Nicole Bégin-Heick

Zinc Supplementation Attenuates Insulin Secretory Activity in Pancreatic Islets of the ob/ob Mouse

The purpose of this study was to establish whether a relationship may exist between the hyperinsulinemia, the exaggerated insulin secretion, and the resistance to insulin characteristic of the obese-hyperglycemic syndrome and the zinc status of the ob/ob mouse. To this end, mice were given control and zinc-supplemented diets, and the effects of zinc supplementation on insulin secretion in vivo and in vitro as well as on glucose tolerance were studied. These data were compared with those obtained with oxytetracycline treatment, which is known to ameliorate the insulin sensitivity and glucose tolerance of these animals. The levels of zinc were measured in several tissues of lean and obese mice and the results show that zinc supplementation attenuated the exaggerated insulin secretion in vivo and in vitro without improving the tolerance to glucose. Zinc levels were significantly higher in the tissues of the obese than of the lean mice, with the exception of bone and pancreas. The results suggest a maldistribution of zinc in the tissues of the obese mouse.

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures.

The subcellular localization of the heterotrimeric G‐proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G‐proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via Gsα were without effect, which was consistent with the localization of Gsα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell‐cell and cell‐extracellular matrix interactions.

Oxytetracycline Treatment Improves the Response to Insulin in the Spontaneously Diabetic (BB) Rat

Chronic oxytetracycline treatment was found to alter the diabetic status of the spontaneously diabetic rat (BB rat). The treatment led to lowered plasma glucose levels in the fed as well as in the fasted state. These results indicate that the oxytetracycline treatment was effective in lowering the insulin requirements as well as in improving the handling of glucose. The effects of the drug are not secondary to the decreased food intake as a food restricted control group did not show the improvements in glycemia or glucose and insulin tolerance. These results are a further indication that oxytetracycline enhances the response of peripheral tissues to insulin and thus favors better control of glycemia.

Do pancreatic islet cells from neonatal rats have surface receptors or sensors for divalent cations

The effects of extracellular divalent cations on the intracellular Ca2+ concentration ([Ca2+]i) in neonatal rat islet cells were investigated to determine whether these cells, like several others, have signal-generating surface cation sensors. Raising the external Ca2+ concentration by 1 mM increments triggered either sustained increases in [Ca2+]i or large sharp [Ca2+]i spikes followed by return to a suprabasal level. The external Ca(2+)-triggered [Ca2+]i responses were abolished by treating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), but not by another phospholipase C inhibitor, U-73,122 (2.5 microM), or the voltage-sensitive Ca2+ channel blockers nifedipine (20 microM) and methoxyverapamil (D600; 50 microM). [Ca2+]i responses were also triggered by barium (Ba2+; 1 mM) and cobalt (Co2+; 1 mM). The Ba2+ responses were also inhibited by neomycin and unaffected by nifedipine or D600 and the Co2+ response required external Ca2+. Therefore, neonatal rat pancreatic islet cells may display divalent cation receptors/sensors on their surfaces. Activation of these putative receptors, which are coupled to neomycin-sensitive, voltage-independent, dihydropyridine-insensitive channels, by Ca2+, Ba2+ or Co2+ would trigger [Ca2+]i responses by opening these channels to admit external Ca2+ into the cell. The physiological function(s) of such cell-surface divalent cation receptors/sensors and the [Ca2+]i surges they generate in pancreatic islet cells is not known.

Comparison of the properties of the ATP-sensitive K+ channels of pancreatic β-cells of lean and obese (ob/ob) C57BL/6J mice

SummaryCultures of pancreatic islet cells from obese and lean mice of the C57BL/6J strain were established and their secretory response to glucose stimulation was measured. Insulin secretion (as % of total cellular insulin content) from the cells of the obese mouse cultures was significantly higher than from lean mouse cells. The properties of the glucose- and ATP-sensitive potassium channels present in these cultured β-cells were compared using the cell-attached and the inside-out configurations of the patchclamp technique. The channels of both types of mouse were indistinguishable in terms of conductance, ionic selectivity, kinetic behavior, voltage dependence or sensitivity to glucose, ATP and ADP. It is concluded that the depolarized state and the hypersecretory response of obese mouse β-cells are not related to an altered behavior of their ATP-sensitive potassium channels.

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes.

