High normal albuminuria is independently associated with aortic stiffness in patients with Type 2 diabetes.

BACKGROUND: High normal albuminuria is associated with higher cardiovascular risk in patients with diabetes. Increased aortic stiffness is an established risk factor of vascular events. However, the relationship between albuminuria within the normal range (0-30 mg/g) and aortic stiffness in patients with Type 2 diabetes is unknown. METHODS: A total of 614 normoalbuminuric subjects with Type 2 diabetes with spot urinary albumin:creatinine ratio ≤ 30 mg/g and estimated glomerular filtration rate ≥ 60 ml min⁻¹ 1.73 m⁻² were included in the study. Aortic stiffness was assessed by carotid-femoral pulse wave velocity. RESULTS: Pulse wave velocity increased progressively with the increase of albumin:creatinine ratio within the normoalbuminuric range (0-30 mg/g). Only 2.6% of the subjects with an albumin:creatinine ratio in the lowest quartile (0.7-3.4 mg/g) were classified as having aortic stiffness (pulse wave velocity ≥12 m/s). In contrast, the proportion of subjects with aortic stiffness increased significantly with the increase of albumin:creatinine ratio level (11.0%, 10.4% and 13.6% in albumin:creatinine ratio quartiles 2, 3 and 4, respectively, P = 0.008). A logistic regression model revealed that the odds of having aortic stiffness were increased by 56% with a 1-SD increase of log albumin:creatinine ratio after adjustment for age, gender, duration of diabetes, HbA1c , blood pressure, HDL and LDL cholesterol, estimated glomerular filtration rate, BMI, usage of renin-angiotensin system antagonists, statins and insulin. CONCLUSIONS: High normal albuminuria is associated with aortic stiffness in patients with Type 2 diabetes, which may in part explain their increased cardiovascular risk.

Associations between pigment epithelium-derived factor, insulin resistance and high density lipoprotein.

AIMS: To measure serum pigment epithelium-derived factor in control subjects with normal fasting glucose, and in subjects with impaired fasting glucose and those with newly diagnosed Type 2 diabetes, before treatment initiation, and to measure pigment epithelium-derived factor prospectively in patients being treated with HDL-raising therapy, niacin. METHODS: We enrolled 89 individuals attending an institutional health screen. Biochemical indices including lipids, homeostasis model assessment-insulin resistance, high-sensitivity C-reactive protein and pigment epithelium-derived factor were analysed in fasting blood. To validate the association between HDL and pigment epithelium-derived factor, we analysed samples from a separate study cohort with low HDL, followed up for 12-weeks while on niacin treatment. Secreted pigment epithelium-derived factor from 3T3-L1 adipocytes, after HDL treatment (24-h), was measured using Western blot analysis. RESULTS: Mean (± sd) serum pigment epithelium-derived factor was significantly higher in subjects with impaired fasting glucose [13.99 (± 3.06) µg/ml] and Type 2 diabetes [12.94 (± 2.61)] µg/ml, compared with control subjects [11.83 (± 2.85) µg/ml (P = 0.014)]. In multivariate analyses, serum pigment epithelium-derived factor concentration was associated with BMI (ß = 0.32, 0.007), homeostasis model assessment-insulin resistance (ß = 0.33, P = 0.01) and HDL (ß = -0.24, P = 0.05), after adjustment for age, gender and high-sensitivity C-reactive protein. In the niacin study, on-treatment HDL was an independent determinant of pigment epithelium-derived factor (ß = -0.439, P = 0.033), after adjusting for age, homeostasis model assessment-insulin resistance and treatment. Adipocytes treated with HDL were found to have reduced pigment epithelium-derived factor secretion [24.8% (50 µg/ml), 28.4% (100 µg/ml) HDL; P < 0.05)], compared with the control samples. CONCLUSION: Serum pigment epithelium-derived factor is positively associated with homeostasis model assessment-insulin resistance and negatively associated with HDL. Further studies are needed to understand the mechanism of low HDL and raised pigment epithelium-derived factor and to determine if they are causally related to the pathobiology of insulin resistance.

Defining the chromatin signature of inducible genes in T cells.

BACKGROUND: Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation. RESULTS: To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation. CONCLUSIONS: These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation.

Folic acid prevents and partially reverses glucocorticoid-induced hypertension in the rat.

