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B Draznin

Publications and source records attributed to B Draznin.

At least 73 records · Page 4Linked to original sources
PubMed1990

Postprandial changes in cytosolic free calcium and glucose uptake in adipocytes in obesity and non-insulin-dependent diabetes mellitus.

We evaluated the possible relationship between [Ca2+]i and glucose uptake in the postabsorptive state and postprandially in adipocytes obtained from normal and obese subjects, as well as from patients with non-insulin-dependent diabetes mellitus (NIDDM). Adipocytes isolated from overnight-fasted obese and NIDDM patients revealed high levels of [Ca2+]i (p less than 0.05 vs. control) in association with a decreased insulin-stimulated glucose uptake (p less than 0.05 vs. controls). In obese and NIDDM patients treated with oral hypoglycemic agents, the overnight fasting levels of [Ca2+]i were increased postprandially (p less than 0.05), concomitantly with a further decrease in insulin-stimulated 2-deoxyglucose uptake. Although the precise nature of the relationship between [Ca2+]i in specific insulin target tissues and diminished insulin action remains unknown, it is clear that high levels of [Ca2+]i may contribute to the development of insulin resistance.

Adipose Tissue↗
PubMed1989

In vivo administration of interleukin-1 inhibits glucose-stimulated insulin release.

Recombinant interleukin-1 beta (IL-1 beta) was administered intraperitoneally for 3 days to normal C57BL/6ByJ (B6) mice. The islets from IL-1-treated and control animals were isolated and glucose-stimulated insulin secretion studied in the perifusion system. The total islet insulin content and the ultrastructure of the islets isolated from the animals treated with IL-1 did not differ from those seen in control animals. However, glucose-stimulated insulin release was significantly impaired after 3 days of in vivo administration of IL-1, either 3 micrograms/animal/day or 0.3 micrograms/animal/day. The administration of IL-1 inhibited an acute phase of glucose-induced insulin release, whereas neither basal insulin secretion nor insulin release from 10-30 min of perifusion with glucose was impaired. There was an only partial (27%) and non-significant restoration of the insulin secretory response to glucose stimulation 4 days after discontinuation of IL-1 treatment. We conclude that IL-1 administered in vivo is capable of adversely affecting pancreatic islet response to glucose stimulation. After 3 days of administration, these changes are confined to the process of insulin release, with the islet cell morphology and total insulin content being unaffected.

Animals↗
PubMed1989

Mechanism of insulin resistance induced by sustained levels of cytosolic free calcium in rat adipocytes.

We have recently provided evidence that elevated levels of cytosolic free Ca2+ ([Ca2+]i) decreased insulin-stimulated glucose uptake in isolated rat adipocytes. To investigate the mechanism of Ca2+ action, we examined the effects of elevated levels of [Ca2+]i on insulin binding, autophosphorylation, and tyrosine kinase activity (TKA) of insulin receptors as well as basal and insulin-stimulated cellular distribution of glucose transporters. The latter was assessed by cytochalasin-B binding to plasma membrane and cytosolic fractions. Elevated concentrations of [Ca2+]i were maintained by incubating adipocytes with a depolarizing concentration of K+ (40 mM). Basal nonstimulated glucose uptake was not altered by increased levels of [Ca2+]i. Adipocytes with higher [Ca2+]i (220 +/- 15 nM) showed 30% reduction in insulin-stimulated 2-deoxyglucose uptake compared with control cells ([Ca2+]i, 140 +/- 18 nM). Moreover, adipocytes with higher levels of [Ca2+]i demonstrated an approximately 10% reduction in autophosphorylation and TKA of insulin receptors without a change in insulin binding. Both basal and insulin-stimulated distributions of glucose transporters were unaffected by sustained levels of [Ca2+]i. The effects of elevated [Ca2+]i were not mimicked by protein kinase-C activation. These observations suggest that 1) elevated or sustained levels of [Ca2+]i impair insulin-stimulated glucose uptake; and 2) Ca2+-induced impairment appears to reside at the postbinding steps of insulin action and probably interferes with the TKA of insulin receptors and the intrinsic activity of glucose transporters.

Adipose Tissue↗
PubMed1988

Intracellular calcium, insulin secretion, and action.

