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Mitochondrial metabolism plays an integral role in glucose-stimulated insulin secretion (GSIS) in β-cells. In addition, the diabetogenic role of glucagon released from α-cells plays a major role in the etiology of both type 1 and type 2 diabetes because unopposed hyperglucagonemia is a pertinent contributor to diabetic hyperglycemia. Titrating expression levels of the mitochondrial protein mitoNEET is a powerful approach to fine-tune mitochondrial capacity of cells. Mechanistically, β-cell–specific mitoNEET induction causes hyperglycemia and glucose intolerance due to activation of a Parkin-dependent mitophagic pathway, leading to the formation of vacuoles and uniquely structured mitophagosomes. Induction of mitoNEET in α-cells leads to fasting-induced hypoglycemia and hypersecretion of insulin during GSIS. MitoNEET-challenged α-cells exert potent antiapoptotic effects on β-cells and prevent cellular dysfunction associated with mitoNEET overexpression in β-cells. These observations identify that reduced mitochondrial function in α-cells exerts potently protective effects on β-cells, preserving β-cell viability and mass.

Hyperglucagonemia is a hallmark in obesity and type 2 diabetes (T2DM). Suppression of glucagon signaling improves glycemic control in T2DM. We evaluated glucagon homeostasis in lean and obese mice and people. Discordant with the canonical rise in glucagon with fasting, our studies show that fasting (4, 8, 16 and 24 h) caused a progressive decrease in serum glucagon in diet-induced obese, hyperglucagonemic mice (P Disclosure J.H. Stern: None. G.I. Smith: None. R.H. Unger: None. S. Klein: Stock/Shareholder; Self; Aspire Bariatrics. Consultant; Self; Pfizer Inc.. Research Support; Self; Merck & Co., Inc., Johnson & Johnson Services, Inc., REMD Biotherapeutics. P.E. Scherer: None.

Since the discovery of glucagon’s opposing actions to insulin, drugs targeting the inhibition of glucagon action have been pondered. In recent years, several attempts to generate small molecules or antibodies that impair glucagon action have been pursued as potential therapeutics for type 2 diabetes. In the current issue of Diabetes Care , Kazda et al. (1) summarize the outcomes of the phase 2a and phase 2b clinical trials of LY2409021, a small-molecule glucagon receptor antagonist (GRA). This is the largest and longest trial for safety and efficacy of a GRA ever performed. Importantly, LY2409021 does not produce side effects on cholesterol homeostasis that have impeded the progress of other small-molecule GRAs. Here, we place their success in perspective and discuss the advantages and concerns relating to glucagon-based therapeutics as this line of drugs comes closer than ever to achieving their clinical potential. In 1922, the first children with type 1 diabetes were treated with an insulin-containing pancreatic extract, preventing ketoacidosis and an insidious death. In addition to the discovery of insulin, the crew of Banting, Best, and Collip observed glucagon action, as they had noticed in their preclinical studies in canines that some of their crude insulin preparations would raise glucose levels in the dog briefly before glucose was lowered (2,3). This glucose-raising peptide was termed “glucagon” (4) and subsequently purified and identified as a 29–amino acid peptide (5). In 1959, the development of the radioimmunoassay made it possible to quantify the two major glucoregulatory hormones, insulin (6) and glucagon (7). It was quickly established that glucagon was in fact a true hormone responsible for maintaining the glucose supply to the brain via increased glycogenolysis and gluconeogenesis. It has since been demonstrated that every form of diabetes is associated with hyperglucagonemia, the suppression of which eliminates hyperglycemia (8 …

OBJECTIVE To evaluate racial/ethnic differences in pancreatic triglyceride (TG) levels and their relationship to β-cell dysfunction in humans. RESEARCH DESIGN AND METHODS We studied black, Hispanic, and white adults who completed three research visits: screening and an oral glucose tolerance test; frequently sampled intravenous glucose tolerance tests for evaluation of β-cell function and insulin resistance; and proton magnetic resonance spectroscopy for evaluation of pancreatic and hepatic TG levels. RESULTS Pancreatic TG levels were higher in Hispanics and whites than in blacks (P = 0.006). Hepatic TG levels were highest in Hispanics (P = 0.004). Compensatory insulin secretion and disposition index were higher in blacks (P = 0.003 and P = 0.024, respectively). Insulin sensitivity was comparable between Hispanics and blacks and was lower than in whites (P = 0.005). In blacks, compensatory insulin secretion increased steeply with small increments in pancreatic TG levels (R2 = 0.45, slope = 247). In whites, the range of pancreatic TG levels was higher, and the slope was less steep than in blacks (R2 = 0.27, slope = 27). In Hispanics, pancreatic TG levels were similar to those of whites, but compensatory insulin secretion was described by a combination of pancreatic and hepatic TG levels and visceral fat mass ( R2 = 0.32). CONCLUSIONS In a multiethnic sample of adults with mild obesity and without diabetes, we found striking ethnic differences in the levels of pancreatic TGs and in the relationship between pancreatic TGs and β-cell dysfunction. Our data implicate pancreatic TG content measured by proton magnetic resonance spectroscopy as a noninvasive novel biomarker for pancreatic β-cell dysfunction, especially in the Hispanic population.