BACKGROUND: To investigate the effect of folic acid on the increased pressure in rats treated with either adrenocorticotropic hormone (ACTH) or dexamethasone (Dex), and to further investigate the role of tetrahydrobiopterin (BH(4)) in any effect of folic acid by comparing the effect of BH(4) with that of folic acid in Dex hypertension. METHODS: Male Sprague-Dawley (SD) rats were treated with saline, subcutaneous ACTH (0.2 mg/kg/d) or Dex (10 microg/rat/d). Folic acid (0.04 g/L drinking) or BH(4) (10 mg/kg/d intraperitoneally) was started before (prevention) and during (reversal) glucocorticoid treatment. RESULTS: Saline, BH(4), vehicle for BH(4), or folic acid alone did not change systolic blood pressure (BP). Systolic BP was increased by ACTH and Dex. Folic acid, but not BH(4), prevented the development of hypertension caused by ACTH and Dex treatment. The ACTH and Dex hypertension were partially reversed by folic acid. The BH(4) increased plasma total biopterin concentrations. The Dex decreased plasma NOx concentrations but had no effect on plasma biopterin concentrations. The ACTH and Dex increased plasma F(2)-isoprostane concentrations and decreased serum homocysteine concentrations compared with control but had no effect on serum folate concentrations. Folic acid increased serum folate concentrations compared with control but had no effect on homocysteine concentrations. CONCLUSIONS: Folic acid prevented and partially reversed both ACTH and Dex hypertension in rats without modifying the increase in plasma F(2)-isoprostane concentrations. Given that BH(4) failed to prevent ACTH or Dex hypertension, folic acid is unlikely to be acting through increased BH(4) production. The precise mechanism for the BP-lowering effect of folic acid in this model of hypertension remains to be determined.

Apocynin but not L-arginine prevents and reverses dexamethasone-induced hypertension in the rat.

BACKGROUND: Dexamethasone (Dex)-hypertension in rats is associated with increased oxidative stress. We investigated effects of the NAD(P)H oxidase inhibitor apocynin and the nitric oxide (NO) precursor L-arginine on Dex-hypertension to determine the relative roles of NAD(P)H oxidase and uncoupling in the reactive oxygen species (ROS) generation and hypertension. METHODS: Male Sprague-Dawley rats (n = 10/group) received Dex (20 microg/kg/day subcutaneously) or saline (vehicle) for 14 days. In a prevention study, rats received 4 days of apocynin treatement (1.5 mmol/L in drinking water) followed by Dex/saline for 12 days. In reversal studies, apocynin or L-arginine was given from day 8 to 14. Systolic blood pressure (SBP) was measured by tail cuff, and thymus weight was used as a marker of glucocorticoid activity. RESULTS: Administration of Dex increased SBP (104 +/- 3 to 122 +/- 3 mm Hg, P < .01, mean +/- SEM) and decreased thymus and body weight (P' < .05). Apocynin alone had no effect on SBP, BW, or thymus weight. Apocynin prevented (122 +/- 4 Dex, 111 +/- 3 mm Hg Apocynin+Dex, P' < .05) and reversed Dex-hypertension (130 +/- 4 to 116 +/- 4 mm Hg, P < .01). L-arginine did not reverse Dex-hypertension. CONCLUSIONS: In male SD rats, apocynin but not l-arginine prevented and reversed Dex-hypertension, suggesting that NAD(P)H oxidase-mediated superoxide production but not endothelial nitric oxide synthase uncoupling is important in Dex-hypertension.

Role of eicosanoids in biosynthesis and secretion of insulin.

PURPOSE: The role of icosanoids, which are products formed from the metabolism of arachidonic acid, in pancreatic islet-cell function was investigated. PROCEDURES: Secretion and biosynthesis of insulin and glucagon, biosynthesis of icosanoids, and biosynthesis of enzymes and proteins necessary for icosanoid synthesis were studied in vitro, using perfused rat pancreas, isolated and incubated islets, and insulin-secreting islet-cell lines. FINDINGS: Certain exogenous prostaglandins stimulated the secretion of insulin and glucagon; leukotrienes stimulated insulin but not glucagon release. Leukotrienes inhibited glucose-induced insulin release, but promoted insulin biosynthesis. Islet cells produced prostaglandins. Although the production of leukotrienes in islet cells could not be demonstrated conclusively, glucose-responsive biosynthesis of 5-lipoxygenase and 5-lipoxygenase-activating protein was considered evidence for leukotriene synthesis. Inhibitors of prostaglandin or leukotriene biosynthesis attenuated hormone secretion. CONCLUSION: Icosanoids produced in islet cells are involved in signal-transduction, in the form of a fine-tuning amplification of biosynthesis or secretion of insulin and glucagon in response to nutrient stimuli.

Diacylglycerol inhibits potassium-induced calcium influx and insulin release by a protein kinase-C-independent mechanism in HIT T-15 islet cells.