Changes in cytosolic free calcium concentration [( Ca2+]i) constitute an important element of signal transduction in various cells. These changes either reflect alterations in calcium (Ca2+) fluxes or result from mobilization of intracellular Ca2+ stores. In pancreatic islet cells, an increase in [Ca2+]i is critical for secretagogue-induced insulin release. Thus, glucose evokes a rapid increase in [Ca2+]i, primarily by stimulating Ca2+ influx. Under physiologic conditions, glucose may also promote mobilization of intracellular Ca2+ stores by virtue of stimulating membrane phospholipid hydrolysis and formation of inositol triphosphate, a potent stimulus for Ca2+ mobilization. This action of glucose requires the presence of extracellular Ca2+. The magnitude of change in [Ca2+]i may not parallel the level of insulin release, suggesting that the role of [Ca2+]i in the process of insulin release must be considered in concert with other cellular mechanisms. The role of [Ca2+]i in promoting insulin action is a subject of continuous controversy. Recent observations that chelation of intracellular Ca2+ with quin-2 diminishes insulin action (and that of insulin mimetics) support the role of Ca2+ in mediating the insulin-generated signal. Insulin has also been demonstrated to increase [Ca2+]i in adipocytes in close association with its effect on 2-deoxyglucose uptake. Finally, in both pancreatic islet cells and adipocytes, high concentrations of either extracellular or intracellular Ca2+ inhibit cellular responsiveness. The optimal concentrations of cytosolic Ca2+ appear to be within the 140 to 350 nM range. When Ca2+ concentrations are too low or too high, the ability of pancreatic islets and insulin target cells to respond appropriately to physiologic stimuli is significantly diminished. Impaired cellular Ca2+ homeostasis (either primary or secondary to other cellular lesions) may represent a crucial and identical link in the pathogenesis of impaired insulin secretion and in the pathogenesis of impaired insulin action.

Adipose Tissue↗
PubMed1988

Cyclosporin-induced inhibition of insulin release. Possible role of voltage-dependent calcium transport channels.

The exposure of normal pancreatic islets to cyclosporin-A (1 microgram/ml) for 24 hr resulted in significant inhibition of glucose-induced (16.7 mM) insulin release from 197 +/- 14 microU/10 islets/15 min (control) to 103 +/- 14 microU/10 islets/15 min (Cy-A-treated islets; P less than 0.001). Cy-A did not alter insulin release in the presence of non-stimulatory (1.7 mM) or submaximally effective glucose concentrations (9.2 mM). In parallel experiments, Cy-A reduced glucose-stimulated increases in cytosolic free calcium concentrations, [Ca2+]i (217 +/- 15 nM without and 137 +/- 3 nM with Cy-A in the presence of 16.7 mM glucose, P less than 0.01). To better define the site of Cy-A action, we studied its effect on insulin release and increases in [Ca2+]i induced by either K+ (50 mM), which promotes Ca2+ influx via voltage-dependent Ca2+ channels, or by forskolin (20 microM), dibutyryl cyclic AMP (1 mM) or arachidonic acid (49 microM), all of which stimulate mobilization of intracellular Ca2+ stores. Cy-A significantly inhibited K+-induced changes (203 +/- 13 nM without and 77 +/- 6 nM with Cy-A, respectively, P less than 0.001), but not those induced by forskolin, dibutyryl cyclic AMP or arachidonic acid. These observations suggest that Cy-A inhibits insulin release by interfering with Ca2+ influx via voltage-dependent calcium channels.

Animals↗
PubMed1988

Insulin and glucose modulate protein kinase C activity in rat adipocytes.

In the presence of 1 mM glucose, insulin (10 ng/ml) increases both catalytic and receptor-binding properties of adipocyte cytosolic protein kinase C (PKC). Preincubation of adipocytes with 10 mM glucose raises basal PKC catalytic activity and prevents further stimulation of this enzyme by insulin. The effect of hyperglycemia is likely to be mediated by direct conversion of glucose into diacylglycerol. Thus, an incorporation of 14C-glucose into diacylglycerol is enhanced 10-fold in the presence of 10 mM glucose. These observations indicate that, in normal adipocytes, both insulin and glucose activate PKC; hyperglycemia eliminates the ability of insulin to stimulate this enzyme, thereby interfering with insulin action.

Adipose Tissue↗
PubMed1988

Glyburide increases cytosolic-free calcium concentrations in normal rat pancreatic islet cells.