OBJECTIVE To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency. RESEARCH DESIGN AND METHODS We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr−/−) mice and wild-type (Gcgr+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction. RESULTS Gcgr+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower. CONCLUSIONS We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.

The objective of this study was to determine whether the late failure of β-cells in islets transplanted via the portal vein is caused by excess insulin-stimulated lipogenesis and lipotoxicity and, if so, whether the damage can be prevented by reducing lipogenesis surrounding the islets. Based on the premise that high portal vein levels of nutrients and incretins would stimulate hyperinsulinemia, thereby inducing intense lipogenesis in nearby hepatocytes, normal islets were transplanted into livers of syngeneic streptozotocin-induced diabetic recipients. Hydrolysis of the surrounding fat would flood the islet grafts with fatty acids that could damage and destroy the β-cells. Reducing lipogenesis by leptin or caloric restriction should prevent or reduce the destruction. After a rise after transplantation, insulin levels gradually declined and hyperglycemia increased. Four weeks after transplantation mRNA of the lipogenic transcription factor, sterol regulatory element–binding protein-1c (SREBP-1c) and its lipogenic target enzymes were elevated in livers of these recipients, as was triacylglycerol content. Positive oil red O staining for lipids and immunostaining for SREBP-1 were observed in hepatocytes surrounding islets with damaged β-cells. Leptin-induced lipopenia prevented and caloric restriction reduced steatosis, hyperglycemia, and apoptotic β-cell destruction. Excessive SREBP-1c–mediated lipogenesis, induced in hepatocytes by insulin hypersecretion, is followed by β-cell destruction in the grafts and reappearance of diabetes. Graft failure is prevented by blocking lipogenesis. The results suggest that strict antilipogenic intervention might improve outcomes after human islet transplantation.

Overaccumulation of fat in pancreatic islets of obese ZDF fa/fa rats is believed to cause beta-cell failure and diabetes. Previously, we demonstrated that ZDF islets have an increased capacity to esterify fatty acids imported via the circulation. Here we examine the capacity of ZDF islets to synthesize fatty acids de novo. Compared with age-matched wild-type (+/+) control islets, acetyl CoA carboxylase (ACC) mRNA was fivefold and sixfold higher and fatty acid synthetase (FAS) was fourfold and sevenfold higher in prediabetic and diabetic ZDF islets, respectively. Incorporation of label from [14C]glucose into lipids was 84% higher in ZDF islets and was not suppressed normally by fatty acids. Chronic hyperleptinemia, induced by adenoviral transfer of leptin cDNA, reduced ACC and FAS mRNA in +/+ islets by 93 and 80%, respectively, but did not decrease the high ACC and FAS expression in islets of fa/fa rats. Recombinant leptin cultured with islets isolated from +/+ rats lowered ACC and FAS expression by 66 and 47%, respectively, but had no effect in fa/fa islets. We conclude that de novo lipogenesis in islets is controlled by leptin and remains low in leptin-responsive islets. It is increased in leptin-insensitive fa/fa islets, contributing to the fat overload that leads to beta-cell dysfunction and diabetes.

Leptin was overexpressed in the liver of normal Wistar rats by infusing recombinant adenovirus containing the cDNA encoding leptin. Plasma leptin levels rose to 12–24 ng/ml (vs.

We sought to explore the emerging concept that malonyl-CoA generation, with concomitant suppression of mitochondrial carnitine palmitoyltransferase I (CPT I), represents an important component of glucose-stimulated insulin secretion (GSIS) by the pancreatic β-cell (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long-chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802–5810, 1992). Accordingly, pancreases from fed rats were perfused with basal (3 mM) followed by high (20 mM) glucose in the absence or presence of 2 mM hydroxycitrate (HC), an inhibitor of ATP-citrate (CIT) lyase (the penultimate step in the glucose → malonyl-CoA conversion). HC profoundly inhibited GSIS, whereas CIT had no effect. Inclusion of 0.5 mM palmitate in the perfusate significantly enhanced GSIS and completely offset the negative effect of HC. In isolated islets, HC stimulated [1-14C]palmitate oxidation in the presence of basal glucose and markedly obtunded the inhibitory effect of high glucose. Directional changes in 14C incorporation into phospholipids were opposite to those of 14CO2 production. At a concentration of 0.2 mM, 2-bromostearate, 2-bromopalmitate and etomoxir (all CPT I inhibitors) potentiated GSIS by the pancreas and inhibited palmitate oxidation in islets. However, at 0.05 mM, etomoxir did not influence insulin secretion but still caused significant suppression of fatty acid oxidation. The results provide more direct evidence for a pivotal role of malonyl-CoA suppression of CPT I, with attendant elevation of the cytosolic long-chain acyl-CoA concentration, in GSIS from the normal pancreatic β-cell. They also raise the possibility that the tested CPT I inhibitors act, in part, by generating non-metabolizable acyl-CoA species that mimic the effects of natural acyl-CoAs in triggering insulin release.

