Adenoviral mediated hepatocyte growth factor gene attenuates hyperglycemia and beta cell destruction in overt diabetic mice

Hepatocyte growth factor (HGF) is a potent mitogen and promoter of proliferation of insulin producing beta cells of pancreatic islets. To study the role of HGF, an adenoviral vector carrying the human HGF (Ad.hHGF) gene was transfected into the streptozotocin-induced diabetic mice and evaluated the effect on the blood glucose metabolism and the insulin-secreting beta cells of pancreatic islets. Ad.hHGF gene transfection resulted in amelioration of hyperglycemia and prolongation of survival period in the diabetic mice. Concomitantly adenoviral- mediated hHGF gene therapy slightly increased serum insulin concentration and the expression of insulin in the pancreatic islet. Although the proliferation of beta-cell mass was not noticeable, the beneficial effect of HGF is significant to an almost deteriorated pancreatic islets. Taken together, these data suggest that the Ad.hHGF gene therapy into diabetic mice may prevent the further destruction and present as a beneficial remedy for type 1 diabetic patients.     

Angiotensin II receptor blocker attenuates overexpression of vascular endothelial growth factor in diabetic podocytes

VEGF expressed in glomerular podocytes, is known to increase vascular permeability to macromolecules. Angiotensin II can stimulate the release of VEGF, and the protective effects of angiotensin II antagonist against diabetic glomerular injury suggest that the angiotensin II-induced VEGF is an important pathogenetic mechanism in the development of proteinuria during diabetic nephropathy although this mechanism is not fully understood. In this study, the changes of VEGF expression was examined in the experimental diabetic nephropathy to determine whether these changes were modified by renoprotective intervention by blockers of angiotensin II receptors. The streptozotocin- induced diabetic rats were treated with L-158,809, a blocker of angiotensin II receptors, for 12 weeks. Age-matched rats with L-158,809 served as controls. RT-PCR and immunohistochemistry were used to assess and quantify gene and protein expression of VEGF. A progressive increase in urinary protein excretion was observed in diabetic rats. Glomerular VEGF expression was significantly higher in diabetic rats than in the control groups, with a significant reduction in glomerular VEGF expression and proteinuria in L-158,809- treated diabetic rats. VEGF mRNA was also significantly higher in diabetic kidneys than in the control groups, with a significant reduction in VEGF mRNA in L-158,809-treated diabetic kidneys. These results demonstrates that VEGF expression is significantly increased in diabetic podocytes, and angiotensin II receptor antagonist attenuated these changes in VEGF expression and prevented the development of proteinuria in vivo. Attenuation of increased VEGF expression in podocytes could contribute to the renoprotective effects of angiotensin II receptor antagonists in diabetic nephropathy.     

Gold nanoparticles as carriers for efficient transmucosal insulin delivery

Nanomaterials have gained tremendous importance in biology and medicine because they can be used as carriers for delivering small molecules such as drugs, proteins, and genes. We report herein the binding of the hormone insulin to gold nanoparticles and its application in transmucosal delivery for the therapeutic treatment of diabetes mellitus. Insulin was loaded onto bare gold nanoparticles and aspartic acid-capped gold nanoparticles and delivered in diabetic Wistar rats by both oral and intranasal (transmucosal) routes. Our principle observations are that there is a significant reduction of blood glucose levels (postprandial hyperglycemia) when insulin is delivered using gold nanoparticles as carriers by the transmucosal route in diabetic rats. Furthermore, control of postprandial hyperglycemia by the intranasal delivery protocol is comparable to that achieved using the standard subcutaneous administration used for type I diabetes mellitus, thus showing considerable promise for further development.     