We have attempted to delineate the effect of glyburide on the regulation of cytosolic-free calcium concentrations, [Ca2+]i, in normal rat pancreatic islet cells. In the presence of extracellular calcium (1 mmol/L), glyburide increased [Ca2+]i from 70 nmol/L to 260 nmol/L in a dose-dependent manner. The maximal effect was seen at a concentration of 2 mumol/L with half-maximal stimulation observed at .25 mumol/L. The effect of glyburide (.25 mumol/L) was inhibited 90% by the calcium channel blocker, verapamil (30 mumol/L). At a maximally effective concentration of glyburide (2 mumol/L), the inhibitory effect of verapamil was only 17%. In the absence of extracellular calcium, glyburide increased [Ca2+]i from 55 nmol/L to 107 nmol/L, indicating its ability to mobilize intracellular calcium stores. These results correlated well with the ability of glyburide (2 mumol/L) to stimulate insulin secretion both in the presence (from 38 +/- 5 mumol/L/10 islets to 131 +/- 28 microU/10 islets) and in the absence (from 49 +/- 4 microU/10 islets to 93 +/- 7 microU/10 islets) of extracellular Ca2+. The present observations suggest that glyburide promotes calcium influx via voltage-dependent calcium channels and may mobilize intracellular calcium stores.

Animals↗
PubMed1988

Exocytosis in normal anterior pituitary cells. Quantitative correlation between growth hormone release and the morphological features of exocytosis.

We have used high-pressure freezing techniques to study exocytosis in rat anterior pituitary cells. The cells were either unstimulated or exposed to 1 nM growth hormone releasing factor (GRF) for 10 min before ultrarapid freezing. The magnitude of growth hormone (GH) release was then correlated with the number of exocytotic events observed with freeze-fracture electron microscopy. High-pressure freezing of unfixed and uncryoprotected specimens permits cryofixation of samples up to 1 mm diam (0.5 mm thick) without ice crystal damage, and arrests exocytotic events within 10 ms. Our studies comparing conventionally fixed specimens with those prepared by high-pressure freezing confirm that areas of intramembrane particle clearing at potential exocytotic sites are an artifact of conventional fixation and/or cryoprotection techniques. The cells exposed to 1 nM GRF released approximately fivefold more GH than did unstimulated cells. Morphologically, we have observed a 3.3-fold increase in the number of exocytotic events in GRF-stimulated cells, 33.7 events/100 micron2 compared with 10.4 events/100 micron2 for unstimulated cells. In additional experiments, we studied the effects of two inhibitors of GRF-induced exocytosis, somatostatin and sodium isethionate. Both compounds elicit the same response, a parallel decrease in exocytotic events and in secreted product. We conclude that high-pressure freezing, combined with freeze-fracture and freeze-substitution processing techniques, is an excellent tool for studying the morphological aspects of exocytosis. In the present investigation, it has allowed us to quantitatively relate the biochemistry and morphology of exocytosis in anterior pituitary cells.

Animals↗
PubMed1988

Possible role of cytosolic free calcium concentrations in mediating insulin resistance of obesity and hyperinsulinemia.

Insulin- and glyburide-stimulated changes in cytosolic free calcium concentrations [( Ca2+]i) were studied in gluteal adipocytes obtained from six obese women (139 +/- 3% ideal body wt) and six healthy, normal weight age- and sex-matched controls. Biopsies were performed after an overnight fast and twice (at 3 and 6 h) during an insulin infusion (40 mU/m2 per min) (euglycemic clamp). In adipocytes obtained from normal subjects before insulin infusion, insulin (10 ng/ml) increased [Ca2+]i from 146 +/- 26 nM to 391 +/- 66 nM. Similar increases were evoked by 2 microM glyburide (329 +/- 41 nM). After 3 h of insulin infusion, basal [Ca2+]i rose to 234 +/- 21 nM, but the responses to insulin and glyburide were completely abolished. In vitro insulin-stimulated 2-deoxyglucose uptake was reduced by insulin and glucose infusion (25% stimulation before infusion, 5.4% at 3 h, and 0.85% at 6 h of infusion). In obese patients, basal adipocyte [Ca2+]i was increased (203 +/- 14 nM, P less than 0.05 vs. normals). The [Ca2+]i response demonstrated resistance to insulin (230 +/- 23 nM) and glyburide (249 +/- 19 nM) stimulation. Continuous insulin infusion increased basal [Ca2+]i (244 +/- 24 nM) and there was no response to either insulin or glyburide at 3 and 6 h of study. Rat adipocytes were preincubated with 1-10 mM glucose and 10 ng/ml insulin for 24 h. Measurements of 2-deoxyglucose uptake demonstrated insulin resistance in these cells. Under these experimental conditions, increased levels of [Ca2+]i that were no longer responsive to insulin were demonstrated. Verapamil in the preincubation medium prevented the development of insulin resistance.

Adipose Tissue↗
PubMed1988

Relationship between cytosolic free calcium concentration and 2-deoxyglucose uptake in adipocytes isolated from 2- and 12-month-old rats.