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.

Previous studies indicate that diets rich in digestible carbohydrates improve glucose tolerance in nondiabetic individuals, but may worsen glycemic control in NIDDM patients with moderately severe hyperglycemia. The effects of such high-carbohydrate diets on glucose metabolism in patients with mild NIDDM have not been studied adequately. This study compares responses to an isocaloric high-carbohydrate diet (60% of total energy from carbohydrates) and a low-carbohydrate diet (35% of total energy from carbohydrates) in 8 men with mild NIDDM. Both diets were low in saturated fatty acids, whereas the low-carbohydrate diet was rich in monounsaturated fatty acids. The two diets were matched for dietary fiber content (25 g/day). All patients were randomly assigned to receive first one and then the other diet, each for a period of 21 days, in a metabolic ward. Compared with the low-carbohydrate diet, the high-carbohydrate diet caused a 27.5% increase in plasma triglycerides and a similar increase in VLDL-cholesterol levels; it also reduced levels of HDL cholesterol by 11%. Plasma glucose and insulin responses to identical standard breakfast meals were studied on days 4 and 21 of each period, and these did not differ significantly between the two diets. At the end of each period, a euglycemic hyperinsulinemic glucose clamp study with simultaneous infusion of [3-3H]glucose revealed no significant changes in hepatic insulin sensitivity; and peripheral insulin-mediated glucose disposal remained unchanged (14.7 ± 1.4 vs. 16.5 ± 2.3 μM · kg−1 · min−1 on the high-carbohydrate and low-carbohydrate diets, respectively). We conclude that in patients with mild NIDDM, high-carbohydrate diets do not improve glycemic control nor insulin sensitivity, and they raise plasma triglyceride and VLDL-cholesterol concentrations and reduce HDL-cholesterol levels, which may not be desirable.

Groups of 27 nondiabetic Pima Indians, 34 nondiabetic Caucasians, and 12 diabetic Pima Indians with recent onset of their disease received an arginine infusion to determine if (1) nondiabetic Pima Indians and Caucasians had a similar glucagon response to arginine and (2) diabetic Pimas had excessive glucagon response to arginine as reported in other racial groups. The fasting glucagon levels in the three groups were not significantly different. During arginine monochloride infusion (5 mg./kg./minute for 40 minutes) the diabetic Pimas had glucagon levels significantly higher at 10 minutes and at all sampling points thereafter than the normo-glycemic Pimas. Plasma insulin levels also increased during the infusion but, notably, never differed significantly between these two groups. There was no significant difference in the glucagon levels at any sampling point between the nondiabetic Pimas and Caucasians. The differences in glucagon levels between the nondiabetic and diabetic Indians are similar to those differences reported between diabetic and nondiabetic subjects of other racial origins.

Electron microscopy using antiglucagon or antiglicentin antisera and the protein A-gold (pAg) technique revealed a population of immunoreactive cells in the gastric mucosa of human fetuses. The secretory granules of these cells showed the same ultrastructural characteristics and the same labeling pattern as pancreatic α-granules. These data demonstrate that the stomach of human fetuses contains cells with secretory granules indistinguishable by morphologic and immunocytochemical criteria from the pancreatic Acells.

Twenty-one nondiabetic subjects, their weights ranging from 56 to 165 kg, received an infusion of glucose (420 mg/min), insulin (0.77 mU/kg/min), and somatostatin (500 figlh) for 150 min. A steady state level of plasma insulin and glucose was attained after 90 min. Endogenous insulin secretion, determined by C-peptide measurement, and glucagon secretion remained suppressed throughout the period. With similar steady state levels of plasma insulin (SSPI) maintained in all subjects, the height of the steady state plasma glucose concentration (SSPG) was considered an index of total body sensitivity to insulin-mediated glucose uptake. A positive correlation between SSPG and the degree of obesity, as determined by the body mass index (BMI), was demonstrated (r = 0.70, P < 0.001). No correlation was found between SSPI and BMI. The fasting plasma insulin concentration correlated with BMI (r = 0.82, P < 0.0001) and SSPG (r = 0.80, P < 0.0001). This method provides a simple safe measure of total body insulin resistance over a wide range of obesity and is independent of endogenous insulin secretion.

