Umbilical endometriosis associated with large umbilical hernia. Case report.

Umbilical endometriosis is a rare condition, usually following laparoscopic and surgical procedures involving the umbilicus.Spontaneous umbilical endometriosis occurring without any previous abdominal or uterine surgery is extremely rare. The maximal depth of penetration of the umbilical endometriosis described is up to fascial level. There have been only two cases of endometriosis reported arising within umbilical hernia. The authors report a case of a patient with spontaneous umbilical endometriosis associated with a large umbilical hernia, treated by surgical excision and mesh repair of the abdominal wall. To the best of our knowledge, this is the first described case of the association of umbilical endometriosis with a large umbilical hernia that requires prosthetic mesh repair of the abdominal wall defect.

[Diabetes and research in primary care: how to improve the care of our patients?]. / Diabète et recherche en médecine de premier recours: comment améliorer la prise en charge de nos patients?

Family practitioners are well aware of the guidelines for diabetic care yet they often find it difficult to apply them in practice. Experience from the literature as well as our own research provide guidance on ways to address this problem in Primary care: 1) collaboration with a nurse practitioner for the prevention of micro and macro-angiopathic complications, 2) the use of motivational interviewing techniques to motivate patients to lifestyle changes, 3) multidisciplinary collaboration (with specialists, nurses, colleagues, pharmacists, etc) and the support of information technology. Research within the Swiss academic institutes of Primary care should provide further, more concrete, guidance on ways to apply these different options in Switzerland to improve the quality of care for diabetic patients.

Phenytoin inhibition of insulin release. Studies on the involvement of Ca2+ fluxes in rat pancreatic islets.

The mechanism by which phenytoin inhibits insulin release was studied. Insulin release and 45Ca2+ efflux were measured during perifusion of collagenase isolated rat islets after 2-day maintenance in tissue culture in the presence of a trace amount of 45Ca2+. Islets maintained in the absence of the isotope were used to measure 45Ca2+ uptake over 5 min. Glucose (16.7 mM) induced a biphasic release of insulin, which was accompanied by a biphasic increase in the rate of 45Ca2+ efflux above basal. Phenytoin (80 microM) added during second phase rapidly inhibited insulin release. In contrast, phenytoin added together with glucose failed to affect first phase, but reduced second phase release by 64%. Phenytoin failed to affect basal 45Ca2+ efflux in the presence or absence of Ca2+, nor did it interfere with the inhibitory effect of high glucose on Ca2+ efflux. The drug did not affect basal Ca2+ uptake, but significantly reduced glucose-induced Ca2+ uptake. Glucose utilization was not inhibited by phenytoin. It is suggested that phenytoin inhibits glucose-stimulated insulin release by interfering with Ca2+ uptake via voltage-dependent Ca2+ channels. The pattern of inhibition of insulin release appears to favor this conclusion as the second phase is more dependent on the stimulation of Ca2+ uptake than is the first phase.

Involvement of Ca2+ in the impaired glucose-induced insulin release from islets cultured at low glucose.

Islet culture at a low glucose concentration results in a progressive impairment of glucose-induced insulin release. The role of Ca2+ in this defect was studied by comparing rat islets cultured for 6 days either at 8.3 mM (control) or 2.8 mM glucose. For measurement of 45Ca content and 45Ca2+ efflux, islets were kept in the presence of 45Ca2+ throughout. In islets cultured at 8.3 mM glucose, stimulation with 16.7 mM glucose during perifusion caused a typical biphasic pattern of insulin release paralleled by an increase in the rate of 45Ca2+ efflux. Both effects of glucose were markedly reduced in islets kept at 2.8 mM glucose, despite a similar insulin content. Islet 45Ca content was reduced. Both 45Ca content and insulin release were restored when islets were kept for an additional 24 h at 8.3 mM glucose. Insulin release induced by 3-isobutyl-1-methylxanthine (IBMX) or alpha-ketoisocaproic acid was not impaired, demonstrating that there is no generalized release defect. In contrast, glyceraldehyde- or K+-induced release was decreased. In islets maintained at 2.8 mM glucose, the stimulatory effect of glucose on Ca2+ uptake and the inhibitory effect on Ca2+ efflux (in the absence of Ca2+) were found to be operative. A defect may therefore lie distal to the Ca2+ uptake step involving either the mechanism by which glucose uses cellular Ca or another step yet to be identified.

Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.

New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.

Stimulation of insulin release by glucose in a transplantable rat islet cell tumor.

A subline of an x-ray-induced transplantable rat insulinoma has been studied in vivo and in vitro. Tumors grew rapidly after sc transplantation and were rich in insulin, but contained only small amounts of glucagon and somatostatin. Despite marked basal hyperinsulinemia, iv glucose administration caused a further increase in plasma insulin in tumor-bearing rats. When the pancreas was functionally excluded by ligation of supplying arteries, glucose still elicited a clear insulin response. In vitro, insulin release from perifused tumor fragments was stimulated by the combination of glucose and 3-isobutyl-1-methylxanthine, but not by glucose alone. In contrast, there was a clear stimulation of insulin release by glucose in primary monolayer cultures of tumor cells. This suggests a better functional capacity of the cultured cells compared to that of the tumor fragments. The results indicate that this transplantable rat islet cell tumor is a convenient source of large quantities of functional beta-cells.

Effects of cytokines on rat insulinoma INS-1 cells.

To investigate further the role of cytokines in the pathogenesis of type I insulin-dependent diabetes mellitus, the effects of interleukin-1 beta (IL-1), tumour necrosis factor-alpha (TNF) and gamma-interferon (IFN) were tested on rat insulinoma INS-1 cells. Whereas TNF and IFN had, respectively, a minor or no effect on insulin production, IL-1 caused a time- and dose-dependent decrease in insulin release and lowered the insulin content as well as the preproinsulin mRNA content of INS-1 cells. Both IL-1 and TNF exerted a cytostatic effect, estimated by a decrease in [3H]thymidine incorporation, while only IL-1 decreased cell viability as measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The glutathione content of INS-1 cells was shown to be modulated by the presence of 2-mercaptoethanol in the culture medium, but was not affected by IL-1 or TNF. In conclusion, INS-1 cell culture is considered to be a useful model for studying the effect of cytokines on insulin-producing cells. The differentiated features of these cells will permit several questions to be addressed regarding the mechanism of action of IL-1 and eventually other cytokines, both at the level of gene expression and of intracellular signalling.

Free radical modulation of insulin release in INS-1 cells exposed to alloxan.

Generation of free radicals is thought to mediate the cytotoxic action of alloxan on the pancreatic beta-cell. In this investigation, the early effects of alloxan on cell function were studied. When INS-1D insulinoma cells were exposed to alloxan (1 mM) for 45 min followed by a 3-hr recovery period, the drug increased basal insulin release while abolishing the effect of glucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was added directly after 5 min of alloxan treatment, whereas KCl-induced secretion was only partially inhibited. In perifusion, alloxan caused transient insulin secretion to 50% of the rates obtained with glucose 30 min later. Under these conditions, epinephrine abolished the stimulation due to both agents. Membrane potential and cytosolic calcium concentrations ([Ca2+]i) were recorded to clarify the action of alloxan. Alloxan-induced insulin release correlated with depolarization of INS-1D cells and a rise in [Ca2+]i. Alloxan did not augment [Ca2+]i in the presence of BHA or the absence of extracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca2+]i, whereas verapamil was ineffective. This suggests that alloxan promotes Ca2+ influx through channels distinct from L-type channels, perhaps through non-selective cation channels. Thus, alloxan causes changes in INS-1D cells prevented by antioxidant treatment, suggesting that free radicals may modulate the ionic permeability leading to functional activation.

Improved insulin secretion of cryopreserved human islets by antioxidant treatment.