Antidiabetic properties and mechanism of action of Gynura procumbens water extract in streptozotocin-induced diabetic rats

Gynura procumbens (Lour.) Merr (family Compositae) is cultivated in Southeast Asia, especially Indonesia, Malaysia and Thailand, for medicinal purposes. This study evaluated the in vivo hypoglycemic properties of the water extract of G. procumbens following 14 days of treatment and in vitro in RIN-5F cells. Glucose absorption from the intestines and its glucose uptake in abdominal skeletal muscle were assessed. The antidiabetic effect of water extract of G. procumbens leaves was investigated in streptozotocin-induced diabetic rats. The intraperitoneal glucose tolerance test (IPGTT) was performed in diabetic rats treated with G. procumbens water extract for 14 days. In the IPGTT, blood was collected for insulin and blood glucose measurement. After the IPGTT, the pancreases were collected for immunohistochemical study of β-cells of the islets of Langerhans. The possible antidiabetic mechanisms of G. procumbens were assessed through in vitro RIN-5F cell study, intestinal glucose absorption and glucose uptake by muscle. The results showed that G. procumbens significantly decreased blood glucose levels after 14 days of treatment and improved outcome of the IPGTT. However, G. procumbens did not show a significant effect on insulin level either in the in vivo test or the in vitro RIN-5F cell culture study. G. procumbens also showed minimal effects on β-cells of the islets of Langerhans in the pancreas. However, G. procumbens only significantly increased glucose uptake by muscle tissues. From the findings we can conclude that G. procumbens water extract exerted its hypoglycemic effect by promoting glucose uptake by muscles.     

A simple and rapid method for identifying and semi-quantifying peptide hormones in isolated pancreatic islets by direct-tissue matrix-assisted laser desorption ionization time-of-flight mass spectrometry

We describe a new, simple, robust and efficient method based on direct‐tissue matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry that enables consistent semi‐quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet‐endocrine cells: (α‐cells) glucagon, glicentin‐related polypeptide/GRPP; (β‐cells) insulin I, insulin II, C‐peptide I, C‐peptide II, amylin; (δ‐cells) somatostatin‐14; and (PP‐cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non‐β‐cell‐hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non‐β‐cells. It was also effective in characterizing hormonal phenotype in hemizygous human‐amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide‐hormone discovery by identifying a prominent new Gcg‐gene‐derived peptide (theoretical monoisotopic molecular weight 3263.5 Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide‐ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Improved formulation of W/O/W multiple emulsion for insulin encapsulation. Influence of the chemical structure of insulin

W/O/W multiple emulsions are systems of potential interest in the oral administration of insulin. Although it has been shown that a single oral administration of insulin-loaded W/O/W multiple emulsion to diabetic rats led to the significant decrease of blood glucose levels (Silva Cunha et al., 1998, Int J Pharm 169:33), repeated administrations displayed unpleasant side effects such as diarrhoea and steatosis. These unwanted effects were attributed to the high oil concentration used for their preparation. In the present study, attention was focused on the reduction of oil concentration in the formulation of these systems and on the encapsulation of two different insulins. The physical properties and stability of the multiple emulsions over long periods of time were assessed by conductivity measurements, and granulometric and microscopic analyses. The encapsulation in the inner aqueous phase of two insulins, Umulin and Humalog, differing only by the transposition of one amino acid, had non-negligible effects on the formation and stability of W/O/W multiple emulsions. Both insulins were shown to improve the formation of the multiple emulsions. Circular dichroism studies and surface tension measurements evidenced the contribution of insulin conformation and surface properties in multiple emulsion formation and stability.     

Analysis of free amino acids in islets of Langerhans by high-performance liquid chromatography using pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

A simple high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was developed and used for the analysis of free amino acids in islets of Langerhans; 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) served as pre-column derivatization reagent. Islets of Langerhans were separated from the pancreas of normal and obese rats, treated with pre-cooling methanol-water (80:20, v/v), and ultrasonicated to fragmentize the islets and effect deproteination. Several parameters influencing the derivatization reaction and chromatographic separation were optimized. Amino acid derivatives obtained under optimal conditions were separated on a C18 column with acetonitrile-acetate buffer as mobile phase and detected at 470 nm/540 nm (Ex/Em). Matrix effects were investigated and good linearities with correlation coefficients better than 0.9972 were obtained over a wide range of 0.42-42.11 microM for most of the amino acids. The detection limits (S/N = 3) were within the range of 6.1-51 nM. The precision of the method and recoveries were in the ranges of 1.43-10.76% (RSD%) and 85.07-108.82%, respectively. The analytical results showed that the serine content was markedly higher in normal rats than in obese rats, whereas methionine was of relatively lower content in both normal and obese rats.     