This study was designed to determine if hyperglucagonemia approximating that believed to be present in the portal vein of poorly controlled diabetics can exert metabollcally deleterious effects in insulin-treated diabetics. Three juvenile-type diabetics and one maturity-onset-type patient were given glucagon intravenously at a dose of 6 ng./kg./min. for 24 hours while receiving a continuous infusion of Insulin. This raised their plasma IRG levels from an average of 122 ± 15 pg./ml. on the control day to 594 ± 60 pg./ml. Mean plasma glucose concentration, measured at two-hour intervals around the clock, averaged 200 ± 20 mg./dl. on the control day and increased significantly in all four patients to 293 ± 18 mg./dl. (p < 0.05) during glucagon administration; fasting glucose rose from 138 ± 10 mg./dl. to 201 ± 17 (p < 0.05). Glucose excretion rose significantly from the control value of 36 ± 14 gm. per 24 hours to 152 ± 19 gm. per 24 hours (p < 0.05). The excretion of both urea nitrogen and ketones also increased significantly (p < 0.05) during glucagon administration. The three juvenile-diabetic patients also received a glucagon infusion at a rate of only 3 ng./kg./min., and in each evidence of deterioration in metabolic control was observed. In every experiment, termination of the glucagon infusion was followed by a recession in the metabolic abnormalities. These data indicate that exogenous hyperglucagonemia of the magnitude believed to be present in the portai vein of uncontrolled diabetics can cause metabolic deterioration in diabetics receiving continuous insulin treatment.

The application of immunofluorescence technique with anti-insulin, anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide (PP) antisera to sections of precisely sampled regions of the human pancreas allowed the quantitative evaluation of the total content of these four endocrine cell populations in 13 nondiabetics, in 2 insulin-dependent diabetics (IDDM), and in 2 non-insulin-dependent diabetic subjects (NIDDM) of various age and sex. In nondiabetic subjects, PP-cells appear sex-related. Male individuals have a significantly greater volume of PP-cells than female. In diabetic subjects, the only marked difference as compared with nondiabetics is the reduction of insulin cell volume in IDDM. Other small differences between individual endocrine cell volumes are detectable in both IDDM and NIDDM as compared with nondiabetics, but their significance is at present unclear. The qualitative changes of islet structure accompanying insulin cell reduction in IDDM were not considered in the present Study.

Five tumors associated with the complete glucagonoma syndrome, as well as a series of glucagon-cell adenomas from three patients without this syndrome, were investigated by light and electron microscopy and by immunofluorescence. All tumors associated with the syndrome were large, from 3 to 35 cm along the major axis, and three of them were proved to be malignant. No common histologic arrangement of tumor cells was apparent for the five neoplasms examined. Immunofluorescent staining for glucagon and glicentin was carried out: while most cells were negative, a varying number of scattered cells were positive with both antisera in all tumors except one; three tumors contained more glicentin- than glucagon-immunoreactive cells. Moreover, three tumors were multihormonal, with cells positive for pancreatic polypeptide and/or insulin. Ultrastruc-turally, the secretory granules of cells from these tumors were not typical of those found in A-cells from adult human islets. The glucagon-cell tumors from patients without the syndrome were benign, usually multiple, and were small, with diameters from 0.5 mm to 1 cm. In most cases, the cells from these neoplasms arranged in a characteristic pattern (ribbonlike or “gyriform”). In most tumors, the majority of cells showed both glucagon and glicentin immunofluorescence and the ultra-structural appearance of their secretory granules was similar to that of normal islet A-cells. From the morphologic point of view, therefore, cells from tumors not associated with the glucagonoma syndrome resemble normal glucagon cells more closely than those from tumors associated with the syndrome.

In eleven dogs made hypotensive by means of rapid exsanguination over a period of forty to seventy minutes, mean glucagon rose to a peak of 516 ± (S.E.) 150 pg/ml. and mean glucose to a peak of 341 ± 27 mg. per 100 ml. The hyperglucagonemia induced by exsanguination was substantially reduced, although not abolished, by propranolol infusion, but was not diminished by phentolamine, suggesting that it was largely a β-adrenergic effect. Its possible contribution to survival during shock is considered.

To investigate the effect of secretiti upon islet hormone secretion, highly purified porcine secretin was perfused at 0.0001, 0.001, 0.01, and 0.1 clinical unit (CU)/ml in the isolated normal dog pancreas. The lowest concentration of secretin, a level which is within the physiologic range, significantly suppressed glucagon release and stimulated somatostatin release but was without significant effect on insulin release. At each concentration of secretin tested glucagon was dramatically suppressed and somatostatin was significantly stimulated, both in a dose-dependent manner. B-cell activity was first augmented by the concentration of 0.001 CU/ml (94 pM) of secretin. These data demonstrate that physiologic levels of secretin suppress pancreatic A-cell activity in the isolated dog pancreas.

