bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Aging on the Regenerative Capacity of the Pancreatic B-Cell of the Rat
Authors: Swenne Ingemar
Published: September 2013

Since an increased insulin resistance could be expected to lead to a compensatory B-cell hyperplasia, it may be speculated that human type II diabetes becomes manifest only in those individuals who are unable to respond to an increased insulin demand with a higher rate of B-cell proliferation. To test this hypothesis experimentally, the cell cycle and growth regulation of the pancreatic B cell has been studied in vitro. Islets of fetal, 1-wk, 3-wk, and 3-mo-old rats were isolated and maintained in tissue culture at different glucose concentrations. The proliferating islet cells were synchronized with hydroxyurea and their progression through the cell cycle studied by pulse labeling with 3H-thymidine. From the cell cycle data it was possible to calculate the rate of formation of new B-cells. The cell cycle of the B-cells was similar in all donor age groups. When the glucose concentration of the culture medium was raised from 2.7 to 16.7 mM, the rate of B-cell proliferation increased 2.5-fold in all age groups, but there was no further increase in proliferation at 33.3 mM. At each of the glucose concentrations tested the rate of B-cell formation decreased with increasing age of the donor. Results indicate that only a fraction of the islet cells are capable of entering the cell cycle and undergoing mitosis. This fraction composed about 10% of the fetal islet cells but was less than 3% in the adult islets. The small pool of proliferating cells in adult islets could explain why B-cell multiplication, although present in the aging rat, is insufficient to increase the insulin output to levels at which normal glucose tolerance is maintained. This forms an interesting parallel to the development of type II diabetes in man, in which an inherited low capacity for B-cell regeneration may predispose to the disease.
Modification of Glucose-Induced Insulin Release by Alteration of pH
Authors: Smith Joel S , Pace Caroline S
Published: September 2013

Protons (H+) generated by glucose metabolism have been proposed to serve as a coupling factor between cationic and secretory events in the B-cell. We have examined the influence of alteration of extracellular or intracellular pH (pHo or pH1) on dynamic secretory responses of perifused rat islets to 4.2, 8.4, or 16.7 mM glucose. Reduction of pHo from 7.4 to 7.0 inhibited the secretory response to 16.7, but not 8.4 mM glucose, by 47% during the 30-min period following medium change. Increase of pHo from 7.4 to 7.8 had no influence on the secretory response to glucose. Alteration of pHo had no influence on basal insulin release in the presence of 4.2 mM glucose. Sulfamerazine (5 mM), a permeable weak acid, augmented the secretory response to 8.4 mM glucose by 60% but had no influence on the response to 16.7 mM glucose. In contrast, imidazole (10 mM), a permeable weak base, inhibited the secretory response to both 8.4 (62%) and 16.7 mM (72%) glucose. Another weak base, NH4Cl (20 mM), also inhibited the secretory response to 8.4 (61%) and 16.7 mM (68%) glucose. Alteration of pH1 by sulfamerazine and imidazole did not alter basal insulin release in the presence of 4.2 mM glucose. A comparison of the present findings to those obtained for the influence of pH on glucose-induced electrical activity indicates that alteration of pH1, and not pHo, induces parallel effects on glucose-induced electrical and secretory events.
Insulin Resistance and Insulin Deficiency in Diabetic Retinopathy of Non-insulin-dependent Diabetes
Authors: Maneschi Franco , Mashiter Keith , Kohner Eva M
Published: September 2013

To assess the role of insulin resistance and insulin deficiency in the pathogenesis of diabetic retinopathy in non-insulin-dependent diabetes mellitus, 13 patients with and 12 patients without retinopathy were studied. The glucose clamp technique was used to measure insulin resistance and insulin response to glucose. During the euglycemic clamp, at comparable steady-state levels of glucose and insulin, the mean glucose infusion rate, which indicates the rate of glucose utilization, was lower in the retinopathy group than in the nonretinopathy group (6.1 ± 0.5 versus 8.1 ± 0.7 mg · kg−1 · min−1, P < 0.02). Growth hormone (GH) concentrations were higher in the retinopathy group 8.4 ± 2.5 versus 2.5 ± 0.7 μlU/ml, P < 0.05), but they did not correlate significantly with insulin resistance, expressed as mean glucose turnover. During the hyperglycemic clamp (+7 mmol/L above the fasting plasma glucose), the insulin response in the two groups of diabetics was similar. Increased insulin resistance represents an additional factor, which together with other factors, may be important in the pathogenesis of diabetic microvascular complications.
Effects of Experimental Diabetes on Adrenergic and Cholinergic Receptors of Rat Myocardium
Authors: Williams R Sanders , Schaible Thomas F , Scheuer James , Kennedy Rebecca
Published: September 2013

To more fully characterize the alterations in myocardial adrenergic and cholinergic receptors induced by the diabetic state, we investigated the binding characteristics of (–) [3H] dihydroalprenolol to beta adrenergic receptors (bAR), [3H] prazosin to alpha adrenergic receptors (aAR), and [3H] quinuclidinyl-benzilate to muscarinic cholinergic receptors (MCR) in myocardial membranes derived from rats 8 wk after treatment with streptozotocin. We also studied an equal number of animals from three control groups: free-eating nondi-abetics, pair-weighted nondiabetics, and streptozoto-cin-treated animals treated daily with insulin.

