A cortical band of fine microfilaments is consistently observed in the beta cells of the rat pancreas. Alteration of this cell web by cytochalasin B is associated with an enhancement of glucose-induced secretion of insulin by isolated islets. The microfilamentous web of the beta cell may play an important role in the emiocytosis of insulin secretory granules, by controlling their access to the cell membrane.

https://science.sciencemag.org/content/sci/175/4026/1128.full.pdf

Pancreatic Beta-Cell Web: Its Possible Role in Insulin Secretion

The release of insulin from pancreatic islets requires negative regulation to ensure low levels of insulin release under resting conditions, as well as positive regulation to facilitate robust responsiveness to conditions of elevated fuel or glucose The first phase of release involves the plasma-membrane fusion of a small pool of granules, termed the readily releasable pool; these granules are already at the membrane under basal conditions, and discharge their cargo in response to nutrient and also non-nutrient secretagogues By contrast, second-phase secretion is evoked exclusively by nutrients, and involves the mobilization of intracellular granules to t-SNARE sites at the plasma membrane to enable the distal docking and fusion steps of insulin exocytosis Nearly 40 years ago, the actin cytoskeleton was first recognized as a key mediator of biphasic insulin release, and was originally presumed to act as a barrier to block granule docking at the cell periphery More recently, however, the discovery of cycling GTPases that are involved in F-actin reorganization in the islet β-cell, combined with the availability of reagents that are more specific and tools with which to study the mechanisms that underlie granule movement, have contributed greatly to our understanding of the role of the cytoskeleton in regulating biphasic insulin secretion Herein, we provide historical perspective and review recent progress that has been made towards integrating cytoskeletal reorganization and cycling of small Rho-, Rab- and Ras-family GTPases into our current models of stimulus-secretion coupling and second-phase insulin release

/pdf/mechanisms-of-biphasic-insulin-granule-exocytosis-roles-of-qcrq27mtve.pdf

Mechanisms of biphasic insulin-granule exocytosis – roles of the cytoskeleton, small GTPases and SNARE proteins

A simple perifusion system for in vitro studies on the rate of insulin secretion of isolated rat islets is described. A biphasic pattern of insulin secretion was produced by glucose whereas tolbutamide stimulated only the first phase of insulin secretion. The perifusion system was used to determine the effect of anti-mitotic agents on the biphasic pattern of insulin secretion. Yinblastine and colchicine destroy microtubules whereas deuterium oxide (D2O) produces stabilization and interference with the function of microtubules. Vinblastine (10-4 M) and D2O (100 per cent) inhibited completely the first and second phase of glucose-induced insulin secretion. The inhibitory effect of D2O was reversible and a biphasic pattern of secretion occurred following the replacement of D2O with water. Colchicine (10-3 M) produced significant inhibition of only the second phase of glucose-induced insulin secretion. The first phase of tolbutamide-induced insulin secretion was inhibited by vinblastine and D2O and the inhibitory effect of D2O was reversible. Colchicine did not inhibit the first phase of tolbutamide-induced insulin release. These findings indicate that the microtubular system is involved in both phases of insulin secretion. The first phase of secretion may be due to the release of beta granules already associated with the microtubular system and the second phase could be the result of stored and newlysynthesized granules becoming associated with the system.

Perifusion of Isolated Rat Islets in Vitro: Participation of the Microtubular System in the Biphasic Release of Insulin

Two general categories of ATPases are present in tissue. One group requires Na+ and K+ as well as Mg2+ for maximum enzyme activity and functions in active transport of Na + , K + , and other substances across cell membranes. The second group of ATPases requires Mg2+ or Ca2+ for activation. The biochemical and physiological significances of the Mg2+-Ca2 +-activated enzyme systems are less understood. The one major protein system activated by Mg2+ or Ca2+ which has received extensive study is the actomyosin complex; in this system the hydrolysis of ATP stimulated by divalent cations plays an essential role in muscle contraction and relaxation.

https://science.sciencemag.org/content/sci/179/4072/441.full.pdf

Actomyosin-like protein in brain.

