Tumor necrosis factor-alpha and interferon-gamma inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement.

Nitric oxide has been implicated as one possible mediator of interleukin-1 beta (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN+TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF+IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF+IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.

Growth factor protection against cytokine-induced apoptosis in neonatal rat islets of Langerhans: role of Fas.

Treatment of neonatal rat islets of Langerhans with combined cytokines (interleukin-1beta 10(-10) M, tumour necrosis factor-alpha 10(-10) M, interferon-gamma 5 U/ml) led to extensive cell death, which was potentiated by Fas activation with the anti-Fas cytolytic antibody JO2. Pre-treatment with insulin (25 ng/ml) or insulin-like growth factor-1 (10(-8)M) gave only partial protection against cell killing, but prevented the Fas-mediated component. In the absence of cytokine treatment, Fas-mediated killing was not observed.

DNA replication in mouse pancreatic islets transplanted subcapsularly into the kidney or intraportally into the liver. Influence of unilateral nephrectomy or partial hepatectomy.

3H-thymidine incorporation into DNA (estimated as the autoradiographic labeling index [LI]) was used as an index of DNA replication in syngeneic mouse islets grafted into the kidney or liver 4 weeks before unilateral nephrectomy (UN) or partial hepatectomy (PH). Removal of one kidney resulted in a significantly increased growth of the remaining islet-bearing kidney initiated during the first week after surgery. Seven days after 40% hepatectomy the remaining liver mass corresponded to 91% of that of sham-operated (SO) mice. The induced kidney and liver growth in mice subjected to UN or PH was correlated with an increased LI, which was similar in both organs (3.5 times the LI in kidneys and livers of SO-controls). Pancreatic islets implanted into the organs undergoing compensatory growth also had higher LI values that were less pronounced as compared with the LI measured in the parenchymal cells of the kidney or the liver after UN or PH, respectively. In contrast to the grafted islets, no changes in the LI of the endogenous pancreatic islets were found. To evaluate possible systemic effects of the induced growth stimulation, the livers of UN-mice and the kidneys of PH-mice were investigated. No change in the LI of the liver tissue was observed 3 and 6 days after UN, whereas the kidney cortex showed a significantly increased LI7 days after PH. It is concluded that when exposed to a local, non-specific growth stimulation in vivo pancreatic islets prepared from adult donors are able to respond with an enhanced replicatory activity. The effect is, however, only present when the islets are located in the organs in which growth has been induced.

Comparison of inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans by streptozotocin and methyl and ethyl nitrosoureas and methanesulphonates. Lack of correlation with nitric oxide-releasing or O6-alkylating ability.

We have studied inhibition of glucose-stimulated insulin secretion in islets of Langerhans isolated from adult Sprague-Dawley rats and treated with different alkylating agents. Streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU) all released nitric oxide, as demonstrated by an increase in medium nitrite and cellular cyclic GMP. Methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), which do not possess a nitroso group, did not show evidence of nitric oxide release. All five compounds, however, decreased glucose-stimulated insulin release, suggesting that nitric oxide release was not necessary for the inhibition of secretion. Lack of involvement of nitric oxide was further suggested by the failure of oxyhaemoglobin to reverse STZ and MNU inhibition of insulin secretion. Since ENU was at least as effective as MNU in inhibiting insulin secretion, it appears that alkylation of DNA at the O6 position of guanine may not be involved in this process.

Amino acids and human amniotic fluid increase DNA biosynthesis in pancreatic islets of adult mouse, but this effect is lost following exposure to streptozotocin.

The present study describes the effects of growth hormone (GH), amino acids, and human amniotic fluid on the function and replication of normal and SZ-treated adult mouse pancreatic islets. Thus, mouse islets were exposed in vitro to SZ or vehicle only, and maintained in culture for 7 days in RPMI 1640 containing 11.1 mM glucose and the different supplements described above. Supplementation with amino acids increased the insulin accumulation in the medium, DNA biosynthesis, and polyamine contents in the control islets. In the SZ-treated islets, amino acids increased insulin accumulation in the medium and polyamine contents, but not DNA biosynthesis. Culture of control islets in the presence of 10% human amniotic fluid increased the insulin accumulation in the medium, islet insulin content, total protein and (pro)insulin biosynthesis, and DNA biosynthesis. However, in the SZ-treated islets, the effects of human amniotic fluid were limited to an increase in insulin content and insulin accumulation in the medium. GH significantly increased insulin accumulation in the medium in control, but not SZ-treated islets. In both groups of islets, GH failed to induce a significant increase in thymidine incorporation. It is concluded that amino acids and human amniotic fluid are potent stimulators of DNA biosynthesis in adult mouse pancreatic beta cells, perhaps due to an increase in cellular polyamine contents. However, following exposure to streptozotocin, the islets are not anymore responsive to these stimulators of DNA biosynthesis.