Diabetic hearts demonstrated 27% fewer bAR (P < 0.01) and 31% fewer aAR (P < 0.01) than free-eating controls, without changes in MCR, and without changes in antagonist affinity, agonist affinity, or agonist slope factor (pseudo-Hill coefficient) for any class of receptors. Food restriction had no effect on receptor characteristics, and treatment of diabetic rats with insulin prevented any downregulation of cardiac bAR or aAR.

The parallel decrease in both bAR and aAR suggests that streptozotocin-induced hypothyroidism is not the primary causative factor of bAR downregulation in this model, since hypothyroidism produces upregulation of aAR. Furthermore, the lack of change in cardiac MCR suggests that the adrenergic receptor alterations are not the result of nonspecific abnormalities of protein synthesis in the diabetic heart. Further studies are required to establish the physiologic significance of these receptor alterations, but these data support the hypothesis that altered adrenergic receptor properties may underlie, at least in part, the chronotropic and inotropic abnormalities of cardiac performance that are associated with the diabetic state.
The Adrenergic Contribution to Glucose Counterregulation in Type I Diabetes Mellitus: Dependency on A-Cell Function and Mediation Through Beta2-Adrenergic Receptors
Authors: Feo Pierpaolo De , Bolli Geremia , Perriello Gabriele , Cosmo Salvatore De , Compagnucci Pietro , Angeletti Gabriella , Santeusanio Fausto , Gerich John E , Motulese Mario , Brunetti Paolo
Published: September 2013

In order to assess the adrenergic contribution to hypoglycemie glucose counterregulation in type | diabetes mellitus and to determine whether the adrenergic contribution is mediated through beta1- or beta2-adrenergic receptors, hypoglycemia was induced by an i.v. insulin infusion (30 mU/m2 · min) for 60 min in 11 insulin-dependent diabetic patients (IDDM), 5 with normal plasma glucagon responses and 6 with blunted responses, and also in 7 age-weight-matched nondiabetic subjects. Rates of plasma glucose decrease and postnadir increase, as well as plasma concentrations of free insulin and of counterregulatory hormones, were measured when insulin was infused alone, and when insulin was infused along with propranolol (a beta1- and beta2-adrenergic receptor antagonist) or metoprolol (a selective beta1-antagonist). Postnadir plasma glucose recovery was decreased in IDDM with blunted plasma glucagon responses (21 ± 0.8 µmol. L−1 min−1, P < 0.001), but was normal in patients with normal plasma glucagon responses (30 ± 0.4 versus 33 ± 0.5 μmol · L−1 · min−1 in nondiabetic subjects, P = NS). Postnadir plasma glucose recovery was not affected by either propranolol or metoprolol in normal subjects and in IDDM with normal glucagon responses. However, in IDDM with blunted plasma glucagon responses, postnadir plasma glucose recovery was further decreased by propranolol (14 ± 0.6 µmol. L−1 min1, P < 0.01), but was unaffected by metoprolol (22 ± 0.9 μmol. L 1 min1, P = NS).

These results demonstrate that, as in normal man, adrenergic mechanisms are not essential for normal plasma glucose recovery from hypoglycemia in IDDM with normal plasma glucagon responses. However, in IDDM with blunted plasma glucagon responses, adrenergic mechanisms become important for glucose counterregulation although they do not fully compensate for the blunted plasma glucagon responses. Since propranolol delayed plasma glucose recovery from hypoglycemia in IDDM with blunted glucagon responses but metoprolol had no effect, the adrenergic contribution to glucose counterregulation appears to be mediated through beta2-adrenergic receptors.
Uptake of Bile Acids Into Rat Intestine: Effect of Diabetes Mellitus
Authors: Thomson A B R
Published: September 2013

Diabetes mellitus is associated with the enhanced uptake of several nutrients. The purpose of this study was to use an established in vitro technique to examine the uptake of bile acids into jejunum, ileum, and colon of control (CON) and streptozotocin-diabetic rats (DM). When the bulk phase was stirred to reduce the effective thickness of the unstirred layer, the ileal uptake of 1.5–15 mM cholic (C), glycocholic (GC), taurocholic (TC), chenodeoxycholic (CDC), glycochenodeoxycholic (GCDC), and deoxycholic (DC) acid was similar in CON and DM. The relative values of the maximal transport rates were CDC > GC > GCDC > C > TC = DC, and similar relative values were observed for the Michaelis constants (Km). The values of and Km for each bile acid were similar in CON and in DM. In CON and at pH 7.4 an inverse linear relationship was noted between the number of hydrogen bonds in the bile acids and the natural logarithm of the permeability coefficient (Pd) times the square root of the molecular weight of the bile acid. This slope reflected the incremental change in free energy, δΔFw ← I, associated with the uptake of bile acids; the value of δΔFw ← I was similar for jejunum, ileum, and colon of CON, but was lower in jejunum of DM than CON. Thus, DM is associated with a greater relative permeability of the jejunum but not the ileum or the colon to a series of bile acids. At pH 4.2, the uptake of the bile acids was much greater than at pH 7.4, and the relative ratio of the Pd's of the bile acids was different than the values at pH 7.4, and varied between jejunum, ileum, and colon in CON and DM. Thus, diabetes has a variable effect on the uptake of bile acids, and this effect depends on the bile acid, site, and pH. The kinetics of the ileal active transport system, like those of passive uptake, are dependent upon molecular structure. The finding of different values for of the bile acids suggests that there may be more than one ileal carrier for bile acids, but the properties of these carriers are unaffected by StreptOZOtOCin diabetes in the rat.
Secretin Inhibits Glucagon in the Isolated Perfused Dog Pancreas
Authors: Hisatomi Akitaka , Unger Roger H
Published: September 2013

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.
Degradation of Insulin Receptors in Rat Adipocytes
Authors: Heidenreich Kim A , Berhanu Paulos , Brandenburg Dietrich , Olefsky Jerrold M
Published: September 2013

Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radio-labeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16°C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430–450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons.

