Pancreatic Beta-cell Response Research Articles

Effects of Lowering External Na+ Concentration on Cytoplasmic pH and Ca2+ Concentration in Mouse Pancreatic, .BETA.-Cells: Mechanism of Periodicity of Spike-Bursts.

The periodic spike-burst response of pancreatic beta-cells varies in duration with an increase of external glucose within the range 5-20 mM. To elucidate the mechanism determining the length of spike-burst, we studied the low-Na+ induced change in electrical response to glucose which is similar to the change induced by high glucose. Cytoplasmic pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured by the microfluorometric method under normal and low-Na+ conditions in mouse pancreatic islets. Lowering external Na+ concentration from 135 to 25 mM by replacing Na+ with Tris+ induced progressive alkalinization in islet cells in the presence of 11.1 mM glucose. In contrast, reduction of external Na+ by replacement with Li+ caused intracellular acidification. Both manipulations described above caused a marked increase in [Ca2+]i, suggesting the presence of Na+/Ca(2+)-antiport activity. Although the change in pHi induced by decreasing external Na+ varied in direction depending on the species of cations used for replacing Na+, the pattern of electrical activity consistently changed from the spike-burst type to the continuous spike-generation type without regard for the difference in species of cations replacing Na+. These findings lead to the following hypothesis: A decrease in Na+ influx could cause a decrease in ATP-consumption by Na+/K(+)-pumps that prevents the fall of intracellular ATP concentration. The resultant continuation of high concentrations of intracellular ATP may be responsible for the abolishment of the silent phase.

Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells.

Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.

Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic βcells

Pages C116 and C118: Å. Sjőholm. “Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic betacells.” Page C116, Table 1, the units for [Glucose] should read mM, and page C118, Table 3, the units for [IFN-ggr should read U/ml, as shown in the corrected tables. (The errors were inadvertentlv introduced by the printer at the final stage of proof.) (See PDF)

Glucose modulates glucose transporter affinity, glucokinase activity, and secretory response in rat pancreatic beta-cells

Glucose modulates glucose transporter affinity, glucokinase activity, and secretory response in rat pancreatic beta-cells

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.

Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic beta-cells.

It has been proposed that certain cytokines secreted by islet-infiltrating leukocytes may be involved in the pathogenesis of insulin-dependent diabetes mellitus by participation in beta-cell destruction. In the present study, the impact of various cytokines on replication and long-term insulin secretion by pancreatic beta-cells was investigated. To this end, fetal rat pancreatic islets containing a high fraction of beta-cells were exposed in culture for 1-3 days to interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interferon-alpha (IFN-alpha), and interleukin-6 (IL-6) at different concentrations. It was found that IL-1 beta markedly decreased beta-cell DNA synthesis during the first day of exposure, an effect that vanished after 2 days and was turned into a potent and dose-dependent stimulation by 3 days of exposure. At this latter time point, IL-1 beta also amplified the mitogenicity of growth hormone (GH) and 16.7 mM glucose. In contrast, basal as well as glucose- and GH-stimulated insulin secretion was consistently suppressed by IL-1 beta from days 1-3. IL-1 beta also lowered the islet adenosine 3',5'-cyclic monophosphate (cAMP) content at all time points studied. However, addition of the stimulatory cAMP analogue Sp-diastereomer of adenosine 3',5'-cyclic monophosphothioate or pertussis toxin, which themselves enhanced DNA synthesis and insulin secretion, failed to prevent the inhibitory actions of IL-1 beta on these parameters, making it unlikely that a decrease in cAMP is an important event in transduction of the inhibitory effects of the cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-beta stimulates insulin secretion and blocks mitogenic response of pancreatic beta-cells to glucose

The long-term influence of transforming growth factor-beta (TGF-beta) on replication and insulin secretion by insulin-producing pancreatic beta-cells was investigated. For this purpose, fetal rat pancreatic islets containing a high proportion of beta-cells were isolated and maintained in tissue culture for 3 days at different concentrations of TGF-beta. TGF-beta (5-500 pM) at a glucose concentration of 11.1 mM did not affect the replication of the beta-cells or their insulin content but enhanced secretion of insulin from these cells. TGF-beta (500 pM) counter-acted the mitogenic action of 16.7 mM glucose but failed to affect the glucose-induced increase in islet insulin content or secretion. Growth hormone (GH) also stimulated beta-cell DNA synthesis and insulin secretion, but TGF-beta was unable to prevent these effects. It was found, moreover, that TGF-beta did not prevent the increase in islet polyamine content which occurred in response to glucose or GH, indicating that the effects of TGF-beta are not mediated through this pathway. Addition of neutralizing antibodies to TGF-beta did not affect the mitogenic or secretory responses to glucose or GH, suggesting that TGF-beta does not exert any autocrine or paracrine function in islets.