We reported previously that in pancreatic islet cells, certain diacylglycerols (DGs) evoke increases in cytosolic calcium ([Ca2+]i), mainly by intracellular mobilization. We now examined the effects of DGs on the increase in [Ca2+]i due to Ca2+ influx. In the insulin-secreting HIT T-15 islet cell line, cell membrane depolarization using 40 mM KCl evoked a 2- to 3-fold increase in [Ca2+]i, which lasted several minutes. A cell-permeable DG, 1,2-dioctanoylglycerol (DiC8; 10 microM) induced a 12 +/- 4% rise in [Ca2+]i, which did not occur in the absence of extracellular Ca2+ or in the presence of verapamil; this effect was not protein kinase-C (PKC) dependent, because it was not altered by the addition of the PKC inhibitor staurosporine or by using PKC-depleted cells. When DiC8 was added first, the KCl-induced increase in [Ca2+]i was inhibited in a dose-dependent manner (100% at 10-15 microM DiC8); this effect was PKC independent. At a concentration of 10 microM, other synthetic DGs, 1,2-dihexanoylglycerol (DiC6), 1,2-didecanoylglycerol (DiC10), or 1-oleoyl-2-acetylglycerol, inhibited the KCl-induced rise in [Ca2+]i to 15 +/- 4%, 47 +/- 7%, and 51 +/- 5% of the control value, respectively. R59022 (10 microM), which inhibits DG kinase and causes accumulation of endogenous DGs, inhibited the KCl-induced rise in [Ca2+]i to 2 +/- 0.2% of the control value; this inhibition was not affected by staurosporine. In anchored cells, KCl stimulated insulin release (959 +/- 88 microU/mg protein above the control value); 20 microM DiC6 or DiC8 attenuated KCl-induced insulin release by 68% and 31% of the control value, respectively; DiC10 or 1-oleoyl-2-acetylglycerol had no effect. R59022 inhibited KCl-induced insulin release by 90% of the control value. We conclude that in HIT T-15 cells, DGs may serve as positive and negative modulators of [Ca2+]i, apparently by complex and PKC-independent mechanisms. These divergent actions of DGs on islet cell Ca2+ balance together with the accompanying activation of PKC affect insulin release in a complex manner.

Prevention of insulitis and diabetes onset by treatment with complete Freund's adjuvant in NOD mice.

In studies of immune cell defects in autoimmune diabetes mellitus, we observed that complete Freund's adjuvant (CFA) prevented the onset of diabetes when injected into 8- to 10-wk-old prediabetic nonobese diabetic (NOD) mice. The prevalence of the onset of diabetes in the CFA-injected versus uninjected NOD mice was 2 of 81 (2.5%) vs. 231 of 379 (61%) among females and 2 of 44 (4.5%) vs. 83 of 336 (25%) among males, respectively. The incidence of histologically identifiable insulitis was significantly reduced in CFA-treated prediabetic female NOD mice (18%) compared with the incidence in female age-matched controls (70%). Splenocytes or Mac-(1+)-enriched splenocytes from CFA-treated NOD mice, when cotransferred with splenocytes from diabetic mice, reduced the incidence of diabetes provoked by diabetic splenocytes in vivo. In the spleen, CFA injection induced sustained increases in cell proliferation and an associated major increase in the numbers of an immature cell type that expressed the Mac-1 surface antigen. In CFA-treated NOD mice, lymphocytes derived from the spleen failed to respond in vitro to stimulation by the mitogen concanavalin A or by anti-CD3. When cocultured, Mac-1+ cells, enriched from the splenocytes of CFA-treated mice, suppressed concanavalin A- or anti-CD3-induced proliferation of T lymphocytes derived from either the spleen or thymus of untreated NOD mice. Therefore, treatment with CFA prevents the development of diabetes, and concomitantly, insulitis while stimulating the generation of splenic suppressor cells that are capable of suppressing diabetogenic T-lymphocyte function in vivo and in vitro.

Dioctanoylglycerol regulation of cytosolic Ca2+ by protein kinase C-independent mechanism in HIT T-15 islet cells.