Subsequent to photoaffinity labeling of insulin receptors at 16°C, adipocytes were incubated at 37°C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37°C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h. This loss of radiolabeled receptors was not observed at 16°C and was inhibited by chloroquine (200μM) and cycloheximide (3.5 μM). Under conditions in which approximately 6% of the receptors were photoaffinity-labeled, occupancy of the remaining receptors with insulin had no effect on the degradation of photolabeled receptors. The results suggest that photoaffinity-labeled insulin receptors on viable adipocytes are initially degraded via an endocytotic lysosomal pathway. This pathway of degradation is very rapid and appears to be different from that mediating the basal turnover of unoccupied receptors.
The Effect of Chloroquine on the Binding, Intracellular Distribution, and Action of Insulin on Isolated Mouse Pancreatic Acini
Authors: Iwamoto Yasuhiko , Roach Eileen , Bailey Anne , Williams John A , Goldfine Ira D
Published: September 2013

The effects of chloroquine on the binding, intracellular distribution, and action of insulin were studied in isolated pancreatic acini prepared from diabetic mice. Chloroquine had three effects on these cells. First, chloroquine altered cellular morphology by inducing an increase in the number and size of autophagic vacuoles and vesicles in the Golgilysosomal region. Second, chloroquine, in a dose-dependent fashion, increased the amount of 125I-insulin associated with acini. A detectable effect of chloroquine was seen at 10 μM, and a maximal effect was seen at 30–100 μM where cell-associated insulin was more than doubled. Employing electron microscope autoradiographs, this accumulation of hormone was observed in the Golgilysosome area of the pancreatic acinar cell. Third, chloroquine had selective effects on the action of insulin. Preincubation with chloroquine had no effect on basal [3H]2-deoxy-D-glucose uptake, but in a dose-dependent fashion it decreased the stimulatory effect of insulin on this function; at 100 μM chloroquine, the effect of insulin was abolished. In contrast, chloroquine had negligible effects on the stimulation of 2-deoxy-D-glucose uptake into acini by cholecystokinin. Chloroquine in dose-dependent fashion partially inhibited basal [3H]leucine incorporation into acinar cell protein, but in contrast to its effects on 2-deoxy-D-glucose uptake, the drug had no effects on the stimulation of this function by insulin. These data indicate, therefore, that: (1) chloroquine alters the morphology of pancreatic acini by increasing the size and number of autophagic vesicles and vacuoles in the Golgilysosomal area; (2) chloroquine increases the accumulation of insulin into these structures; and (3) chloroquine selectively blocks the action of insulin on 2-deoxy-D-glucose uptake.
Calmodulin-Binding Proteins in a Cloned Rat Insulinoma Cell Line
Authors: Nelson Teresita Yap , Oberwetter James M , Chafouleas James G , Boyd A E
Published: September 2013

Calmodulin concentrations were measured in isolated hamster isiets or in a cloned rat insulin-secreting cell line RIN-m-5F treated with high glucose. There was no change in the cellular calmodulin content of either islets or RIN-m-5F cells despite increases of insulin concentrations in the media. Treatment of cells with the anti-calmodulin drug W13 inhibited insulin-stimulated glucose release, whereas a small effect on insulin accumulation in the media was observed with W12, the dechlorinated, less active analogue of W13. At the lowest dose tested (30 μM) the effect of W13 on insulin accumulation in the media was completely reversible. To further investigate the possible role of calmodulin in insulin secretion, calmodulin-binding proteins in subcellular fractions of the RIN-m-5F cells were identified using a gel overlay technique. Ca2+-dependent binding of 125I-calmodulin was observed to cystosolic proteins with apparent M, = 125,110, 56, 52, and 34 K (kilodalton). This binding was completely displaceable with unlabeled calmodulin, whereas only partial displacement was observed when the homologous Ca2+binding protein of skeletal muscle, troponin C, was used. Four proteins with Mr similar to the histones bind calmodulin in a Ca2+-independent manner. 125I-calmodulinxalmodulin binding protein interactions were inhibited in a dose-dependent manner by the anti-calmodulin drug, W13. Little effect was observed with the analogue W12. These data demonstrate that glucose-stimulated insulin secretion is not associated with alterations in total calmodulin levels and suggest that the inhibition of insulin secretion by the anti-calmodulin drug, W13, may be through inhibition of the association of calmodulin to calmodulin-binding proteins in the cell.
Glucose Stimulation of Beta-Cell DNA Replication in the Intact Rat and in Pancreatic Islets in Suspension Culture: Effects of Alpha-Ketoisocaproic Acid, Dibutyryl Cyclic AMP, and 3-Isobutyl-1-Methylxanthine in the In Vitro System
Authors: Logothetopoulos John , Valiquette Nancy , Cvet David
Published: September 2013