We have examined the relationship between insulin-stimulated 2-deoxyglucose uptake and cytosolic free calcium concentrations, [( Ca2+]i), in adipocytes isolated from 2- and 12-month-old rats. The basal rates of glucose uptake and the levels of cytosolic Ca2+ were only minimally reduced in 12-month-old animals. In contrast, insulin-stimulated glucose up-take and [Ca2+]i were significantly decreased in older adipocytes at all insulin concentrations (P less than 0.01). When the rate of glucose uptake was plotted as a function of [Ca2+]i, insulin-stimulated glucose uptake was almost identical in older and younger animals at any given level of [Ca2+]i. Similar to insulin, glyburide and K+ increased [Ca2+]i in both younger and older adipocytes. However, glyburide- and K+-elicited responses were lower in older rats (P less than 0.01). The effects of insulin, glyburide, and K+ on [Ca2+]i are mediated via voltage-dependent Ca2+ channels. Thus, the present observations suggest an impairment in either function and/or availability of the voltage-dependent Ca2+ channels in older animals. This was supported by the finding of reduced [3H]nitrendipine binding in adipocytes isolated from older animals (6.5% vs. 3.3% in 2- and 12-month-old rats, respectively; P less than 0.01). The results of these experiments indicate that the postreceptor changes in adipocyte responsiveness to insulin in aging may involve inadequate increases in [Ca2+]i. The latter probably occurs as a result of decreased availability and/or function of the voltage-dependent calcium channels.

Adipose Tissue↗
PubMed1987

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes.

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.

Adipose Tissue↗
PubMed1987

Unmasking of arachidonate-induced insulin release by removal of extracellular calcium. Arachidonic acid mobilizes cellular calcium in rat islets of Langerhans.

Exogenous arachidonic acid does not stimulate insulin release in Ca++-containing medium, but a potent effect was unmasked by extracellular Ca++ depletion. This secretion met several criteria of exocytotic release. It did not require the oxygenation of arachidonate or its esterification into islet membranes, but was potentiated by the presence of 16.7 mM glucose such that 33 microM arachidonate could reverse the inhibitory effects of extracellular Ca++ removal on glucose-induced insulin secretion. Arachidonic acid alone stimulated a rise in intracellular Ca++ concentrations in dispersed islet cells (measured by the fura-2 technique) equal to that induced by 16.7 mM glucose in normal medium. Arachidonic acid may be a critical coupling signal in normal islets.

Animals↗
PubMed1987

Insulin and glyburide increase cytosolic free-Ca2+ concentration in isolated rat adipocytes.

We investigated the effect of insulin and a hypoglycemic sulfonylurea agent glyburide on cytosolic free-Ca2+ concentrations [( Ca2+]i) in isolated rat adipocytes. Both insulin and glyburide increased [Ca2+]i in a dose-dependent manner. Half-maximal effects were seen at 0.5 ng/ml of insulin and 0.5 microM glyburide. Nifedipine (25 microM), a Ca2+-channel blocker, inhibited the effect of both agents. The effect of insulin on [Ca2+]i was 40 and 70% potentiated by ambient glucose concentrations at 180 and 300 mg/dl, respectively. Depolarizing doses of potassium (40 mM) induced an increase in cytosolic Ca2+ that was also inhibited by nifedipine. It is suggested that both insulin and glyburide increase cytosolic free Ca2+ levels at least in part by promoting Ca2+ influx through voltage-dependent Ca2+ channels.

Adipose Tissue↗
PubMed1987

Cytosolic free-calcium concentrations in normal pancreatic islet cells. Effect of secretagogues and somatostatin.

We have assessed the effect of somatostatin on glucose-, potassium-, forskolin-, and dibutyryl cAMP-induced changes in cytosolic free [Ca2+] in normal rat pancreatic islet cells with the new Ca2+ indicator fura 2. The cytosolic free [Ca2+] in islet cells incubated with nonstimulatory concentrations of glucose (30 mg/dl) ranged from 54 to 64 nM. In the presence of extracellular Ca2+ (1 mM), glucose (300 mg/dl) rapidly increased the cytosolic free [Ca2+] to a level of 90-110 nM. In the absence of extracellular Ca2+, glucose failed to increase the cytosolic free [Ca2+], which remained at a level of 55-60 nM. Somatostatin inhibited glucose-induced increases in cytosolic free [Ca2+] in a dose-dependent manner (maximal inhibition was 34%). Half-maximal inhibition was observed at 10(-9) M somatostatin, which correlated well with somatostatin binding to islet cells (Kd = 2.6 X 10(-10) M). Potassium (50 mM) rapidly increased the cytosolic free [Ca2+] to 110-120 nM, and its effect was not influenced by the presence of somatostatin. Forskolin (20 microM) and dibutyryl cAMP (1 mM) rapidly increased cytosolic free Ca2+ both in the presence and absence of extracellular Ca2+. More than 80% of the overall increase in cytosolic free-Ca2+ levels could be accounted for by the mobilization of intracellular Ca2+ stores. Somatostatin effectively blocked the forskolin effect (32% inhibition) but not the dibutyryl cAMP-induced effect. Somatostatin appears to inhibit secretagogue-induced increases in cytosolic free [Ca2+] by interfering with cAMP production and probably with Ca2+ transport across the cell membrane.