Cytoplasmic microtubules and their functions

The beta cell of the islets of Langerhans has been shown to contain a microtubular-microfilamentous system. The possible role of this system in the insulin secretory process was investigated by examining the effect upon glucoseinduced insulin secretion of mitotic spindle-inhibitors and microtubules-stabilizers. Inhibition of secretion was observed when the pieces of pancreas were pre-incubated for ninety minutes with either colchicine (0.01 to 1.0 mM) or vincristine (0.01 mM) prior to measurement of their secretory response to a high glucose concentration. Significant reduction of glucose-induced secretion was also noticed in thepresence of D 2 O (25 per cent or more, v/v), 2-methyl-2,4-pentanediol (0.5 to 1.0 per cent, v/v) and ethanol (1.0 per cent, v/v). The inhibitory effect of D 2 O was reversible. The stimulant action of glucose upon both calcium uptake and insulin biosynthesis by isolated islets was inhibited by mannoheptulose, but unaffected by either vincristine or D 2 O, suggesting that the latter agents did not interfere with glucose metabolism in the beta cell. These results suggest that the integrity of the microtubular-microfilamentous system is required for glucose to exert its normal stimulant action upon insulin secretion. It is hypothesized that glucose-induced calcium uptake by the beta cell could trigger the release of insulin by causing the contraction of such system, favoring the migration of the secretory granules and their extrusion in the process of emiocytosis.

The Stimulus-secretion Coupling of Glucose-induced Insulin Release: V. The Participation of a Microtubular-microfilamentous System

The HLA-DR genotypes of 158 new type I diabetic probands from simplex families are compared with those of 43 multiply affected sibships. There were no significant differences in the gene frequencies of the insulin-dependent diabetes mellitus (IDDM)-associated alleles, DR3 and DR4, and whereas the DR3/4 heterozygotes were as frequent among simplex probands as among the first affected of multiplex sibships, subsequently affected sibs displayed lower frequencies of this genotype in this as well as in previously reported samples, indicating that the excessive risk associated with DR3/4 heterozygosity depends on the order of affection and thus on environmental factors. It is proposed that the penetrance of the susceptibility gene is enhanced by epistatic effects of this genotype and that this enhancement is strongest under conditions of low environmental liability. Thus, the excessive risk for DR3/4 individuals appears to depend on secondary interactions between DR and the environmental factors that trigger the onset of this disease and does not in itself indicate the existence of distinct susceptibility alleles in coupling with these genes, i.e., of genetic heterogeneity or overdominance.

Excess of DR3/4 in Type I Diabetes: What Does It Portend?

The gene frequencies, haplotype relative risks, and zygotic assortments of HLA-DR in three ethnically defined samples of insulin-dependent diabetes mellitus (IDDM) patients were determined in a prospective family study. Although DR3 and DR4 were positively associated with IDDM in the probands of 123 northern European, 94 Ashkenazi Jewish, and 49 New York Hispanic families, significant excess of DR*3/4 heterozygotes was observed only among the probands from families of northern European ancestry. There was also a significant decrease in the frequency of Bw62,DR4 haplotypes derived by northern European patients from their mothers compared with their fathers. This difference, together with data reported in the literature, suggests that the expressivity of the susceptible genotype(s) in IDDM patients may be modified by protective maternal effects associated with Bw62,DR4 and probably other DR4 haplotypes. Samples of IDDM patients from populations with high frequencies of these modifiers should have different DR-gene frequencies contributed by fathers and mothers, capable of accounting for the observed Hardy-Weinberg disequilibrium. We postulate that, because the mechanism of action of these modifiers is distinct from that of the susceptibility gene, the difference must be considered in devising strategies for elucidation of the mode of inheritance of the disease and for understanding the molecular nature of the susceptibility.

Mary O Walker

Papers

The Stimulus-secretion Coupling of Glucose-induced Insulin Release: V. The Participation of a Microtubular-microfilamentous System

Excess of DR3/4 in Type I Diabetes: What Does It Portend?

No Excess of DR*3/4 in Ashkenazi Jewish or Hispanic IDDM Patients