DNA replication in pancreatic beta-cells was compared in intact rats maintained hyperglycemie by continuous glucose infusion up to 10 days and in rat islets maintained in suspension culture in RPM11640 medium up to 12 days. Replicative activity was evaluated by counting the proportion of labeled beta-cell nuclei after injection or addition of [3H]thymidine. In both experimental systems, DNA replication initially was markedly stimulated by high glucose; then it subsided, even though high levels of glucose were maintained. Culture of pancreatic islets in suspension permitted the study of various treatments on beta-cell DNA replication. Alpha-ketoisocaproic acid, a nonglycolytic substrate for the beta-cell, stimulated DNA replication. 3-Isobutyl-1-methylxanthine (IBMX), in the presence of 4 mM glucose, was a potent stimulator. In many instances the proportion of labeled beta-cell nuclei in islets cultured with IBMX exceeded that obtained with 25 mM glucose. Dibutyryl-cyclic AMP stimulated DNA replication but was not as effective as IBMX. Although the effect of IBMX markedly decreased during the second week of culture, IBMX was much more effective than 25 mM glucose in maintaining DNA replication persistently above basal (4 mM glucose) levels.
Reduced Solubility of Short-Acting Soluble Insulins when Mixed with Longer-Acting Insulins
Authors: Nolte Martha S , Poon Violet , Grodsky Gerold M , Forsham Peter H , Karam John H
Published: September 2013

Using insulins from three manufacturers, we examined the recovery by radioimmunoassay of short-acting soluble insulin when mixed with long-acting insulin as a function of the ratio of the mixture and the time of premixing. In ratios of 1:2, 1:3, and 1:5 (short- to long-acting insulin), all Novo, Nordisk, and Lilly short-acting insulins tested showed a significant loss of solubility when mixed with the respective company's long-acting insulin either for less than 75 s or for 20 min before centrifugation.

In ratios of 1:1, Novo's Actrapid (regular) with Monotard (lente) and Lilly's regular with lente showed no significant loss of solubility when pre-mixed for less than 75 s, and the regular insulin also showed no significant loss when pre-mixed for 20 min. However, when Lilly's regular was mixed with either NPH or ultralente in a 1:1 ratio, a significant loss of solubility of the short-acting insulin occurred regardless of time [as was also found with Nordisk's Velosulin (regular) with Insulatard (NPH)].

When Lilly regular was incubated with Lilly lente in ratios of 1:3 for <75 s, 20 min, 4 h, and 24 h before centrifugation, there was a progressive loss of solubility. In contrast, with the same ratios and times of premixing, Lilly regular when mixed with Lilly NPH showed a rapid initial loss of solubility that plateaued by 20 min before centrifugation.

Supernatants from the centrifugation of Lilly's long-acting insulins were assayed and found to contain only 0.67–1.5% of the total insulin content. When Lilly regular was mixed in a 1:3 ratio with these supernatants, there was a small but significant loss of solubility.

These in vitro data indicate that short-acting insulins may lose solubility when mixed with long-acting insulins as the proportion of the latter increases and the time Of pre-mixing iS prolonged.
Abnormalities of Ambulatory 24-hour Heart Rate in Diabetes Mellitus
Authors: Ewing D J , Borsey D Q , Travis P , Bellavere F , Neilson J M , Clarke B F
Published: September 2013

Twenty-one normal subjects and 64 diabetics with varying severity of autonomic damage underwent 24-h ambulatory EKG monitoring. No diabetics had the “sick sinus syndrome,” and the frequency of arrhythmias was no higher than in the normal subjects. The diabetics had higher mean hourly heart rates, and with increasing autonomic damage there was reduction in diurnal heart rate variation. The mean waking and sleeping heart rates were higher in the diabetics. The maximum heart rates were not significantly different, but the minimum heart rates were significantly higher in the diabetics. These previously unrecognized abnormal 24-h heart rate patterns provide further evidence of damage to the heart rate-controlling mechanisms in diabetes mellitus.
Prevention of the Glucose Intolerance of Thiazide Diuretics by Maintenance of Body Potassium
Authors: Helderman J Harold , Elahi Dariush , Andersen Dana K , Raizes Gary S , Tobin Jordan D , Shocken Dauglas , Andres Reubin
Published: September 2013

The effect of thiazide diuretics on the glucose tolerance of seven normal men in whom potassium loss was prevented with supplementation was studied using the glucose clamp technique. An initial control 2-h hyperglycemic clamp was performed to create a square wave of hyperglycemia 125 mg/dl above basal. At 1 h, 40 g glucose/m2 body surface area was ingested. Serial insulin (IRI) and gastric inhibitory polypeptide (GIP) levels were measured as well as the level of glucose infusion necessary to maintain the stable hyperglycemic level. After the initial study, subjects were placed on a 10-day course of 100 mg hydrochlorothiazide and 80 meq potassium per day. Subjects were monitored for dietary potassium intake, urinary potassium, and sodium losses, and the replacement of potassium adjusted accordingly. A repeat glucose clamp was done on day 10. When potassium losses were prevented, thiazides induced no alterations in glucose tolerance, beta-cell sensitivity to glucose, GIP-cell sensitivity to glucose, beta-cell sensitivity to GIP, or tissue sensitivity to insulin. Two control studies in which hypokalemia was allowed to ensue after hydrochlorothiazide ingestion revealed a diminution in glucose tolerance, a consequence of diminished pancreatic beta-cell response to glucose. We conclude that the thiazide effect on glucose tolerance is a consequence of the resultant hypokalemia that the diuretic may create.
Differences at the Receptor and Postreceptor Levels Between Human Omental and Subcutaneous Adipose Tissue in the Action of Insulin on Lipolysis
Authors: Bolinder Jan , Kager Lars , Östman Jan , Arner Peter
Published: September 2013