Role of polyamines in mitogenic and secretory responses of pancreatic beta-cells to growth factors

We have investigated the effects of glucose and the polypeptide growth factor growth hormone (GH), platelet-derived growth factor (PDGF), insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor alpha (TGF alpha) on the polyamine content, in relation to proliferation and insulin secretion and content, of pancreatic beta-cells. Fetal rat pancreatic islets containing a high proportion of beta-cells were cultured for 3 days with growth factors. beta-cell replication was significantly increased by glucose, GH, and PDGF plus IGF-I in parallel with increased islet polyamine contents. In contrast, neither EGF nor TGF alpha influenced the islet DNA synthesis rate, polyamine content, insulin content, or insulin accumulation in culture medium. When the increased polyamine content evoked by growth-promoting agents was prevented by inhibitors of polyamine synthesis, elevated DNA synthesis rates persisted or were even augmented. However, subcellular fractionation analysis of islet homogenates revealed that the nuclear polyamine content was not affected by the inhibitors. On the other hand, islet insulin content and glucose-regulated insulin release were decreased by polyamine synthesis inhibitors. Glucose oxidation rates remained unchanged, suggesting that inhibitors were not toxic to islet cells. We conclude that prevention of increases in total cellular content of polyamines in response to glucose, GH, or PDGF plus IGF-I does not prevent mitogenicity of these growth factors. However, when their synthesis is inhibited normal levels of polyamines seem to be maintained in the cell nucleus, an event that may be sufficient to permit a mitotic signal to be translated into a proliferative response.

Plasma C-peptide response to oral glucose load in hyperthyroidism

Aim of the present study was to evaluate the pancreatic beta cell response to oral glucose load in a group of patients with hyperthyroidism. For this purpose plasma C-peptide at fasting and after a 100 g oral glucose load was measured in 8 newly-diagnosed untreated hyperthyroid patients with fasting normoglycemia, and 8 sex-, age-, and weight-matched healthy controls. As compared to healthy subjects, patients with hyperthyroidism showed higher plasma glucose levels (incremental area 5405 +/- 742 vs 2729 +/- 539 mg/dl x 180 min, p less than 0.05), and slightly reduced plasma C-peptide concentrations (incremental area 166 +/- 12 vs 182 +/- 36 pmol/ml x 180 min, p = NS) following oral glucose load. The ratios between plasma C-peptide and plasma glucose incremental areas were lower in hyperthyroid patients than in controls (3.66 +/- 0.85 vs 10.41 +/- 3.08, p less than 0.05). These data suggest that hyperthyroidism is characterized by a decreased pancreatic beta cell response to oral glucose load.

Circulating somatostatin after oral glucose in hypothyroidism

The response of circulating somatostatin-like immunoactivity (SLI) to oral glucose and its relation to other pancreatic islet cell hormones were studied in 10 hypothyroid subjects before and after treatment. None of the patients suffered from diabetes mellitus or obesity. Compared with normal controls, the hypothyroid subjects had higher fasting and stimulated SLI levels but lower fasting pancreatic glucagon levels. Integrated glucose and insulin responses following glucose ingestion were normal, but the peak insulin response was delayed to 120 min suggesting impaired pancreatic beta-cell response to oral glucose. On the other hand, the peak response of plasma C-peptide was higher probably because of a reduction in metabolic clearance. In both hypothyroid subjects and controls, a significant correlation was found between the maximal increment of SLI and the maximal decrement of glucagon following oral glucose. In conclusion, plasma SLI is increased in hypothyroidism. The changes in SLI may be due to either an increased hormonal secretion or a reduced metabolic clearance in hypothyroidism. This elevated SLI might contribute to the slower gastrointestinal motility observed in hypothyroidism. Our data also suggest that the reduction in glucagon secretion may be secondary to the increase in circulating SLI.

Postreceptor effects of sulfonylurea on skeletal muscle glycogen synthase activity in type II diabetic patients