Functional response of a limited beta-cell mass to long-term hyperglycemia.

250 syngeneic islets were implanted either beneath the kidney capsule or into the liver of diabetic LEW 1.A rats to investigate the functional response of a limited mass of beta-cells to long-term hyperglycemia. The number of islets, per se, was expected to be insufficient to reverse the hyperglycemia. All animals were characterized by a substantial body weight gain. Unexpectedly, 29% of the rats with a subrenal and 40% of the animals with a portal islet graft normalized their plasma glucose in 64 +/- 13 and 75 +/- 12 days, respectively. Depending on the glycemic state of the recipients, there was an elevation of the graft insulin content after 120 days over the level at transplantation. The responsiveness of the implanted islets to different secretagogues was tested either in vitro by static incubation of the prepared grafts from the kidney or in situ by perfusion of the islet-containing liver. Grafts of normoglycemic rats showed a pronounced response, although the biphasic profile of the hormone release was lost. In principle, grafts exposed permanently to a hyperglycemic environment have kept their responsiveness, although the insulin outflow was considerably lower. The functional viability of the islets was not influenced by the site of transplantation. Long-term hyperglycemia does not necessarily result in destruction and loss of beta-cells even when their total mass is already limited, but it obviously impairs their functional responsiveness.

Functional alterations in the rat kidney induced either by diabetes or high protein diet.

The aim of this study was to compare the effect of long-term diabetes with that of a long-term high protein diet in vivo on the kidney function and in vitro on cellular parameters of isolated glomeruli of BB rats. Four groups of rats were investigated: Group 1 = normoglycaemic (N) rats > 250 days old; group 2 = age-matched diabetic (D) rats with a diabetes duration of more than 150 days; group 3 = BB rats which were fed a protein diet of 8% (low protein = LP) and group 4 = rats which received a high protein (HP) diet (32%) for more than 80 weeks. From 24-h-urine samples albumin, urea, creatinine and electrolyte excretion were estimated. At the end of the study the total kidney weight was determined and glomeruli were isolated for in vitro experiments. Hyperglycaemic and HP fed rats were characterised by a significantly increased excretion of albumin, urea and different electrolytes compared to normoglycaemic or LP fed animals. Creatinine excretion was neither affected by hyperglycaemia nor HP diet. HP and D caused a significant increase in total kidney weight when compared to the LP and N group, respectively (N: 1.79 +/- 0.08; D: 2.33 +/- 0.09; LP: 2.26 +/- 0.18; HP: 3.28 +/- 0.46 g; p < 0.05). The increased kidney weight of diabetic and HP rats correlated well with a significant enhanced DNA content of isolated glomeruli (N: 3.41 +/- 0.15; D: 4.45 +/- 0.19; LP: 4.18 +/- 0.35; HP: 6.40 +/- 0.62 micrograms/1000 glomeruli; p < 0.02). In addition, glomeruli obtained from either D or HP fed BB rats incorporated significantly more 3H-thymidine into their DNA indicating an elevated rate of DNA synthesis. The results demonstrate that HP diet caused markedly altered kidney function and induced cellular changes of glomeruli which are interpreted as enhanced proliferative processes. These alterations are comparable to those associated with diabetes mellitus. An unbalanced high protein diet represents a considerable risk factor for the development of functional and structural impairments of the kidney and should absolutely be avoided in patients suffering from diseases which are known to be associated with kidney alterations.

Circadian variations in blood pressure and heart rate in diabetes prone and resistant rat strains compared with spontaneously hypertensive rats.

Circadian blood pressure (BP), heart rate (HR) and motor activity (MA) of nondiabetic (nd) and spontaneously diabetic (d) BB/OK rats were compared with that of spontaneously hypertensive rats (SHR). In addition a diabetes-resistant and non-hypertensive rat strain (LEW.1W) was monitored for the same parameters. Systolic and diastolic BP (SBP, DBP), HR and MA were measured telemetrically. In d BB rats, the 24 h mean value of SBP (132 +/- 0.15 mm-Hg) was significantly increased compared to nd BB rats (125 +/- 0.18 mmHg). No differences were found in DBP between d and nd BB rats (93 +/- 0.13 v.s. 94 +/- 0.15 mmHg). Both, d and nd BB rats were significantly different in SBP and DBP to that of SHR (155 +/- 0.19 and 110 +/- mmHg). Nondiabetic BB rats did not significantly differ from LEW.1W rats in SBP and DBP (125 vs. 123 mmHg and 94 vs. 94 mmHg). The heart rate was lowest in diabetic BB rats compared with all other strains. Compared to nd BB the diabetic rats had an altered daily rhythm in BP. The results demonstrate that the diabetic BB rats develop circadian variations in BP and HR similar to those observed in hypertensive rats.