The possible existence of regional differences in the antilipolytic action of insulin in human adipose tissue was investigated in vitro. Insulin-induced inhibition of glycerol release and insulin binding, measured in terms of receptor number, receptor affinity, and dissociation rates, were determined in omental and subcutaneous adipose tissue segments and isolated fat cells of 16 nonobese subjects who were undergoing elective abdominal surgery but were otherwise healthy. The sensitivity of the antilipolytic effect of insulin was higher in subcutaneous than in omental adipose tissue; the half-maximal effect was obtained with 1 and 3 μU/ml of insulin, respectively (P < 0.01). Responsiveness of the antilipolytic effect of insulin was threefold higher in the subcutaneous than in the omental region (P < 0.005). Insulin receptor affinity was significantly higher in subcutaneous than in omental fat cells, but there was no difference in receptor number (about 300,000 sites/cell). 125I-insulin dissociated more rapidly from omental than from subcutaneous adipocytes in both the absence and the presence of excess native insulin. The data suggest that significant regional differences exist in the antilipolytic action of insulin in man; omental fat being less responsive than subcutaneous fat. The difference involves the insulin receptor affinity, which is caused at least partly by variations in the insulin dissociation rate but is also due to differences in insulin action at the postreceptor level.
Abnormal Glucose Counterregulation in Insulin-dependent Diabetes Mellitus: Interaction of Anti-Insulin Antibodies and Impaired Glucagon and Epinephrine Secretion
Authors: Bolli Geremia , Feo Pierpaolo De , Compagnucci Pietro , Cartechini Maria G , Angeletti Gabriella , Santeusanio Fausto , Brunetti Paolo , Gerich John E
Published: September 2013

To evaluate the roles of counterregulatory hormones and insulin antibodies in the impairment of plasma glucose recovery from hypoglycemia in diabetes mellitus, and to assess the relationship between the glucagon response and duration of the disease, 21 insulin-dependent diabetic patients and 10 nondiabetic subjects were studied. The diabetics consisted of 5 patients with recent onset of diabetes (< 1 mo); 11 with 2.6 ± 0.3 (mean ± SEM) yr duration of diabetes, 5 of whom had insulin antibodies; and 5 patients with long-term diabetes (21 ± 3 yr), insulin antibodies, and autonomic neuropathy. During insulin-induced hypoglycemia (28 mU/m2 × min for 60 min) in patients with recent-onset diabetes, plasma free insulin, glucose, and counterregulatory hormone concentrations did not differ from those of nondiabetic subjects. In patients with insulin antibodies, the disappearance of insulin after insulin infusion was delayed, and both restitution of normoglycemia and plasma glucagon response were blunted compared with patients without antibodies. When glucagon was infused (80–130 ng/m2 × min) during hypoglycemia in diabetics with impaired glucagon responses in order to simulate normal glucagon responses, plasma glucose recovery was normalized in patients without antibodies but not in those with antibodies. In patients with long-standing diabetes, restitution of normoglycemia was further impaired and this was associated with an absent plasma glucagon response and a diminished plasma epinephrine response. Plasma glucagon responses to hypoglycemia were inversely correlated to the duration of diabetes (r = −0.943; P < 0.0005). It is concluded that impaired A-cell secretion is the predominant mechanism for the delayed glucose recovery after hypoglycemia in diabetic patients without insulin antibodies and normal epinephrine responses. Slowed disappearance of insulin due to the presence of insulin antibodies further delays the restoration of normoglycemia. Patients with long-standing diabetes and autonomic neuropathy exhibit decreased epinephrine secretion, which leads to an additional retardation of glucose recovery. Since plasma glucagon and epinephrine responses to hypoglycemia were normal at the onset of diabetes but diminished in long-term diabetes, it appears that the impaired glucagon and epinephrine responses to hypoglycemia are acquired defects that develop subsequent to B-cell failure.
Effect of Age and Diet on Insulin Secretion and Insulin Action in the Rat
Authors: Reaven E , Wright D , Mondon C E , Solomon R , Ho H , Reaven G M
Published: September 2013