To elucidate the subcellular mechanism of action of sulfonylurea on glucose utilization of skeletal muscle, we studied nine newly diagnosed patients with type II (non-insulin-dependent) diabetes. Examinations were performed before and after 8 wk of gliclazide therapy. Gliclazide treatment was associated with improved glycemic control and enhanced pancreatic beta-cell responses to meal stimulation. During euglycemic insulin clamps, insulin-inhibited endogenous glucose production was improved after gliclazide therapy. Moreover, mean (+/- SE) glucose disposal rate increased from 3.2 +/- 0.7 to 4.8 +/- 0.8 and from 7.9 +/- 0.9 to 10.4 +/- 0.9 mg.kg-1.min-1 at in vivo plasma insulin levels of approximately 75 and approximately 320 mU/L, respectively. In addition, insulin-receptor function and glycogen synthase activity were analyzed in skeletal muscle biopsies obtained in seven patients. The biopsies were obtained during basal insulinemia and hyperinsulinemia (approximately 320 mU/L) before and after treatment. Insulin receptors purified with wheatgerm agglutinin showed unchanged insulin-binding properties and unchanged receptor kinase function with respect to basal and insulin-stimulated phosphorylation of exogenous peptide poly(Glu80Tyr20). Gliclazide treatment had no effect on the maximal activities of glycogen synthase. Moreover, in biopsies obtained at basal insulinemia, the half-maximal activation constant for glucose 6-phosphate (A0.5) was identical before and after therapy (0.54 +/- 0.05 vs. 0.54 +/- 0.05 mM, respectively, NS). However, in biopsies obtained at hyperinsulinemia, A0.5 was 0.30 +/- 0.05 vs. 0.20 +/- 0.02 mM before and after gliclazide therapy, P less than .04.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin-releasing hormone (TRH): Insulin-like action on glucoregulation

Normoglycemia is maintained through a complex interplay between the glucoregulatory hormones, glucagon, epinephrine. the glucocorticoids and insulin [l, 21, and it is under the controlling influence of various neurotransmitters and peptides acting upon the endocrine pancreas, the liver or at the level of the central nervous system (CNS) [3-6]. Most of the hormones and neural factors implicated in glucoregulation are called into play in response to hypoglycemia resulting from diminished supply or increased expenditure of metabolic fuel, and they act to normalize glucose levels by directly or indirectly promoting sugar production [7]. For example, glucagon is released from alpha-cells in the pancreas whenever plasma glucose levels fall below normal, and it stimulates glucose production in the liver through direct action [ 1,8] or, additionally, by acting within the CNS [9]. Similarly, epinephrine is released from the adrenal medulla in response to hypoglycemia, and it promotes glucose production through direct action on muscle or liver [ 1, 101 as well as indirectly, through CNS action [ll]. Other factors, such as cholecystokinin, bombesin, the opioid peptides and corticotropin-releasing factor (CRF), influence glucoregulation in part by modulating pancreatic function and in part by indirectly stimulating glucose production through CNS action [4, 12-151. In contrast to the multitude of apparently redundant counterregulatory mechanisms set in motion by hypoglycemia, only one peptide hormone, insulin. serves to counteract hyperglycemia. Insulin is released from pancreatic beta-cells in response to high glucose, and it normalizes sugar levels partly by stimulating glucose utilization in muscle and adipose tissues and partly by inhibiting glucose output from the liver [16, 171. In addition, insulin can lower circulating glucose levels by indirectly modulating the production and utilization of sugar through action in the CNS [lS-211. Thyrotropin-releasing hormone (TRH) is a hypothalamic tripeptide (pGlu-His-Pro-NH,) implicated in the regulation of pituitary thyrotropin and prolactin synthesis and secretion [22,23]. In addition. it is known for its many extrapituitary actions. Most

Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells.

Glucose is a well-known stimulus of proinsulin biosynthesis. In purified beta cells, the sugar induces a 25-fold increase in the synthesis of insulin immunoreactive material over 60-min incubation. Autoradiographic analysis of the individual cells shows that this effect is achieved via dose-dependent recruitment of pancreatic beta cells to biosynthetic activity. Recruitment of beta cells is also seen in isolated islets exposed to glucose. The sigmoidal dose-response curve for glucose-induced proinsulin biosynthesis thus reflects a heterogeneous responsiveness of pancreatic beta cells rather than a progressively increasing activity of functionally homogeneous cells. Dose-dependent recruitment of functionally diverse cells may be a ubiquitous mechanism in tissue function.

Impaired β-cell Responses Improve when Fasting Blood Glucose Concentration is Reduced in Non-insulin-dependent Diabetes

Pancreatic beta-cell responses to a controlled intravenous glucose stimulus in 18 untreated non-insulin-dependent diabetic patients were compared with those in seven healthy control subjects. The patients' first-and second-phase responses were only 10 to 20 per cent of those of the normal subjects. However, when normal subjects had been hyperglycaemic for two hours, their first-phase responses were similar to those of the patients. Six patients were subsequently treated by diet alone, six by diet and tolbutamide and six by diet and metformin. There was no improvement in first-phase responses after treatment, but second-phase responses doubled. Improved responses were seen with all treatments, and correlated with the fall in fasting blood glucose concentration during treatment. This study supports the view that hyperglycaemia impairs beta-cell function and suggests that first-phase responses are more sensitive to hyperglycaemia than second-phase responses.