Meticulous metabolic management of diabetes has become a feasible therapeutic option now being offered to increasing numbers of diabetics in the hope of delaying or preventing diabetic complications. This hope, while still untested by rigorous clinical trials, is nevertheless supported by a considerable body of scientific and clinical data (see ref. 1 for a review) and could well represent the first truly significant therapeutic advance since the discovery of insulin. Recently, however, several reports of unexpected deaths have occurred in patients “tightly” controlled with open-loop insulin delivery devices. At least some of these deaths are believed to have been directly or indirectly attributable to hypoglycemia. These reports, together with new understanding of the hormonal and metabolic lesions in treated and untreated type I diabetes, have raised legitimate questions as to the safety of efforts to normalize glycemia, whether by continuous subcutaneous insulin infusion or by multiple injections. In this review, the potential long-term benefits of meticulous glucoregulation will be weighed against the short-term risks, particularly the risk of hypoglycemic brain damage, and guidelines will be proposed in the hope of reducing the risk-benefit ratio of such regimens to an absolute minimum.

To determine the effect of an increase in insulin levels within the range occurring under physiologic conditions on the protein- and acid-induced release of splanchnic somatostatin, insulin was infused in dogs for 1 h following the intragastric instillation of a neutral protein load (20% liver extract at pH 7), a weak stimulus of somatostatin-like immunoreactivity (SLI), and after an intragastric HCl, a strong stimulus of SLI release, instilled 30 min later. Insulin levels between 50 and 60 microunits/ml significantly reduced the rise in peripheral venous SLI levels elicited by the acid load from a mean integrated incremental value of 1705 +/- 182 pg/ml in controls to 840 +/- 312 in the insulin-infused group (P less than 0.05). Prevention of the insulin-induced hypoglycemia and the secondary rise in glucagon, a known stimulus of pancreatic somatostatin secretion, by means of a concomitant infusion of glucose, did not modify the reduction in acid-induced increase in plasma SLI concentration associated with hyperinsulinemia. Insulin-glucose infusion significantly lowered the SLI in the pancreaticoduodenal vein, and in the gastroepiploic vein draining the antrum (P less than 0.02; P less than 0.05), but not in the short gastric veins draining the fundus of the stomach in response to the acid load. It is concluded that physiologic elevation of insulin levels causes significantly reduced response of SLI to an intragastric acid load in dogs. This reduction is explained by a diminished increment of SLI in the venous effluent of the pancreas and the antrum.

Chronic clamping of plasma glucose levels at greater than or equal to 250 mg/dl in four partially depancreatized but previously nondiabetic dogs was followed within 2 wk by persistent hyperglycemia and glycosuria of less than or equal to 500 g/day, ketonuria, and weight loss. Three of the four dogs required daily insulin injections to control these catabolic manifestations. There was no evidence of spontaneous improvement of the severe diabetic state during the 39-69 days of observation after discontinuation of intravenous glucose infusion. Impairment of intravenous glucose tolerance, loss of the insulin response to glucose and arginine, fasting hyperglucagonemia, exaggerated glucagon responsiveness to arginine, and a significant reduction in sensitivity to insulin were characteristic of all diabetic dogs. Morphometric analysis of the endocrine pancreas revealed a profound reduction in the number and size of identifiable islets of the hyperglycemic dogs compared with islets from their own pancreases resected months earlier and with those from pancreatic remnants of eight subtotally depancreatized control dogs that had not been subjected to chronic hyperglycemic clamping. The reduction in number and size of islets of the hyperglycemic dogs was largely the consequence of depletion of insulin-containing cells and was similar to that of dogs with long-standing alloxan-induced diabetes. In the eight control dogs, clinical evidence of diabetes did not develop during a follow-up period of 193-296 days. In this group, there was no evidence of diminution of intravenous glucose tolerance, of the insulin response to glucose or arginine, or of insulin sensitivity as determined by an acute hyperinsulinemic hyperglycemic clamp. The number and size of islets and number of beta-cells in pancreatic remnants from these dogs did not differ morphometrically from those of the pancreatic segment that had been resected. We conclude that in subtotally depancreatized but nondiabetic dogs, maintenance of constant hyperglycemia of greater than or equal to 250 mg/dl by means of intravenous glucose infusion causes a severe, persistent, and often insulin-requiring diabetic state that does not occur in the absence of the hyperglycemia.