The viability of islets of Langerhans prior to grafting is believed to influence the clinical outcome of islet transplantation. To determine whether oxidative stress occurs during the isolation-purification procedure as well as during tissue culture and cryopreservation, we have measured the glutathione redox state (GSH/GSSG) of islets. Human islets were purified by standard techniques from organ donors, cultured, and cryopreserved. Glucose-induced insulin release was monitored in parallel during static incubations to assess the function of the islets. Cultured human islets responded by a 2.2-fold increase in insulin release to a glucose challenge. After cryopreservation the hormonal response was lower. Immediately after islet isolation the GSH/GSSG ratio was 25.2 +/- 5.2, and it increased slightly to 32.0 +/- 6.1 after 1-3 days in tissue culture. The GSH/GSSG decreased significantly after cryopreservation to 12.2 +/- 3.4, suggesting that the freezing and thawing procedures imposed oxidative stress on the islets. To explore this hypothesis further, cryopreserved islets were treated with the antioxidant butylated hydroxyanisole (BHA). Islets exposed to BHA showed an improved glucose-induced insulin release and had an increased insulin content. BHA also protected the islets when they were exposed to alloxan, a free radical generating agent. However, after cryopreservation, BHA treatment did not modify the glutathione redox state. Although the BHA effect could not be explained merely by a change in the glutathione redox state, it is not precluded that redox changes of other cell components ameliorate the glucose sensitivity of the beta cells. Further studies will be needed to determine possible ways of improving islet cryopreservation with antioxidant treatments and particularly, to validate the present observations by in vivo experiments in the context of clinical islet transplantation.

[Android-type obesity and gynecoid-type obesity]. / Obésité de type androïde et obésité de type gynoïde.

There are several types of obesity, and the metabolic conditions associated with these phenotypes are also heterogeneous. Obesity of the male (android) type shows a dominant visceral and upper thoracic distribution of adipose tissue, whereas in the feminine (gynecoid) type adipose tissue is found predominantly in the lower part of the body (hips and thighs). Android obesity is clearly a cardiovascular risk factor, more so than gynecoid obesity. Hereditary factors contribute significantly to the occurrence of this pathology in families, although environmental factors play a role in its development. Android obesity is associated with metabolic anomalies which also characterize the syndrome X: resistance to insulin, arterial hypertension and dyslipidemia. The predisposition of individuals with android obesity to become diabetic rests in part on genetic and in part on environmental factors. Hyperinsulinemia and a high flux of free fatty acids act at the level of liver and endocrine pancreas to increase resistance to insulin and to decrease insulin secretion, two determining factors for type II diabetes. Other functional anomalies have been involved to explain android obesity such as dysregulation of adrenocortical and sexual steroids or a global derangement of stress mechanisms. No significant proof, however, seems to support either one of these hypotheses.

Effect of 2-mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells.

The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-mercaptoethanol for survival in tissue culture. The effect of 2-mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.

[Injuries in Vojvodina 1989]. / Prikaz traumatizma u Vojvodini 1989.

Trauma has all sociomedical characteristics and importance which line up it in the leading problems of the contemporary medical pathology. Injuries are among the first five groups of the diseases in the structure of overall outpatient's morbidity (general medicine 4.05%, occupational medicine 7.57%, pediatric medicine, 1.84% and health service for pupils 4.96%) and are a significant cause of hospitalization. Concerning the very thorough and all-inclusive follow up of all aspects of trauma it is necessary to establish the regional register of injury and intoxication.

Islet cell metabolism is reflected by the MTT (tetrazolium) colorimetric assay.

Insulin secretion depends critically on glucose metabolism. We investigated whether a rapid viability test could be established for assessing glucose metabolism in insulin secreting cells. The MTT (C,N-diphenyl-N'-4,5-dimethyl thiazol-2-yl tetrazolium bromide) colorimetric assay (reduction of tetrazolium salt to formazan) was applied to rat islets and rat insulinoma cell lines. It was found that the rate of formazan production correlated with glucose oxidation and glucose utilization at glucose concentrations which also stimulated insulin secretion. In differentiated insulinoma INS-1 cells, salt reduction paralleled the insulin release at glucose concentrations of up to 8.3 mmol/l. The glucose-induced formazan production in INS-1 cells and islets was abolished by exposure to the Beta-cell cytotoxic agents, streptozotocin or alloxan. The MTT assay thus provides a convenient tool for the rapid assessment of Beta-cell metabolism and viability.