Glucose regulation in non-insulin-dependent diabetes mellitus: Interaction between pancreatic islets and the liver

The degree of fasting hyperglycemia in patients with non-insulin-dependent diabetes mellitus is dependent on the rate of hepatic glucose production. The basal rate of hepatic glucose production is increased in patients with non-insulin-dependent diabetes mellitus, and there is a positive correlation between hepatic glucose production and fasting glucose levels. Diminished secretion of insulin, impaired hepatic sensitivity to insulin's effects, or a combination of these factors could contribute to the elevated hepatic glucose production in patients with non-insulin-dependent diabetes mellitus. The relationship between insulin secretion and hepatic glucose production is regulated by a closed feedback loop operating between glucose levels and pancreatic beta cells. Although fasting insulin levels are usually comparable between patients with non-insulin-dependent diabetes mellitus and normal subjects, insulin secretion is markedly impaired in non-insulin-dependent diabetes mellitus in relation to the degree of hyperglycemia present. In fact, the degree of fasting hyperglycemia in a given patient with non-insulin-dependent diabetes mellitus is closely related to the degree of impaired pancreatic beta-cell responsiveness to glucose. Such findings suggest that impaired insulin secretion leads to increased hepatic glucose production, which raises the plasma glucose level. The resulting hyperglycemia helps to maintain relatively normal basal insulin output. Chronic sulfonylurea drug therapy of patients with non-insulin-dependent diabetes mellitus enhances pancreatic islet sensitivity to glucose, leading to increased insulin secretion, suppression of hepatic glucose production, and a decline in the steady-state fasting glucose level.

C-Peptide responses to glucose load in maturity-onset diabetes of the young (MODY).

Pancreatic beta cell responses were measured in obese and nonobese maturity-onset diabetes of the young (MODY) patients by estimating serum immunoreactive insulin (IRI) and C-peptide (CP) during oral glucose tolerance testing (OGTT). The serum CP responses were generally low in MODY patients and more pronounced in obese patients. The IRI responses were heterogenous; some patients had normal and others low responses. It was observed that in several patients there was a relatively higher IRI concentration in comparison with the CP values, as indicated by low CP and normal IRI values. This is suggestive of altered metabolic fates of insulin and CP in the MODY patients.

Prevention of the glucose intolerance of thiazide diuretics by maintenance of body potassium.

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.

Development of the Biostator Glucose clamping algorithm.

The "glucose clamping" technique has been proposed as a method for the early detection of a beginning derangement of glucose homeostasis and thus for the possible prevention of maturity-onset diabetes. This technique interrupts the physiological glucose-insulin relationship by placing a patient's blood glucose concentration under an investigator's control, for quantification of the pancreatic beta-cell response during hyperglycemic clamps and of sensitivity of body tissue to exogenous insulin during normoglycemic clamps. We report the development of a glucose clamping algorithm for use with the Biostator glucose-controlled insulin-infusion system (Horm. Metab. Res., Suppl. 8: 23-33, 1977). This algorithm adds simplicity and precision to the glucose clamping procedure and reduces operator effort to a minimum. We describe the early development of the algorithm with a model system and report evaluations made during animal studies and preliminary investigations with human subjects.

Insulin responses to glucose in non-insulin-dependent diabetic subjects with and without the chlorpropamide-alcohol flush: effect of salicylate and naloxone.

We examined the role of endogenous opiates and/or prostaglandins on the abnormal insulin secretion characteristic of some non-insulin-dependent diabetic subjects. A group of chlorpropamide-alcohol flush positive (CPAF+) and a group of flush negative (CPAF-) non-insulin-dependent subjects were compared as to their pancreatic beta-cell responses to intravenous glucose tolerance tests before and after sodium salicylate infusion, and before and after naloxone infusion. There was no difference in mean insulin secretion (either first or second phase) between CPAF+ versus CPAF- groups. Both groups increased their insulin secretion with salicylate infusion, and both had a small decrease with naloxone infusion. There was no correlation between chlorpropamide-alcohol flushing and beta-cell response to glucose.

The mechanism of abnormal pancreatic beta cell response to food following acute hypoglycaemia in man.

Following acute insulin-induced hypoglycaemia, an abnormal pattern of insulin secretion in response to a meal has been demonstrated in six healthy volunteers. This is characterized by an initial impairment in insulin secretion and late hyperinsulinaemia. Postprandial gastrointestinal hormone levels were normal following hypoglycaemia in these subjects. In four other subjects, the administration of intravenous glucose prior to the meal partially reversed the abnormal pattern of secretion. In two patients with a total pre-ganglionic sympathectomy, the pattern of blood glucose, plasma insulin and C-peptide was similar to that observed following hypoglycaemia in normal subjects. It is unlikely that an abnormal entero-insular axis or an elevation of plasma catecholamine levels are primarily responsible for this phenomenon. This effect of hypoglycaemia on postprandial insulin secretion may be caused by glucopenia of the pancreatic beta cells.