Momordica charantia Extract Protects against Diabetes-Related Spermatogenic Dysfunction in Male Rats: Molecular and Biochemical Study

More than 90% of diabetic patients suffer from sexual dysfunction, including diminished sperm count, sperm motility, and sperm viability, and low testosterone levels. The effects of Momordica charantia (MC) were studied by estimating the blood levels of insulin, glucose, glycosylated hemoglobin (HbA1c), testosterone (TST), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in diabetic rats treated with 250 and 500 mg/kg b.w. of the total extract. Testicular antioxidants, epididymal sperm characteristics, testicular histopathology, and lesion scoring were also investigated. Testicular mRNA expression of apoptosis-related markers such as antiapoptotic B-cell lymphoma-2 (Bcl-2) and proapoptotic Bcl-2-associated X protein (Bax) were evaluated by real-time PCR. Furthermore, caspase-3 protein expression was evaluated by immunohistochemistry. MC administration resulted in a significant reduction in blood glucose and HbA1c and marked elevation of serum levels of insulin, TST, and gonadotropins in diabetic rats. It induced a significant recovery of testicular antioxidant enzymes, improved histopathological changes of the testes, and decreased spermatogenic and Sertoli cell apoptosis. MC effectively inhibited testicular apoptosis, as evidenced by upregulation of Bcl-2 and downregulation of Bax and caspase-3. Moreover, reduction in apoptotic potential in MC-treated groups was confirmed by reduction in the Bax/Bcl-2 mRNA expression ratio.     

Modulation of pancreatic β-cells in neonatally streptozotocin-induced type 2 diabetic rats by the ethanolic extract of Momordica charantia fruit pulp

Effective doses of the Momordica charantia fruit pulp (MCF) ethanolic extract on pancreatic β-cells modulation in neonatally streptozotocin-induced type 2 diabetic rats were studied. Diabetic rats (n=8) were treated with MCF extract (400 mg kg(-1) day(-1)) or glibenclamide (5 mg kg(-1)) for 28 days. Control rats (n=11) and untreated diabetic rats (n=8) received only water. Fasting glucose, serum insulin (by ELISA) and β-cell function (HOMA %B by homeostasis model assessment) were measured. β- and α-cells were identified by immunostaining, nuclei by DAPI, and β-cell size and number by morphometry. Significant improvement of fasting blood glucose, serum insulin and β-cell function was observed with the MCF extract for the diabetic rat model. The islet size, total β-cell area and number of β-cells were increased to almost double in the diabetic rats treated with MCF extract as compared to the untreated diabetic rats. The number of α-cells did not change significantly. Insulin granules in β-cells were notably reduced in diabetic islets as compared to control islets. However, extract-treated diabetic rat β-cells were abundant with insulin granules, which was comparable to non-diabetic control islets. The modulation of pancreatic β-cells may be involved in the experimental observation of anti-diabetic effects of M. charantia extract.     

Two-color electrophoretic immunoassay for simultaneous measurement of insulin and glucagon content in islets of Langerhans

A multianalyte CE competitive immunoassay using two-color detection was developed to measure insulin and glucagon in islets of Langerhans. Insulin was quantified with FITC-insulin (Ins*) and anti-insulin antibodies (Ins Ab) and glucagon was quantified with Cy5-glucagon (Glu*) and anti-glucagon antibodies (Glu Ab). A 3 mW Ar(+) laser at 488 nm and a 25 mW laser diode at 635 nm were used to excite FITC and Cy5, respectively. Fluorescence was split with a half-silvered mirror and passed through a 520 +/- 20 nm bandpass filter or a 663 nm longpass filter for the detection of insulin and glucagon, respectively. The two-color detection format enabled independent quantitation of both analytes even with concentrations of insulin immunoassay reagents 20-fold higher than glucagon reagents. Simultaneous calibration curves were generated and used to determine insulin and glucagon content in islets of Langerhans. Amounts of insulin and glucagon were 56.6 +/- 3.2 and 1.0 +/- 0.5 ng/islet, respectively. LODs were 7 nM insulin and 3 nM glucagon. The assay will be applicable to fast monitoring of multiple peptides secreted from islets of Langerhans and can be applied to other systems for the quantitation of multiple analytes with large differences in concentrations.     

Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans

A capillary electrophoresis competitive immunoassay was developed for the simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for the resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488 nm line of an Ar(+) laser and detected at 520 nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635 nm laser diode module and detected at 700 nm with a separate PMT. Optimum resolution was achieved with a 20mM carbonate separation buffer at pH 9.0 using a 20 cm effective separation length with an electric field of 500 V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3 nM, respectively. This method was used to monitor the simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20 mM glucose for 6h. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11 mM glucose conditions. To further demonstrate the utility of the assay, the Ca(2+)-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for the determination of regulation of secretory processes within islets of Langerhans.     

胰岛素的色谱分析方法

随着糖尿病的发病率不断攀升,有必要建立简单易行的胰岛素的分析方法。胰岛素制剂的纯度检验,胰岛素生产及纯化过程的监控,生物体液及胰岛细胞分泌胰岛素的状况,重组胰岛素结构的测定及活性研究等都离不开胰岛素的测定。而胰岛素的色谱分析是一个年轻的领域。本文按照体内外样品、检测方法和样品制备总结了近年胰岛素的色谱分析方法。     

Effects of Micronutrients on Oxidative Stress and Islet Function in Type 1 Diabetes Mellitus Rats： Relationships to Th2 Type Cytokines, Interleukin-4, and Interluekin-10

To observe the effects of the micronutrients on oxidative and autoimmune destruction of islets so as to prevent a person from the onset and development of type 1 Diabetes Mellitus(T1DM),the interleukin-4 and interleukin-10 expressions of lymphocytes in peripheral blood and spleen of T1DM rats were determined by flow cytometry.GSH-Px activity and MDA level in the rats' pancreas were measured using biochemical methods.The insulin contents in serum and β cell insulin secret storage were tested by RIA and IHC,respectively.There was an increase in the percentages of IL-4 and IL-10 positive lymphocytes in the peripheral blood and spleen of the groups of rats supplemented with various combinations of micronutrients(p<0.01 and p<0.05,respectively);the blood glucose concentration decreased(p<0.05);both the functional β cell in islets and the insulin content in pancreatic tissue increased(p<0.05 and p<0.01);the GSH-Px activity and MDA level of pancreas in the rats enhanced and decreased respectively(p<0.01 and p<0.05).The results suggest that micronutrients may alleviate the islet lesions by upregulating the expressions of IL-4 and IL-10 and lowering oxidative stress in diabetic rats.     

Bioartificial Organs I: Silica Gel Encapsulated Pancreatic Islets for the Treatment of Diabetes Mellitus

The primary objective of this work is to evaluate the potential of silica gel encapsulated pancreatic islets of Langerhans, or islet tissue, as a means by which insulin secretory capacity might be restored to individuals with insulin dependent, type 1, diabetes mellitus. The encapsulation material under investigation is comprised of sol-gel derived silica ceramic that hardens under conditions of pH, salinity, and temperature that are not harmful to living cells and organisms. Preliminary efficacy has been demonstrated by measurement of insulin secretory response of silica gel encapsulated pancreatic isletsin vitro and blood sugar levels of nonobese diabetic micein vivo.     

β-Sitosterol Circumvents Obesity Induced Inflammation and Insulin Resistance by down-Regulating IKKβ/NF-κB and JNK Signaling Pathway in Adipocytes of Type 2 Diabetic Rats

β-sitosterol (SIT), the most abundant bioactive component of vegetable oil and other plants, is a highly potent antidiabetic drug. Our previous studies show that SIT controls hyperglycemia and insulin resistance by activating insulin receptor and glucose transporter 4 (GLUT-4) in the adipocytes of obesity induced type 2 diabetic rats. The current research was undertaken to investigate if SIT could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of SIT (20 mg/kg b.wt) was administered orally for 30 days to high fat diet and sucrose induced type-2 diabetic rats. Metformin, the conventionally used antidiabetic drug was used as a positive control. Interestingly, SIT treatment restores the elevated serum levels of proinflammatory cytokines including leptin, resistin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to normalcy and increases anti-inflammatory adipocytokines including adiponectin in type 2 diabetic rats. Furthermore, SIT decreases sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator–activated receptor-γ (PPAR-γ) gene expression in adipocytes of diabetic rats. The gene and protein expression of c-Jun-N-terminal kinase-1 (JNK1), inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) and nuclear factor kappa B (NF-κB) were also significantly attenuated in SIT treated groups. More importantly, SIT acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that SIT inhibits obesity induced insulin resistance by ameliorating the inflammatory events in the adipose tissue through the downregulation of IKKβ/NF-κB and c-Jun-N-terminal kinase (JNK) signaling pathway.     

Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8?μU/ml. Recoveries ranged between 33.2?% and 51.7?%. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301?μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog?, Novolog?, Apidra?, Lantus?, and Levemir? in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.     

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microg/microgL cDNA and 25 microgL effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.     

In vitro and in vivo testing of glucose-responsive insulin-delivery microdevices in diabetic rats

We have developed glucose-responsive implantable microdevices for closed-loop delivery of insulin and conducted in vivo testing of these devices in diabetic rats. The microdevices consist of an albumin-based bioinorganic membrane that utilizes glucose oxidase (GOx), catalase (CAT) and manganese dioxide (MnO(2)) nanoparticles to convert a change in the environmental glucose level to a pH stimulus, which regulates the volume of pH-sensitive hydrogel nanoparticles and thereby the permeability of the membrane. The membrane is integrated with microfabricated PDMS (polydimethylsiloxane) structures to form compact, stand-alone microdevices, which do not require tethering wires or tubes. During in vitro testing, the microdevices showed glucose-responsive insulin release over multiple cycles at clinically relevant glucose concentrations. In vivo, the microdevices were able to counter hyperglycemia in diabetic rats over a one-week period. The in vitro and in vivo testing results demonstrated the efficacy of closed-loop biosensing and rapid response of the 'smart' insulin delivery devices.     

Non-ideal behaviour of silica-based stationary phases in trifluoroacetic acid-acetonitrile-based reversed-phase high-performance liquid chromatographic separations of insulins and proinsulins.

Several C18 stationary phases were found to behave non-ideally when insulins and proinsulins were eluted with shallow acetonitrile gradients in 0.1% trifluoroacetic acid, resulting in poor peak shapes or no elution at all. With triethylammonium phosphate or ammonium sulphate as buffer components, the insulins and proinsulins were eluted with excellent peak shapes, presumably owing to better masking of residual silanol groups on the stationary phases. Similar use of trifluoroacetic acid-acetonitrile gradients on the less hydrophobic C4 or C3 stationary phases resulted in excellent peak shapes. The difficult separation of rat proinsulin I and II, which are important for the study of rat insulin biosynthesis, was only achieved with two different stationary-mobile phase combinations.     

The stimulatory effect of IL-1beta on the insulin secretion of rat pancreatic islet is not related with iNOS pathway

Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to destroy pancreatic beta-cells, and thought to be involved in the pathogenesis of type I diabetes mellitus. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1beta may impair an islet function in rodents. Inhibition of iNOS may protect against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. To examine the role of NO in the IL-1beta treated cells, rat islets were treated with various concentrations (0, 0.5, 5, 50, 500 pmol/L) of IL-1beta with or without NG-monomethyl-L-arginine (NMMA; a competitive inhibitor of nitiric oxide synthase) for 2 or 6 h. Insulin secretion was stimulated in islets treated with 5, 50, and 500 pmol/ L of IL-1beta for 2 h and 0.5 pmol/L for 6 h, respectively. The stimulatory effect of IL-1beta on the insulin secretion of rat islets was not prevented by NMMA. Nitrate concentration was increased in a time- and concentration-dependent manner. Nitrate production was inhibited by NMMA. iNOS mRNA expression was increased at concentrations more than 5 pmol/L of IL-1beta in a dose dependent manner. iNOS mRNA was detectable after 2 h and peaked at 6 h but decreased after 24 h. These results suggested that the stimulatory effect of IL-1beta on the insulin secretion of rat islets is independent of iNOS-related NO production of IL-1beta and the enzyme activity of nitric oxide synthase.