Interactions of Ca2+, Mg2+, and Na+ in regulation of insulin release from rat islets.

The effects of glucose and ionic modifications on unidirectional Ca2+ efflux and insulin release has been studied. Rat pancreatic islets were isotopically equilibrated with 45Ca2+ for 2 days and then perifused at 10(-8) M Ca2+ to allow for strict interpretation of 45Ca2+ efflux. Under these conditions 16.7 mM glucose inhibited Ca2+ efflux but did not stimulate insulin release. Removal of Mg2+ from the buffer markedly stimulated Ca2+ efflux that was counteracted by glucose. The omission of Na+ decreased basal Ca2+ efflux by 30% at 10(-8) M Ca2+, thus demonstrating the importance of Na-Ca countertransport for Ca2+ extrusion. Like glucose, Na+ omission or the addition of ouabain attenuated Ca2+ efflux stimulated by Mg2+ removal. Glucose may interfere with Na-Ca countertransport because the actions of 16.7 mM glucose and Na+ omission were not additive. At 10(-8) M Ca2+, glucose elicited insulin release only when both 1) loss of cellular calcium was minimized by prior inhibition of Ca2+ efflux (Na+ omission or ouabain), and 2) Ca2+ mobilization was favored by Mg2+ removal. Under these conditions (in contrast to normal Ca2+), insulin release was not accompanied by increased Ca2+ efflux. Thus, unidirectional Ca2+ measurements do not permit the detection of Ca2+ mobilization in intact islets because glucose may concomitantly inhibit Ca2+ extrusion.

Cobalt inhibition of insulin release: evidence for an action not related to Ca2+ uptake.

Insulin release, Ca2+ fluxes, and adenosine 3',5'-cyclic monophosphate (cAMP) levels were measured under various conditions to test the selectivity of cobalt (Co2+) actions in pancreatic islets. Insulin release was measured simultaneously either with 45Ca2+ uptake or 45Ca2+ efflux from rat islets maintained 2 days in tissue culture. Glucose (16.7 mM)-stimulated insulin release was inhibited by Co2+ (0.25, 0.5 mM) without concomitant inhibition of glucose-stimulated 45Ca2+ uptake. At higher concentrations of Co2+ (1.0, 2.5 mM) glucose- as well as K+-stimulated insulin release were inhibited in parallel with 45Ca2+ uptake. In the presence or the absence of external Ca2+, Co2+ (0.5 mM) also decreased 3-isobutyl-1-methylxanthine-stimulated insulin release, which does not depend on the stimulation of Ca2+ uptake. Co2+ did not alter islet cAMP levels. Co2+ also did not affect the unidirectional basal 45Ca2+ efflux measured in the absence of Ca2+ but completely overcame the stimulated 45Ca2+ efflux evoked by the removal of external Mg2+. Under the latter conditions, 50, but not 5 microM verapamil, decreased the stimulated 45Ca2+ efflux. It is concluded that Co2+ is able to inhibit insulin release and lower Ca2+ efflux without apparent interference with Ca2+ uptake. This suggests that Co2+, in addition to its well-known inhibition of Ca2+ uptake, may exert its action at a distal step in the insulin release process.

Calmodulin in various insulin secreting cell types.

The involvement of the Ca2+ binding protein, calmodulin, in the regulation of insulin release was studied. Calmodulin was measured in isolated rat islets, rat insulinoma cells, the insulin secreting cell line (RINm5F) and in islets isolated from normal and diabetic Chinese hamsters. Total content of calmodulin was determined by a radioimmunoassay using a rabbit anti-calmodulin serum and was found to lie in the range of 4 to 7 micrograms/ml protein. When rat islets were maintained in tissue culture for 6 days at 2.8 or 8.3 mM glucose, the content of calmodulin of the two groups was similar. Likewise there was no difference in calmodulin content between islets from normal and diabetic hamsters. This study suggests that a variation of the total cellular calmodulin does not play a role in the process of insulin secretion.