Phosphatidylinositol kinase in rat pancreatic islets: subcellular distribution and sensitivity to calcium

Plasma membrane of pancreatic islets contains a calcium sensitive phosphatidylinositol kinase. This enzyme catalyzes the first reaction in the pathway leading to the production of inositol trisphosphate, which is believed to cause a redistribution of intracellular calcium. Since the activity of this enzyme is inhibited by calcium (K0.5 = 10 microM), a loss of calcium from plasma membrane (the site of PI kinase) may be necessary for activation of the enzyme in vivo.     

Postnatally disturbed pancreatic islet cell distribution in human islet amyloid polypeptide transgenic mice

OBJECTIVE: Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth. RESULTS: Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation. CONCLUSIONS: Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

Tissue, cellular, and subcellular distribution of 241Pu in the rat testis

The distribution and localization of 241Pu in rat testes were determined by quantitative autoradiography. Rats were given an intravenous injection of 241Pu citrate and tissues were collected 1 week later. The tissue distribution of 241Pu was determined by light microscope autoradiography. Significant concentrations of 241Pu were observed in the interstitial tissue but not in seminiferous tubules. The cellular distribution and subcellular localization of 241Pu were determined by electron microscope autoradiography. Within the interstitial tissue, 241Pu was concentrated in macrophages. There was no preferential localization of 241Pu in any other interstitial tissue components. Within interstitial tissue macrophages, 241Pu was concentrated in the lysosomal system of the cell. Other organellar compartments of the cell did not preferentially incorporate 241Pu. The association of 241Pu with the macrophage lysosomal system may explain the long retention times of Pu in testes as observed in experimental studies.     

Regional, cellular, subcellular distribution of neurospecific protein-hormonal complexes

A sensitive radioimmunological assay (RIA) has been developed to detect the tissue specificity and subcellular localization of three specific protein-hormonal complexes (PHC) of the hypothalamus which have a regulatory function in the brain and visceral organs. Using the highly specific rabbit antisera to beef PHC an order of increasing immunoreactivity in different areas of CNS is as follows: hypothalamus, cerebellum, occipital cortex. The PHC-like immunoreactivity (IR) is found in the neurosecretory granules of the hypothalamo-neurohypophyseal system (about 30%) and in the synaptosomal fraction (70%). In the myelin, mitochondrial and nuclear fractions the PHC-like IR is not revealed. IR of PHC is 1000-fold higher in the brain than in visceral organs (heart, skeletal muscles, adrenals, pancreas and blood serum). A possible role of the PHC as markers of the neuroendocrine cells is discussed.     

Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium

The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2-acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP-diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.     

The pancreatic islet endothelial cell: emerging roles in islet function and disease

The pancreatic islets are one of the most vascularized organs of the body. This likely reflects the requirements of the organ for a rich supply of nutrients and oxygen to the tissue, as well as the need for rapid disposal of metabolites and secreted hormones. The islet endothelium is richly fenestrated to facilitate trans-endothelial transport of secreted hormones, has a unique expression of surface markers, and produces a number of vasoactive substances and growth factors. The islet endothelial cells play a critical role in the early phase of type 1 diabetes mellitus by increasing the expression of surface leucocyte-homing receptors, thereby enabling immune cells to enter the endocrine tissue and cause beta-cell destruction. Following transplantation, pancreatic islets lack a functional capillary system and need to be properly revascularized. Insufficient revascularization may severely affect the transport properties of the islet endothelial system, resulting in a dysfunctional islet graft.     

The pancreatic islet endothelial cell: emerging roles in islet function and disease

The pancreatic islets are one of the most vascularized organs of the body. This likely reflects the requirements of the organ for a rich supply of nutrients and oxygen to the tissue, as well as the need for rapid disposal of metabolites and secreted hormones. The islet endothelium is richly fenestrated to facilitate trans-endothelial transport of secreted hormones, has a unique expression of surface markers, and produces a number of vasoactive substances and growth factors. The islet endothelial cells play a critical role in the early phase of type 1 diabetes mellitus by increasing the expression of surface leucocyte-homing receptors, thereby enabling immune cells to enter the endocrine tissue and cause beta-cell destruction. Following transplantation, pancreatic islets lack a functional capillary system and need to be properly revascularized. Insufficient revascularization may severely affect the transport properties of the islet endothelial system, resulting in a dysfunctional islet graft.     

Effect of chronic ethanol consumption on the cellular and subcellular distribution of gamma-glutamyltransferase in rat liver

After 6 weeks of chronic ethanol consumption hepatic gamma-glutamyl-transferase and -hydrolase activities increased compared with pair-fed controls. There was no change in 5'-nucleotidase activity. It was found that the increase in gamma-glutamyltransferase activity occurred exclusively in the parenchymal cells although the principal cellular localisation for this enzyme is the biliary tract in both control and ethanol-fed rats. In both groups of animals the gamma-glutamyltransferase activities were localised by analytical subcellular fractionation techniques to soluble, plasma membrane and canalicular fractions, but the plasma membrane activity was selectively increased in the ethanol-fed rats.     

A computer model of pancreatic islet glycolysis

We have modeled an experiment with perifused pancreatic islet cells using our BIOSSIM language. The experiment and the resulting model are concerned with glucose uptake and glycolysis by the beta-cells of pancreatic islets. Although glycolysis appears to be involved in insulin release, we do not have enough information to represent insulin release in detail. The rapid entry of glucose into the beta-cell is promoted by a carrier having a very high tissue capacity. Phosphorylation of glucose by the low affinity enzyme glucokinase appears to be limiting for glycolysis. The effects of several hexose diphosphate activators of phosphofructokinase are modeled. Model behavior is described. The kinetic parameters of the enzyme submodels are given. Because of the difficulties of preparing large amounts of experimental material, information on pancreatic islet metabolism is limited. This model is a plausible explanation of the experimental results. Recent work on the genetically engineered glucose transporter and glucokinase is discussed.     