To determine the hormonal status of somatostatin in man, plasma levels of approximately 1600-dalton somatostatin-like immunoreactivity (SLI) were measured before and after a mixed meal. Plasma was subjected to gel filtration and the fractions coeluting with somatostatin were concentrated ninefold before radioimmunoassay. In this system the recovery of synthetic somatostatin added to plasma averaged 71 +/- 4.6% (mean +/- SE, N = 5). Synthetic somatostatin infused into normal volunteers gave a dose-related increase in the measured SLI value. Fasting SLI in 13 normal volunteers was 8.0 +/- 0.7 pg/ml (mean +/- SE) and rose to 18.6 +/- 1.5 pg/ml and 20.3 +/- 2.0 pg/ml at 60 and 120 min, respectively, after the meal (P less than 0.001). In seven type I diabetics, the fasting level was 11.5 +/- 1.6 pg/ml and the 60- and 120-min postprandial levels were 21.4 +/- 2.5 and 22.3 +/- 2.9 pg/ml, respectively. The meal-induced rise in approximately 1600 M.W. SLI approximated that produced by infusing somatostatin at 2 microgram/h, a rate that significantly suppressed glucagon levels. These results are therefore consistent with a hormonal role for somatostatin in man.

The Berson Memorial Lecture was established to perpetuate the memory of a great scientist, Dr. Solomon Berson (Figure 1), who died tragically at age 53, just 5 years before Dr. Rosalyn Yalow received the Nobel Prize in Physiology or Medicine for their development of the radioimmunoassay. I am deeply honored to have been selected to be the first Berson Memorial Lecturer, but for me the award has a special significance that transcends the honor itself. This is because, in my eyes, Berson was more than the legendary figure whose brilliance as a scientist, clinician, mathematician, and classical musician won him the admiration and affection of his colleagues. To me, he was a special kind of friend, one who set the course of my research career.

The ability of varying levels of circulating insulin to suppress α- and Β-cell secretion was assessed by plasma glucagon and C-peptide measurement in 6 obese and 6 nonobese subjects maintained in a euglycemic state, with an insulin concentration elevated by 10, 20, or 100 μU/ml above basal levels by a primed-continuous infusion of insulin. The 10-μU/ml increase did not suppress C-peptide levels significantly in either group. However, incremental increases in plasma insulin of approximately 20 and 100 μU/ml above basal suppressed plasma C-peptide by 0.27 ± 0.14 and 0.53 ± 0.07 pmol/ml, respectively, in the obese subjects (14% and 31% of the basal values of 2.20 ± 0.18 and 2.19 ± 0.26 pmol/ml, respectively) and by 0.16 ± 0.06 and 0.17 ± 0.06 pmol/ml in the nonobese subjects (20% and 25% the basal values of 0.74 ± 0.11 and 0.78 ± 0.11 pmol/ml, respectively). Plasma glucagon levels were suppressed to a similar degree in each group in a dose-related manner during both the 20-μU/ ml and 100-μU/ml clamps. We were unable to identify an increment of insulin that suppressed C-peptide and/ or glucagon in one group but not in another. These data demonstrate inhibition of α- and Β-cell secretion by insulin within its physiologic range in both non-obese and obese man, and exclude insulin resistance of α- and β-cells in obese individuals. However, despite their much higher insulin levels before and during the insulin infusions, obese subjects had C-peptide levels that, at all times, were 3–3.5-fold higher than those of the nonobese subjects. This is consistent with a greater number and/or a higher secretory activity of β-cells in the obese subjects.

To determine if the abnormal A-cell function of human diabetes can be corrected by insulin therapy, 10 patients with juvenile-type and 10 with adult-onset types of diabetes were aggressively treated with insulin for up to 10 days. Glucose, immunoreactive glucagon (IRG), and, where possible, insulin were measured in plasma specimens obtained at two-hour intervals for periods of up to 10 days. These patients were compared with nine nondiabetics who were studied similarly. During a two-day “uncontrolled” period, marked hyperglycemie, glycosurie, and elevated IRG concentrations were present in both groups of diabetic patients. “Improved control” was achieved in the juvenile-type diabetic group with an average insulin dose of 115 ± 24 U. per day. Such therapy significantly reduced the mean plasma glucose (p < 0.001) and IRG (p < 0.05) concentrations when compared with the “uncontrolled” period, but both remained above (p < 0.05) the values seen in the nondiabetk subjects. “Improved control” was achieved in the maturity-onset group of patients with an average insulin dose of 160 ± 24 U. per day. This dose was associated with a mean plasma-insulin value of 106 ± 26 μU. per milliliter, significantly greater (p < 0.05) than that of the nondiabetk: group. Mean plasma glucose (p < 0.001) and IRG (p < 0.02) concentrations were reduced significantly as compared with the “uncontrolled” period; the mean IRG level was the same as that of the nondiabetics, but the mean plasma glucose levels remained significantly greater (p < 0.001) than in the nondiabetics. The results indicate that aggressive administration of insulin by conventional means does significantly reduce the average IRG level in juvenile-type and adult-type diabetics. However, despite the large doses of insulin and unphysiologically high concentrations of circulating insulin in the juvenile-diabetic group, IRG remained significantly above that of the nondiabetics. In the adult-onset group, although it was reduced to normal, the IRG level can be viewed as above normal relative to the persistent hyperglycemie and hyperinsulinemia.