The effects of aging on various aspects of insulin secretion and action were studied in male Sprague-Dawley rats, maintained from 1½ to 12 mo of age on conventional rat chow, sucrose-rich, or calorie-restricted diets. In chow-fed rats, islet volume increased as the animals grew from 1½ to 12 mo of age, but glucose-stimulated insulin secretion (per volume islet) declined over the same interval. In addition, in vivo insulin-stimulated glucose utilization fell in these rats. However, the plasma insulin response to an oral glucose challenge was sufficient to prevent frank decompensation of glucose tolerance (presumably due to an increase in total pancreatic endocrine cell mass). All these changes, with the exception of the decline in glucose-stimulated insulin secretion per volume islet, were accentuated by feeding sucrose. Thus, 12-mo-old sucrose-fed rats had larger islets and higher plasma insulin levels in response to an oral glucose challenge, and the rats were more insulin-resistant than chow-fed rats. However, glucose-stimulated insulin release per volume islet was similar in 12-mo-old chow-fed and sucrose-fed rats. In contrast, calorie restriction led to an amelioration in all but one of the age-related changes, i.e., islets from calorie-restricted rats were comparable in size to those of 2-mo-old rats, the animals had lower plasma insulin levels in response to an oral glucose load, and they were less insulin resistant than the other two groups of 12-mo-old rats. On the other hand, glucose-stimulated insulin secretion per volume islet was similar to that of the other 12-mo-old rats. These results suggest that aging leads to marked changes in both insulin secretion and insulin action. The decline in glucose-stimulated insulin secretion per unit endocrine pancreas appears to be an inevitable consequence of the aging process. In contrast, the age-related changes in islet size, insulin response to a glucose load, and in vivo insulin-stimulated glucose uptake are extremely responsive to variations in amount and kind of calories.
Do Platelets Have Anything To Do With Diabetic Microvascular Disease?
Authors: Colwell John A , Winocour Peter D , Halushka Perry V
Published: September 2013

It has been postulated that abnormal platelet and endothelial function may contribute to microangiopathy in diabetes mellitus. If this proposal is correct, alterations in platelet and endothelial function should be found before the appearance of vascular disease in insulin-dependent patients and in animal models of diabetes mellitus. This appears to be the case for the following: platelet aggregation, increased platelet production of the proaggregatory prostaglandin metabolite thromboxane, decreased endothelial production of the antiaggregatory prostaglandin prostacyclin, and decreased platelet survival. Insulin therapy will return some of these findings to normal. Platelet-plasma interactions that promote platelet aggregation and increased plasma levels of the platelet-specific protein β-thromboglobulin have been reported in insulin-dependent diabetic patients who have not manifested vascular complications as well as in those with vascular complications. It has now been demonstrated in animal models that platelet microthrombi are found in small retinal vessels after months of experimental diabetes.

Collectively, these findings demonstrate that alterations in platelet and endothelial function that favor thrombosis occur early in the diabetic state and may contribute to microvascular disease. There are several ongoing studies of antiplatelet agents in diabetic vascular disease that will provide clinical evidence bearing on the major postulate. Until these and other studies are completed, the platelet-endothelial story remains an attractive hypothesis in the genesis of diabetic microvascular disease.
Studies on the Pathophysiology of Diabetic Retinopathy: The Blood-Retinal Barrier in Diabetes
Authors: Cunha-Vaz José G
Published: September 2013

A review of the pathologic picture of diabetic retinopathy shows that available clinical methods of examination demonstrate the alteration of the blood-retinal barrier (leakage), microaneurysms, capillary closure, preferential channels, preretinal neovascularization and gross extravascular lesions. All of these changes may be shown by fluorescein angiography. The value of this method, however, is fundamentally related to the morphologic demonstration of these lesions and not their quantification. Quantitative evaluation of retinal involvement in diabetes is needed in order to delineate more clearly its natural history, criteria for prognosis, and effect of treatment.

Vitreous fluorophotometry, a quantitative and sensitive method of evaluating the permeability of the blood-retinal barrier, has opened new perspectives for the evaluation of retinal involvement in diabetes. Vitreous fluorophotometry has shown that a disturbance of the blood-retinal barrier, possibly functional, appears in diabetic eyes before any lesion is clinically visible in the fundus, and that there is a close correlation between the severity of the vascular lesions and higher vitreous fluorophotometry readings. Recent studies also indicate an interesting correlation with metabolic control, particularly, glycosylated hemoglobin levels and insulin treatment.

Finally, on the basis of these findings a working hypothesis for the pathogenesis of diabetic retinopathy is presented.
The Stages in Diabetic Renal Disease: With Emphasis on the Stage of Incipient Diabetic Nephropathy
Authors: Mogensen C E , Christensen C K , Vittinghus E
Published: September 2013

Alterations in renal function and structure are found even at the onset of diabetes mellitus. Studies performed over the last decade now allow definition of a series of stages in the development of renal changes in diabetes. Such a classification may be useful both in clinical work and in research activities.

Stage 1 is characterized by early hyperfunction and hypertrophy. These changes are found at diagnosis, before insulin treatment. Increased urinary albumin excretion, aggravated during physical exercise, is also a characteristic finding. Changes are at least partly reversible by insulin treatment.

Stage 2 develops silently over many years and is characterized by morphologic lesions without signs of clinical disease. However, kidney function tests and morphometry on biopsy specimens reveal changes. The function is characterized by increased GFR. During good diabetes control, abumin excretion is normal; however, physical exercise unmasks changes in albuminuria not demonstrable in the resting situation. During poor diabetes control albumin excretion goes up both at rest and during exercise. A number of patients continue in stage 2 throughout their lives.