The subcellular localization of Gsα, Giα1&2, Giα3, and Gβ was studied in primary‐cultured undifferentiated and differentiated, lipid replete, adipose cells. The results show a distinct distribution for each of these G‐proteins and differences between differentiated and undifferentiated cells. All the G‐proteins examined had a cytoplasmic localization; only Giα1 and 2 showed a significant colocalization with the plasma membrane and this only in differentiated cells. Most studies using cells in culture have reported an intracellular localization for G‐proteins, whereas in tissue sections the localization has been reported to be largely with the plasma membrane, with some intracellular localization. The results suggest that the cell‐cell interactions or the specific geometry imposed by culture conditions favor the intracellular compared to peripheral localization of G‐proteins. Alternately, the posttranslational modifications necessary for G‐protein insertion in the plasma membrane may be deficient in cultured cells. J. Cell. Biochem. 65:259–266.

Insulin secretion and intracellular Ca2+ rises in monolayer cultures of neonatal rat β-cells

Abstract Glucose-induced insuline release, glucose-induced rises in intracellular free Ca 2+ concentration ([Ca 2+ ] i ), and voltage-dependent Ca 2+ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca 2+ ] i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca 2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca 2+ ] 2 rise and, like deprivation of extracellular Ca 2+ , prevented the glucose-induced rise in [Ca 2+ ] i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca 2+ channels was demonstrated directly by measuring Ca 2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca 2+ ] 1 after membrane depolarization by 45 mMm K + or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca 2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.

The regulation of adenylate cyclase in liver membranes of lean and obese mice

The modulation of adenylate cyclase by guanosine triphosphate (GTP) and hormones was examined in liver membranes of lean and ob/ob mice, to determine whether a defective regulation of cyclase similar to that found in adipocyte membranes was present. In conjunction with GTP, glucagon was a powerful stimulant of cyclase in both types of membranes. In contrast, GTP alone or in conjunction with isoproterenol and norepinephrine stimulated significantly less in the membranes of the lean than in those of the obese mouse. In addition, low concentrations of norepinephrine elicited an inhibitory response in membranes of the lean mouse, but not in those of the obese. This inhibitory effect of norepinephrine was abolished by the alpha 2-antagonist yohimbine and by treatment with pertussis toxin, but not by propranolol or treatment with cholera toxin. These data show that it is possible to demonstrate inhibitory effects of guanine nucleotides on cyclase in the membranes from lean but not those from obese mice and suggest a defect in inhibitory regulation in the tissue of the obese.

Effect of the genetic background and specific mutation on adenylate cyclase activity in obesity syndromes.

Adenylate cyclase activity and its modulation by guanine nucleotides and isoproterenol were assessed in adipocyte membranes of mice with mutations causing different genetic obesity syndromes. The object was to determine whether the defect in inhibitory modulation observed in the obese (ob/ob) mouse was also present in the diabetes (db/db) mouse. The data show that adipocyte adenylate cyclase in both the ob/ob and the db/db mouse is resistant to activation by isoproterenol. The response to guanosine triphosphate (GTP) differed between the two mutants, such that an inhibitory phase was visible in the db/db but not in the ob/ob membranes. Moreover, pertussis toxin attenuated the inhibitory effect of GTP and significantly stimulated cyclase activity in the db/db but not in the ob/ob membranes. The data show that the two mutations affect the expression of adenylate cyclase activity via different mechanisms.

Insulin Secretion in the Obese (ob/ob) Mouse: The Effect of Oxytetracycline on Insulin Release

The effect of oxytetracycline (OTC) pretreatment on the response of the ob /ob mouse to insulin secretagogues in vivo and in vitro was investigated. With glucose loading in vivo, the peak glucose was twofold greater and the insulin levels threefold greater in obese than in lean mice. After OTC treatment, there was no significant difference in insulin levels between lean and obese mice although the peak glucose level was still 1.5 times as high. Glucagon increased plasma glucose 2.5-fold and plasma insulin 20-fold in the obese as compared with lean mice. After OTC treatment, the glycemie response of the obese was indistinguishable from that of the lean control. The insulin levels, while higher than those of lean mice, were only 25% of those found in the untreated obese. Aminophylline produced an 8- and a 20-fold increase in peak glucose and insulin levels, respectively, as compared with lean mice. In the OTC-treated obese mice, the injection of aminophylline produced a slower rise in plasma glucose than in the obese controls, but the levels were not significantly different from those of the untreated obese mice at 90 min. On the other hand, the insulin levels attained a plateau at a value which was one-fifth that found in the control obese group. In vitro, isolated islets from obese mice showed an exaggerated response to the secretagogues. Pretreatment with OTC attenuated this response. The fraction of insulin released at 10 mM glucose was less than one-fourth that in the obese controls. With glucagon added, the response was only one-eighth, and with aminophylline, one-half as great in the OTC-treated than in the obese control. The effects of OTC cannot be attributed to the effects of the drug on food consumption, since obese mice food restricted to the intake of the OTC-treated obese mice showed either no improvement or much smaller Changes.