Animals↗
PubMed1986

Somatostatin inhibits fusion of pituitary secretion vesicles with the plasma membranes.

Somatostatin has been found to inhibit secretion vesicle fusion with the iodinated (125-I) inside-out plasma membrane vesicles (both were isolated from the anterior pituitaries). This effect of somatostatin was specific and dose-dependent (half-maximal effect at 10(-9) M). Calmodulin (10 microM), but not cyclic AMP, enhanced the fusion process between the two organelles and somatostatin inhibited calmodulin-stimulated fusion. These observations suggest that one facet of somatostatin action on hormone secretion may be its inhibition of secretion vesicle fusion with the plasma membrane.

Animals↗
PubMed1986

Insulin internalization and intracellular protein degradation: a quantitative correlation.

We have recently demonstrated that internalization of insulin is essential for insulin's action upon intracellular proteolysis (Draznin and Trowbridge 1982). In this study we have investigated the quantitative relationship between the rate of insulin internalization and its ability to inhibit intracellular proteolysis. We have used the acidification technique to separate surface bound 125I-insulin (sur) from internalized ligand (In). The In/Sur ratio plotted as a function of time permits the calculation of the rate of insulin internalization (K-e) (Draznin, Trowbridge and Ferguson 1984). Insulin in a dose dependent manner increased the rate of C14-glucose incorporation into glycogen and inhibited the rate of degradation of intracellular proteins prelabelled in vivo with C14-valine. When insulin internalization was blocked by phenylarsine oxide (10(-5) M), the amount of surface bound ligand and its effect on glucose incorporation into glycogen were unaffected whereas insulin's effect on intracellular proteolysis was markedly diminished. There was a direct and significant correlation between K-e and insulin induced inhibition of intracellular proteolysis (r = .72, P less than .05). The correlation between the amount of internalized insulin and intracellular proteolysis was also significant (r = .84, P less than .01).

Animals↗
PubMed1986

Somatostatin receptors are biologically active before they are inserted into the plasma membrane.

We have examined the biological activity of intracellular somatostatin (SRIF) receptors in cultured rat anterior pituitary cells. We used digitonin-permeabilized cells to introduce free SRIF intracellularly and chloroquine-treated cells to promote intracellular accumulation of SRIF via a receptor-mediated pathway. At a concentration of 0.001%, digitonin (3-min incubation at 37 C) allowed [125I]SRIF to enter the cells without affecting cell viability. Autoradiography of [125I]SRIF demonstrated its association with secretion vesicles (28%), nuclei (25%), and other intracellular organelles. An acid wash technique that removes cell surface-bound ligand revealed that both digitonin-permeabilized cells and chloroquine-treated cells accumulated approximately twice as much intracellular SRIF as did control cells. The biological activities of intracellular SRIF accumulated via two different pathways, receptor mediated and through digitonin-produced pores in the plasma membrane, were different. In chloroquine-treated cells, the accumulation of intracellular SRIF did not result in its additional biological effect. SRIF inhibited GH-releasing factor-induced GH release from 578 +/- 12 to 168 +/- 9 ng/10(6) cells X 30 min, which did not differ from the control value. Cells incubated with digitonin demonstrated normal basal (160 +/- 9 ng/10(6) cells X 30 min) and GH-releasing factor-stimulated GH release (564 +/- 11 ng/10(6) cells X 30 min). However, the inhibitory action of SRIF in these cells was approximately 30% greater (98 +/- 8 ng/10(6) cells X 30 min) than that in either control or chloroquine-treated cells, suggesting that SRIF freely admitted intracellularly produces additional biological activity. These observations confirm the presence of the intracellular receptors and suggest that these receptors exist in a biologically active form.

Animals↗