Microcarriers: A new approach to pancreatic islet cell culture

Summary Free islet cell suspensions were prepared from isolated fetal rat islets using a modified enzyme dispersion technique. The islet cells were dispensed into a culture flask containing microcarriers (Cytodex) suspended in culture medium RPMI 1640 by a slowly rotating bar magnet. Microscopical examination of the beads showed that the islet cells attached and then progressively proliferated on the surface of the beads as a monolayer. A highly sustained release of insulin from the beads to the medium was observed during the 7 d culture period. The functional viability of the cultured islet cells was further demonstrated by the ability of batches of the cell-coated beads to synthesize insulin and to increase the insulin release in response to an acute challenge (16.7 mmol/l glucose plus 5 mmol/l theophylline). The results suggest that bead microcarriers may provide a new approach to monolayer islet cell culture providing functional monolayers, which can easily be transferred to different test systems and further manipulated. Financial support for the study was received from the University of Uppsala, the Swedish Diabetes Association, Stiftelsen Expressens Prenatalforskningsfond, Nordisk Insulinfond, Stiftelsen Claes Groschinskys Minnesfond, the Swedish Society of Medical Sciences and the Swedish Medical Research Council (Grants No. 12X-109 and 12X-2297). This work was presented in part at the 15th Annual Meeting of the Scandinavian Society for the Study of Diabetes (June 5–7, 1980; Oslo, Norway) and the 16th Annual Meeting of the European Association for the Study of Diabetes (September 24–27th, 1980; Athens, Greece).     

Immunocytochemical localization of relaxin in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state

Relaxin is one of the hormones present during pregnancy and it is synthesized primarily by corpora lutea (CL). Other reproductive tissues including CL of the menstrual cycle may also synthesize this hormone. Very little is known, however, about the cellular and subcellular distribution of relaxin in human CL and dependence of luteal relaxin on the reproductive state. The light and electron microscope immunocytochemical studies described here were undertaken to obtain this information using antisera to porcine and human relaxin. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states or in human hepatocytes. Luteal immunostaining was low in early luteal phase; it increased progressively, reaching the highest level in late luteal phase, and then decreased greatly in corpora albicantia. Term pregnancy CL contained similar immunostaining as early luteal phase CL. Mid luteal phase CL contained more immunostained cells than late luteal phase CL, but the late luteal phase CL contained a greater amount of immunostaining per cell than mid luteal phase CL. The immunogold particles due to relaxin were primarily present in secretory granules and to a small extent in rough endoplasmic reticulum. Quantitation revealed that secretory granules contained a much higher number of gold particles than did rough endoplasmic reticulum. These two organelles from late luteal phase CL contained greater numbers of gold particles than those from mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysosomes and pancreatic islet function

Summary The relation between qualitative and quantitative glucose-dependent alterations of lysosomes in pancreatic islets and the function of the islets was studied. Isolated islets of the mouse were maintained in tissue culture for one week in either 28, 5.5 or 3.3 mmol/l glucose. Insulin biosynthesis, insulin secretion and insulin content of the cultured islets were determined. After culture, the islets were subjected to acid phosphatase cytochemistry and examined by electron microscopy and ultrastructural morphometry. Islets cultured in 28 mmol/l glucose both produced and secreted insulin rapidly. Such islets seemed, however, unable to maintain more than small amounts of granule-stored insulin. Islets cultured at the lower concentrations of glucose displayed a reduced insulin secretion, which apparently resulted in considerable amounts of intracellularly stored insulin. In all cultured islets different types of lysosomes, identified by their acid phosphatase reactivity, could be seen. Dense bodies, i.e., lysosomes characterized by a homogeneous, very fine, particulate content of high density, seemed to predominate at all concentrations of glucose. It is suggested that, in the islets, the dense bodies correspond morphologically to primary lysosomes. Other types of lysosomes with inclusions of various kinds, which were frequent at the two lower concentrations of glucose, may correspond to secondary lysosomes. Morphometry revealed differences between the size distributions of lysosomes in the three experimental groups. Thus, the average lysosomal size was inversely proportional to the concentration of glucose in the culture medium. However, the numerical density of lysosomes was greatest at the highest glucose concentration. The observation of secondary lysosomes, containing material resembling secretory granules, suggests that the increased size and lowered number of lysosomes in islets cultured at low glucose concentrations may depend on a crinophagic process. Such a process, together with insulin biosynthesis and insulin secretion, may be of physiological importance for control of the secretory granule content within the pancreatic B-cell.     

Alpha-melanotropin in the frog brain: regional distribution, quantification and subcellular distribution

The distribution of alpha-MSH containing neurons was studied by immunofluorescence in the brain of the frog Rana ridibunda. Most immunoreactive cell bodies were found in the ventral hypothalamic area. A rich network of fluorescent fibers was observed in the ventral infundibular region, coursing towards the preoptic area and the ventral telencephalon. Some fibers, directed backwards, project into median eminence. By means of a specific radioimmunoassay, the concentrations of alpha-MSH immunoreactive material has been determined in 10 different regions of the brain. The highest concentrations were observed in the infundibular and the preoptic regions. Using the immunogold technique, electron microscopy showed that immunostaining was restricted to 70-100 nm dense core vesicles in positive cell bodies and fibers. These results suggest that, in addition to well known hormonal (melanotropic) activity, alpha-MSH could play the role of a neurotransmitter in the frog brain.