Impact of metabolic activity of beta cells on cytokine-induced damage and recovery of rat pancreatic islets.

The influence of beta cell activity on cytokine-induced functional and structural impairments as well as the ability of those damaged cells to recover were investigated. Rat islets cultured for 4 days in the presence of 5, 10, and 30 mmol/l glucose were exposed to interferon-gamma (IFN, 500 U/ml) and tumor necrosis factor-alpha (TNF, 250 U/ml) for the last 24 h. After cytokine removal islets were allowed to recover spontaneously in culture medium containing 10 mmol/l glucose for a further 7 days. Cytokines significantly inhibited insulin release into culture medium, insulin storage, glucose-stimulated insulin secretion, protein, and DNA synthesis. In the presence of cytokines there was a six- to eightfold increase in nitrite production by the islets. The functional impairments were more pronounced in metabolically stimulated beta cells. In addition, cytokines caused membrane alterations as indicated by increased spontaneous chromium-51 release. The cytokines specifically induced the synthesis of two proteins (72 and 88 kDa, respectively). By immunoblotting, the 72-kDa protein was identified as heat shock protein. After a 1-week recovery period, insulin storage and stimulated insulin secretion of cytokine-treated islets were still significantly diminished. However, protein and DNA synthesis of cytokine-exposed islets returned to pre-exposure levels. In conclusion, high beta cell activity increases islet susceptibility to TNF+IFN. Cytokine-induced, long-lasting, inhibitory effects are primarily directed to beta-cell-specific functions, while general vital cell functions clearly recover after cytokine removal. The induction of certain proteins and the increased protein synthesis and replication rate after cytokine removal might reflect activated repair processes.

Human amniotic fluid obtained from diabetic women. A potent stimulator of islet cell replication.

Recently, human amniotic fluid (HAF) from healthy women was found to stimulate growth and function of pancreatic B-cells. Here, the effect of HAF and serum from healthy probands (HS) was compared with that from probands with gestational (GD), noninsulin-dependent (NIDDM), or insulin-dependent diabetes (IDDM) on islet function and replication. Rat islets were cultured in the presence of either HAF or HS for 7 d. Insulin content and basal insulin release were not different after exposure of the islets to HAF or HS from healthy or diabetic women. In contrast to HS, HAF provoked the islets to deliver significantly more insulin during culture. Additionally, the same islets exhibited a more intense response to a glucose challenge. The degree of HAF-induced insulin release was not influenced by the type of diabetes. HAF and HS from GD and NIDDM women did not influence the islet DNA synthesis in comparison to HAF and HS from healthy pregnant women. However, HAF but not HS from IDDM pregnant women, elicited a significant increase in islet replication. Most effective in stimulating islet cell replication were HAFs from IDDM pregnant women belonging to the White D-type. It was shown that the relatively high concentration of insulin in the HAFs was not directly responsible for the observed increase of the islet DNA synthesis. HAF from women with long-term diabetes is supposed to contain factor(s) that might directly or indirectly enhance islet replication.

The effect of human amniotic fluid on DNA synthesis of rat pancreatic islets in tissue culture.

In this study we investigated the effect of human amniotic fluid (HAF) on the incorporation of 3H-thymidine into cultured rat islets. The addition of 1% HAF (1% HAF, 99% TCM) did not alter the incorporation of labeled thymidine, in comparison to TCM 199 alone (100%). The culture in the presence of 50% HAF (1:1 diluted with TCM, v/v) resulted in a significantly increased DNA synthesis. To exclude any effect of ionic composition or substrate dilution by the HAF, the TCM 199 was also either diluted (1:1) with saline or Hank's balanced salt solution (HBSS). Islets cultured under these conditions did not show any stimulated thymidine incorporation into islet DNA. From these results we conclude that HAF contains an activity which stimulates islet replication. In order to concentrate this stimulatory activity, HAF was tested after it had been lyophilized or evaporated. Independent of the method used, in no case a stimulatory effect of concentrated HAF was found. In contrast, the concentrated form of HAF inhibited the incorporation of labeled thymidine into islet DNA.

3H-thymidine incorporation into islets obtained from prediabetic BB rats.

