), and thus the β-cell loss was underestimated. In contrast, insulin responses to arginine declined markedly after PX. The AIR to arginine obtained at FPG levels declined from 23 ± 3 μU/ml pre-PX to 13 ± 2 μU/ml 6 wk after PX (P = .04). The AIR to arginine obtained at PG levels of ∼250 mg/dl declined even more, from a pre-PX value of 56 ± 7 μU/ml to 21 ± 4 μU/ml 6 wk after PX (P = .02). However, the largest decline in AIR to arginine occurred at PG levels of 550–650 mg/dl (113 ± 13 μU/ml pre-PX vs. 28 ± 7 μU/ml 6 wk after PX, P = .001), thus indicating a marked decrease of maximal glycemic potentiation. Basal levels of glucose and insulin and AIRs to glucose and arginine obtained 1 wk after PX were similar to those obtained 6 wk after PX. The second purpose of the study was to explore mechanisms by which compensation of this β-cell loss occurs and by which hyperglycemia is avoided. One possibility is a reduction of glucagon secretion, but immunoreactive glucagon levels and glucagon secretory responses to arginine measured at three PG levels remained unchanged after PX. Similarly, tissue sensitivity to insulin, measured with euglycemic clamps at two elevated insulin levels, and insulin clearance remained unchanged after PX. Thus, the mechanism for maintenance of euglycemia after partial PX in the dog remains unclear. In summary, a reduction in glycemic potentiation of the insulin response to arginine was found to be the best means of detecting the subclinical β-cell loss induced by a two-thirds PX in dogs.