By means of an exquisitely sensitive and highly specific radioimmunoassay, glucagon has been measured in the plasma of dogs and man for the first time. Its identification in the effluent plasma of the pancreas and the demonstration of alterations in its secretion induced by changes in blood glucose concentration support the view that it is a true hormone with a major role in blood glucose regulation. Glucagon secretion has been shown to rise during all forms of glucose need; this rise is suppressed by glucose refeeding. This favors the concept of glucagon as a hormone of glucose need, the function of which is to maximize hepatic glucose production when food is not available, thereby serving to maintain the flow of glucose to the brain. The current status of efforts to identify a disorder of glucagon secretion in man is reviewed briefly.

The role of pancreatic glucagon in starvation has been difficult to assess in humans because of the nonspecificity of antisera heretofore available for glucagon radioimmunoassay. The development of a relatively specific antispnim for pacreation glucagon has now made possible valid measurements of pancreatic glueagun in human plasma and the effect of total starvation on plasma glucagon was, therefore, re-examined. Ten healthy male volunteers abstained from food for seventy-two hours or longer. During this period the mean level of glucose declined from 86 to 70 mg./100 ml., insulin declined from 10 to 3 μ./ml., while glucagon rose progressively from a mean prestarvation level of 126 μμg./ml. to 157, 189, and 178 μμg./ml. at the end of one, two, and three days' starvation, respectively. The responsiveness of the alpha cells to arginine stimulation was tested at the end of the starvation period by means of a forty-minute infusion of arginine at a rate of 11.5 mg./kg./min. Every one of the five subjects tested exhibited a prompt rise in glucagon secretion which reached a peak of 516 μμg./ml. in thirty minutes, significantly greater than the glucagon response of fed controls. Mean insulin concentration during the arginine infusion rose only 6.8 μU./ml. in contrast to a 29 μU./ml. rise in fed subjects. The biologic effect of the modest rise (about 50 per cent) in glucagon concentration would be exaggerated by the concomitant decline in insulin concentration. The magnitude and promptness of the gjucagon response to arginine suggests that the pancreas contains abundant glucagon after three days of total starvation.

The extremely high levels of glucagon recently observed in dogs with severe alloxan-induced diabetes decline promptly and precipitously to normal as soon as exogenous insulin is infused. This suggests that the normal response of the pancreatic alpha cell to hyperglycemia requires the presence of circulating insulin. To determine if the relative hyperglucagonemia of human diabetics responds similarly to insulin repletion, the plasma glucagon response of ten adult-type diabetic patients to a large, predominantly carbohydrate meal was determined with and without the simultaneous forty-five-minute intravenous infusion of glucagonfree insulin (0.12 to 0.2 U./kg.). The glucagon response to the carbohydrate meal during prompt and supernormal hyperinsulinemia resulting from the infusion did not differ from that of the control meal, i.e. normal suppression of glucagon by hyperglycemia was not restored by the abundance of circulating insulin. To determine if still higher plasma levels of insulin would overcome the hyposuppressibility of the diabetic alpha cell to hyperglycemia, 0.6 U. per kilogram per hour of insulin was infused at a constant rate for two hours together with 0.6 gm. per kilogram per hour of glucose to prevent hypoglycemia. Insulin levels of more than 1,200 μ,U. per milliliter were thus attained. Under these conditions, plasma glucagon declined from a mean preinfusion level of 97 pg./ml. (SEM ± 11) to a nadir of 75 pg./ml. (SEM ± 10) ninety minutes later. This slow, modest, statistically significant (p < 0.01) decline differed strikingly from the response of eight nondiabetic patients given intravenous glucose alone; in these subjects, at a comparable level of hyperglycemia, glucagon declined from a mean fasting level of 90 pg./ml. (SEM ± 8) to 57 pg./ml. (SEM ± 8) within thirty minutes, despite an insulin rise to only 46 μU./ml. It was concluded that in human diabetics the acute restoration of plasma insulin, even to supernormal levels, does not promptly restore to normal the alpha cell responsiveness to hyperglycemia. Simple insulin lack may not, therefore, adequately explain the alpha cell abnormality in human diabetes.