Stage 3, incipient diabetic nephropathy, is the forerunner of overt diabetic nephropathy. Its main manifestation is abnormally elevated urinary albumin excretion, as measured by radioimmunoassay. A level higher than the values found in normal subjects but lower than in clinical disease is the main characteristic of this stage, which appeared to be between 15 and 300 μg/min in the baseline situation. A slow, gradual increase over the years is a prominent feature in this very decisive phase of renal disease in diabetes when blood pressure is rising. The increased rate in albumin excretion is higher in patients with increased blood pressure. GFR is still supranormal and antihypertensive treatment in this phase is under investigation, using the physical exercise test.

Stage 4 is overt diabetic nephropathy, the classic entity characterized by persistent proteinuria (>0.5 g/ 24 h). When the associated high blood pressure is left untreated, renal function (GFR) declines, the mean fall rate being around 1 ml/min/mo. Long-term antihypertensive treatment reduces the fall rate by about 60%7 and thus postpones uremia considerably.

Stage 5 is end-stage renal failure with uremia due to diabetic nephropathy. As many as 25% of the population presently entering the end-stage renal failure programs in the United States are diabetic. Diabetic nephropathy and diabetic vasculopathy constitute a major medical problem in society today.
Capillary Basement Membranes in Diabetes
Authors: Williamson J R , Kilo C
Published: September 2013

In discussions of vascular complications of diabetes the fact that capillary basement membranes are, in general, thickened (CBMT) in poorly controlled diabetics is no longer at issue. However, three important questions concerning the pathophysiologic significance of CBMT remain unanswered: (1) How and why do capillary basement membranes thicken in diabetes? (2) What is the functional significance of capillary basement membrane changes in diabetes? (3) What is the nature of the relationship of CBMT to other forms of diabetic vascular disease; in particular, is CBMT observed in tissues amenable to needle biopsy, i.e., skeletal muscle, useful in identifying individuals at high risk for developing clinically significant retinopathy, nephropathy, or atherosclerotic vascular disease?

In this survey, we will consider the nature of capillary basement membrane changes in diabetes and subsequently address the above questions.
Decreased Response of Mobile Calcium in Pancreatic Islets of Fasted Rats to Glucose Stimulation and Calcium Manipulation
Authors: Wolters G H J , Pasma A , Konijnendijk W
Published: September 2013

The effect of fasting on mobile calcium in the B-cells of rat pancreatic islets was investigated in view of a possible role of calcium in the fasting-induced impairment of insulin secretion. Mobile calcium (GBHA-Ca), an ionized or readily ionizable calcium fraction, was determined histochemically with glyoxal bis (2-hydroxyanil). Fasting (24–72 h) strongly decreased the GBHACa content of islets in situ (55–60%).

Incubation of isolated islets at 2.5 mM glucose in the presence of 2.5 mM Ca2+ resulted after 15 min in stable GBHA-Ca levels, which were 25% lower in fasted than in fed islets. Glucose (15 mM) caused the GBHA-Ca content of fed islets to decrease rapidly and to rise again after 30 min. These changes did not occur after 24 or 72 h of fasting. GBHA-Ca appeared not to be displaceable with La3+.

At 2.5 mM glucose, withdrawal of Ca2+ rapidly reduced GBHA-Ca in fed and fasted islets. Glucose (15 mM) inhibited this rapid fall, and this inhibitory effect was particularly evident in fed islets.

Washing and preincubation in the absence of Ca2+ (2.5 mM glucose) largely depleted fed and fasted islets of GBHA-Ca. Reintroduction of Ca2+ at 2.5 mM glucose only partially restored the GBHA-Ca levels of fed and fasted islets. By contrast, 15 mM glucose restored the characteristic pattern of GBHA-Ca in fed islets as seen in nondepleted islets, but in fasted islets at lower levels as previously seen.

Thus, fasting decreased the GBHA-Ca content and its response to glucose stimulation. It is suggested that GBHA-Ca, which is presumably mainly localized in the secretory granules, plays a role in the initiation of insulin secretion.
Elevated Intestinal Disaccharidase Activity in the Streptozotocin-Diabetic Rat Is Independent of Enteral Feeding
Authors: Schedl H P , Al-Jurf A S , Wilson H D
Published: September 2013

Specific and total activities of the disaccharidases, sucrase, maltase, and lactase are increased in mucosa of the small intestine of the streptozotocin diabetic rat. Because disaccharidases are essential for terminal digestion of carbohydrate, and disaccharidase deficiency is a common clinical problem, understanding the mechanisms regulating disaccharidase activity is important. In normal animals, disaccharidase activities are determined by route of feeding and are decreased by parenteral feeding. The indirect exocrine, endocrine, neurocrine, and paracrine functions of the gastrointestinal tract that are dependent on feeding via the gut are greatly decreased in parenteral as compared with enteral feeding. Hormone secretion by the gut and the pattern of response after feeding may be abnormal in diabetes and might be regulatory for disaccharidases. We tested the hypothesis that the elevated intestinal disaccharidases in diabetes are dependent on enteral feeding. Streptozotocin-injected rats (diabetics) and vehicle-injected rats (controls) were fed rat chow ad libitum for 4 days. A subset of control and diabetic animals was then killed to determine disaccharidase activity of the jejunum at the start of pair-feeding the elemental diet. The remaining animals were fed 60 cal/day of glucose, amino acid (Travasol), and electrolyte solution either intragastrically or intravenously for 4 days. Specific and total activities of disaccharidases were greater in diabetics than in controls under all feeding conditions. In controls, the pattern of activity of disaccharidase specific activity was initial > intragastric > intravenous. In diabetics, disaccharidase specific activities did not differ among groups. In both controls and diabetics, mean mucosal mass was high-est initially; intermediate with intragastric feeding; and lowest with intravenous feeding. In both controls and diabetics, total disaccharidases decreased from initial to intragastric to intravenous. We conclude: (1) disaccharidase specific activity in controls is sensitive to feeding route and nature of diet, but is nearly independent of these factors in diabetics; (2) total disaccharidase activities respond to feeding stimuli in parallel with changes in mucosal mass in both controls and diabetics; and (3) the lack of feeding effect on the elevated specific activities of disaccharidases in diabetes suggests that this elevation is a response to the diabetic state and is independent of enteral factors such as luminal nutrition and gastrointestinal hormones.
Growth Hormone Stimulates Islet B-Cell Replication in Neonatal Rat Pancreatic Monolayer Cultures
Authors: Rabinovitch Alexander , Quigley Carol , Rechler Matthew M
Published: September 2013