DNA synthesis, measured as 3H-thymidine incorporation, was estimated in islets obtained from BB rats before the onset of diabetes and compared with the replication rate of islets obtained from age-matched BB rats having never developed hyperglycaemia. Using a biopsy procedure, splenic pancreas was removed from both 65 and from 80 day old diabetes prone BB rats. After isolation by a modified collagenase method pancreatic islets were incubated with labeled thymidine. The animals submitted to biopsy were followed up until they reached an age of 200 days. The diabetes incidence of the BB rats biopsied at day 65 and at day 80 was comparable (35% and 31%). In both groups approximately 140 mg of splenic pancreas were necessary to isolate 70-90 islets, regardless whether the animals become diabetic or not. Using a retrospective experimental design, the thymidine incorporation into islets obtained from prediabetic (rats which developed hyperglycaemia) and from normoglycaemic BB rats were comparatively analysed. The DNA synthesis of pancreatic islets obtained from 65 day old prediabetic BB rats is not different from that measured in islets from normoglycaemic BB rats. A similar replicatory behaviour was also observed using islets from prediabetic and normoglycaemic 80 day old BB rats. From our results we seek to conclude that a prediabetic state is not related to a diminished replicatory activity of pancreatic islets.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of serum concentrations and sources on DNA synthesis and insulin secretion of cultured islets.

We investigated the effect of different sera, as newborn calf serum, fetal calf serum, rat serum and human serum at various concentrations on biofunctions of cultured pancreatic islets. Islets isolated from newborn LEW. 1 W rats were maintained at 37 degrees C in TCM 199 containing 10 mmol/l glucose and either 1%, 10% or 50% newborn calf serum (NCS), fetal calf serum (FCS), serum obtained from adult syngeneic rats (RS) or different batches of human serum (HS). While exposure of islets to increasing concentrations of NCS significantly inhibited the islet DNA synthesis as well as the glucose stimulated insulin secretion after 2, 4 and 8 days of culture, HS at different concentrations failed to inhibit both, the islet DNA synthesis and the secretory response to glucose at each time point investigated. The action of FCS, RS and 2 further batches of HS on islet DNA synthesis was analysed by short-term culture. The supplementation of the medium with 50% FCS and RS resulted in a marked decrease of H-thymidine incorporation into islet DNA similar to that observed with 50% NCS. In contrast, exposure of islets to different batches of HS never resulted in an inhibition of DNA synthesis. Moreover, the 50% concentration of HS 465 caused a significant stimulation of thymidine incorporation.

The effect of human amniotic fluid in supporting long-term survival of pancreatic islets in tissue culture.

Pancreatic islets obtained from newborn Lewis rats were kept free-floating in TCM 199 either in the presence of 50% human amniotic fluid (HAF), 50% Hank's balanced salt solution (HBSS) or 10% fetal calf serum (FCS) for up to 14 days. The culture, in the presence of HAF, resulted in a good islet viability as demonstrated by islet number, islet insulin content and insulin release into the medium. Contradictory results were obtained when HBSS was used as the medium supplement. Moreover, the islets cultured in HAF-supplemented medium are characterized by a marked replicatory activity as reflected by the incorporation of labeled thymidine into islet DNA and gradual increase in DNA content with the progression of culture time. Even if compared to DNA synthesis of islets cultured under so called standard culture conditions as e.g. supplementation of 10% FCS to the medium, 50% HAF but not yet 10% HAF was found to stimulate the islet replication twofold already after 4 days of culture. It was also demonstrated that FCS has no additional effect on HAF-stimulated DNA synthesis. These findings support the view that HAF-enriched culture medium may have a use in supporting the long-term survival of well preserved endocrine pancreatic tissue.

Human amniotic fluid stimulates DNA synthesis of rat pancreatic islets.

Collagenase isolated pancreatic islets from newborn Lewis rats were used to study the effect of human amniotic fluid (HAF) on DNA synthesis measured as incorporation of 3H-thymidine into islet DNA and as estimation of labeling index. We investigated individual samples of HAF obtained from pregnant women between the 28th and 39th week of gestation. Since there were no significant differences of HAF obtained from different gestational weeks on islet DNA synthesis, the following experiments were carried out with pooled HAF. When islets were cultured for 2 days in TCM 199 containing 10 mmol/l glucose the addition of 50% HAF resulted in a significantly increased DNA synthesis in comparison to islets cultured in TCM 199 supplemented to 50% with Hank's balanced salt solution (HANK). When extending the culture period from 2 to 4 days the stimulatory effect of 50% HAF was well preserved. Islets cultured in HANK are characterized by a declined DNA-content, which was prevented in the presence of HAF. There was a good correlation between incorporation of 3H-thymidine and the percentage of labeled nuclei, estimated on hematoxylin/eosin-stained autoradiograms of islets cultured for 4 days either in TCM 199 alone, in the presence of 50% HANK or 50% HAF, indicating that the measured incorporation of labeled thymidine into islet DNA was representative for islet replication.