Recent questions concerning the role of glucagon as a hormone of “glucose need” prompted this evaluation of glucose-glucagon relationships. To obviate certain problems relating to the sensitivity and specificity of the glucagon radioimmunoassay of peripheral venous plasma, glucagon was assayed in pancreatic venous effluent plasma obtained from conscious dogs with indwelling catheters in the pancreaticoduodenal vein. The effect of change in glucose concentration upon the pancreaticoduodenal vein glucagon concentration was determined. Insulin induced hypoglycemia was uniformly associated with a rise in pancreaticoduodenal vein glucagon concentration to a peak of 5.7 mμg./ml. at thirty minutes. The hyperglucagonemia continued intermittently during the hypoglycemic period but fell promptly during hyperglycemia induced by rapid injection of glucose. Hyperglycemia, induced in fifteen dogs by glucose infusion, was accompanied by a decline of glucagon from 2.7 to 1.8 mμg./ml. Cataglycemia, induced by rapid termination of a glucose infusion, produced a rise of glucagon in three of six dogs. Stimulation of glucagon secretion by hyperaminoacidemia was blocked by hyperglycemia induced by glucose infusion. The effect of starvation upon twenty-four-hour secretion of glucagon could not be determined in these studies. Wide variations in pancreaticoduodenal vein glucagon concentration made meaningful comparison of day-to-day changes impossible. A statistically significant negative correlation was observed between rapidly induced change in plasma glucose concentration and change in pancreaticoduodenal vein glucagon levels. The findings are compatible with the view that glucagon is a hormone of glucose need, which probably functions in the moment-to-moment regulation of blood glucose homeostasis and the maintenance of the so-called “normal” blood glucose limits.

Rapid endoportal injections of glucagon in a dose (1 μg.) which does not elevate thearterial glucagon concentration beyond the physiologic range caused a prompt rise in both mean arterial glucose and pancreaticoduodenal insulin concentration to a peak six minutes later. Hyperglycemia of similar magnitude produced by intravenous glucose infusion failed to elicit, as great a rise in insulin release. With smaller doses of glucagon a more variable response was observed; however, in some dogs doses of glucagon which were too small to cause a significant change in arterial glucagon concentration did cause a rise in both glucose and insulin levels. It is concluded that glucagon administered in physiologic doses via-the portarvenous system elicits a greater rise in insulin release than can be attributed to the concomitant hyperglycemia and, as suggested by others, does possess the qualifications of a potentiator of insulin secretion.

The present studies were designed to obtain further information concerning the chemical interrelationships of the fractions of glucagon-like immunoreactivity (GLI) present in gastrointestinal tissues, their biologic activity, and their immunochemical and electrophoretic properties. Chromatography of canine jejunal extracts on a Bio-Gel P-10 column separated GLI into two fractions; one, designated Peak II, eluted with the glucagon-I-131 marker (molecular weight 3,500), while the other, designated Peak I, appeared before the insulin-I-131 marker (molecular weight >6,000). Neither rechromatography, nor incubation in 8 M urea, acetic acid, or mercaptoethanol changed the elution volume of Peak I; however, trypsin caused a major loss of immunoreactivity and a shift of residual immunoreactivity to the postglucagon-I-131 zone. Peak I was devoid of glycogenolytic activity in the perfusedrat liver, as was its tryptic product, while Peak II was consistently as active as pancreatic glucagon. Immunoprecipitated Peak II was also active, proving that the immunoreactive fraction was responsible for its biologic action. Unlike pancreatic glucagon, Peak I, Peak II, and the tryptic product of Peak I diluted disproportionately with the less specific antiserum; however, Peak II could not be differentiated from pancreatic glucagon by means of polyacrylamide gel disc electrophoresis. It is concluded that (1) Peak I does not consist of Peak II bound noncovalently either to itself or to another protein; (2) Peak I and its tryptic product are devoid of glycogenolytic activity while Peak II has the approximate activity of pancreatic glucagon; (3) the glycogenolytic and immunoreactive components in the eluate containing Peak II are probably on the same molecule; (4) both Peak I, its tryptic product, and Peak II are immunologically different from pancreatic glucagon.

Near-normal glucoregulation was maintained in five patients with juvenile-onset diabetes mellitus for 4–5 wk with a preprogrammed, continuous, subcutaneous insulin infusion using a portable battery-powered infusion pump. This form of therapy significantly lowered immunoreactive glucagon (IRG) levels below those observed while on conventional insulin treatment at several times during the 24-h profile. The maximum IRG levels were also reduced in all five subjects. Thus, a flexible system of insulin delivery, as is provided by certain open-loop pump systems, can overcome inappropriate glucagon secretion that occurs with conventional insulin therapy.

The effect of insulin on the glucagon response to intravenous arginine was studied in eight juvenile-type and six adult-onset diabetics. In the juvenile-type diabetics, concomitant administration of insulin significantly blunted the glucagon response from a mean maximal rise of 310 ± 54 pg./ml. to only 184 ± 39 pg./ml. (p < 0.01), about the same as in nondiabetics. In the adult-onset patients, however, insulin had no effect, the mean maximal rise being 250 ± 50 pg./ml. without insulin and 307 ± 71 pg./ml. with insulin (N.S.). This study demonstrates that in juvenile-type diabetics concomitant administration of supraphysiologic quantities of insulin can reduce the exaggerated glucagon response to intravenous arginine to normal, whereas in the adult-type group, it has no apparent effect.