A possible role for growth hormone (GH) in stimulating islet B-cell replication was examined in neonatal rat pancreatic monolayer cultures. Addition of ovine GH (1000 ng/ml) to serum-free medium for 2 days resulted in a significant increase (+114%) in [3H]thymidine labeling of B-cells in aldehyde-thionine-stained autoradiographs, and a similar increase in the B-cell mitotic index. The stimulatory effects of GH on islet B-cell replication were greatest in serum-free medium, unaffected by glucose concentrations (2.8–16.7 mM), and not accompanied by any stimulation of insulin release. The threshold concentration of GH for a significant stimulatory effect on B-cell replication was 30 ng/ml. The insulin-like growth factor, multiplication stimulating activity (MSA), also effectively stimulated B-cell replication. Although some effects of GH are mediated by the insulin-like growth factors (IGFs), the effects of GH on B-cell replication did not appear to be mediated by IGFs since (1) the maximal effect of GH (+156%) was significantly > that of MSA (+91%); (2) the combination of maximal stimulatory concentrations of GH and MSA produced an additive effect; and (3) a significant effect of GH on B-cell replication was observed as early (8 h) as that produced by MSA. These results suggest that GH can stimulate islet B-cell replication directly, and that this effect may not depend on production of either insulin-like growth factors.
Pancreatic Endocrine Cell Fractions in Erythroblastosis Fetalis
Authors: Milner R D G , Dinsdale F , Wirdnam P K , van Assche F A
Published: September 2013

Pancreatic sections from 21 cases of rhesus disease and 20 control newborn infants of 30–40-wk gestational age were stained by the immunoperoxidase method for insulin, glucagon, somatostatin, and pancreatic polypeptide (PP). The fractional area occupied by each cell type was estimated, taking note of whether the gland contained PP-rich (ventral lobe) or PP-poor islets (dorsal lobe). In the PP-rich part of the pancreas, the volume fraction of all four endocrine cell types was significantly greater in the rhesus cases than in the controls. No difference was found between the two groups in the PP-poor part of the gland. The results show that abnormal development of the PP-rich part of the pancreas occurs in erythroblastosis fetalis. The localization of the changes to one part of the pancreas may explain some of the earlier conflicting reports on this topic.
Kinetics of Insulin Receptor Biosynthesis and Membrane Insertion: Relationship to Cellular Function
Authors: Marshall Stephen
Published: September 2013

A method was developed for maintaining isolated rat adipocytes in primary culture in a viable state for several days. Using this method, the kinetics of insulin receptor biosynthesis and membrane incorporation were assessed by inactivating (>95%) cell-surface insulin receptors with trypsin and then incubating cells in medium at 37°C for various times up to 44 h. The insertion of nascent receptors, into the plasma membrane was monitored by measuring the recovery of specific 125l-insulin binding activity. After trypsinization, insulin binding activity recovered at a linear rate with half of the original cell-surface receptor complement replenished by 24 h (6000 receptors or 2% of total surface receptors/h). This recovery was most probably mediated by de novo receptor synthesis, since no recovery of specific insulin binding was seen when cells were pretreated with cycloheximide for 3 h. Under conditions where protein synthesis was blocked immediately after trypsinization, however, a lag of 2–3 h was observed before receptor incorporation was inhibited. This lag suggests that insulin receptors are processed and routed within the cell for 2–3 h before being inserted into the plasma membrane. In support of this hypothesis it was found that the intracellular pool of insulin receptors (10% of the total receptor complement) could be depleted by about 80% by treating cells with cycloheximide for 3 h. Thus, it appears that most, if not the entire, intracellular receptor pool in adipocytes consists of newly synthesized receptors en route to the cell surface.

Insulin, after binding to cell-surface receptors, triggers various biologic actions in adipocytes such as glucose transport and endocytosis of insulin-receptor complexes. To determine whether nascent receptors are functional immediately on insertion, we compared the recovery of insulin binding activity after trypsinization with the ability of these cells to respond to insulin with an increase in glucose uptake and an augmented rate of insulin internalization. These data revealed a high correlation between recovery of binding and biologic responsiveness. Thus, these data indicate that immediately upon insertion, or shortly thereafter, receptors become functional and are able to mediate the biologic actions of insulin.