The effect of activators of protein kinase C (PKC) on cytosolic concentration of free Ca2+ [( Ca2+]i) was assessed in insulin-secreting islet cell line HIT T-15. Dioctanoylglycerol (DiC8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) evoked activation of PKC. Basal [Ca2+]i was 65-160 nM. DiC8 induced triphasic increases in [Ca2+]i; phase 2 was the most prominent and consistent one. With 25-150 microM DiC8, [Ca2+]i increased in a dose-dependent manner during phase 2; half-maximal stimulatory dose was 53 microM. TPA did not evoke any increase in [Ca2+]i. Staurosporine, sphingosine, and H7, which are inhibitors of PKC, did not block DiC8-induced rise in [Ca2+]i. DiC8-induced rise in [Ca2+]i was also seen in cells that had been depleted of PKC by prior exposure to TPA. DiC8-induced rise in [Ca2+]i still occurred in the presence of the Ca(2+)-channel blocker verapamil or when the extracellular Ca2+ had been reduced from 2.5 mM to 30 nM by EGTA. Three immediate metabolites of DiC8, monooctanoylglycerol, octanoate, and glycerol, did not evoke any change in [Ca2+]i. Monooleoylglycerol and R59022, which induce increases in endogenous diacylglycerol (DAG) by inhibiting DAG kinase, evoked increases in [Ca2+]i. DiC8 did not cause any change in inositol 1,4,5-trisphosphate levels. DiC8 evoked biphasic increases in insulin release; the second-phase increase in [Ca2+]i preceded the late phase of insulin secretion. Exogenous DAGs should be used with caution in assessing PKC function. Changes in the generation in DAGs must be included among the mechanisms by which Ca2+ homeostasis is regulated in islet cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate.

We have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, we considered the possibility that 3-phosphoglycerate may act as a coupling factor, and we searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [gamma-32P]ATP and appropriate test substances and were subjected to NaDodSO4/PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of a 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion.

Lipoxygenase-generated icosanoids inhibit glucose-induced insulin release from rat islets.

Lipoxygenase-pathway metabolites of arachidonic acid are produced in pancreatic islets. They are are implicated in insulin release, since nonselective inhibitors of lipoxygenases inhibit glucose-induced insulin release. We studied the interplay in insulin release between glucose and selected icosanoids formed in 5-, 12- and 15-lipoxygenase pathways. Effects on immunoreactive insulin release of 10(7) to 10(6)-12-(R)-HETE, 12-(S)-HETE, hepoxilin A3, lipoxin B4, LTB4 or LTC4 were tested individually in 30-min incubations of freshly isolated young adult Wistar rat pancreatic islets, in the presence of 5.6 mM or 23 mM glucose. Basal insulin release (at 5.6 mM glucose) was stimulated by LTC4 and hepoxilin A3 (304% and 234% of controls at 5.6 mM glucose alone, respectively), inhibited by 12-(S)-HPETE (56%), and was not affected by 12-(R)-HETE, 12-(S)-HETE, lipoxin B4 or LTB4 (111%, 105%, 106% and 136%, respectively). Insulin release evoked by 23 mM glucose (190-320%) was inhibited (50-145%) by all icosanoids tested, except LTC4 (162%). We conclude that, among the lipoxygenase products tested, only leukotrienes and hepoxilin are candidates for a tonic-stimulatory influence on basal insulin release. Since glucose promotes icosanoid formation in islets, the observed inhibition of glucose-induced insulin release by lipoxygenase products suggests the existence of a negative-feedback system.

Insulin release in RINm5F cells and glyceraldehyde activation of protein kinase C.

In the insulin-secreting, glucose-insensitive islet cell subclone RINm5F, the distribution of protein kinase C (PKC) activity in the cytosol and membrane fractions was determined, and the activation of the enzyme, as reflected in its translocation to the membrane fraction, was characterized in conjunction with insulin release. DL-Glyceraldehyde (15 mM) evoked a rapid redistribution of PKC from the cytosol to the membrane fraction; insulin release increased concomitantly. When monitored over 5 min with 15 mM glyceraldehyde, membrane stabilization of PKC reached a maximum at 30 s and decreased thereafter; insulin release occurred at a high rate for the first 15 s and diminished thereafter. With 2-20 mM glyceraldehyde, a dose-dependent increase in membrane stabilization of PKC occurred and was accompanied by a matching increase in insulin release. Exogenous 1,2-dioctanoyl-sn-glycerol (100 microM) induced a rapid membrane stabilization of PKC and concomitant stimulation of insulin release. Glucose (15 mM) failed to evoke any redistribution of PKC or release of insulin. Depletion of total PKC activity by 95% induced by 18-h incubations with 2 microM 12-O-tetradecanoylphorbol-13-acetate resulted in a 67-91% reduction in glyceraldehyde-induced insulin release. We conclude that in the RINm5F islet beta-cell subclone 1) the rapid activation of PKC, which occurs in response to the administration of glyceraldehyde, a nutrient secretagogue, plays an amplifying role in the initiation of stimulated insulin release; and 2) the failure of the activation of PKC may be responsible for the insensitivity to glucose.

Fluctuations in basal plasma levels of pancreatic polypeptide in monkeys and humans.

We reported that significant rapid oscillations occur in basal plasma levels of insulin, glucagon, and glucose in rhesus monkeys and humans. We searched for evidence for similar spontaneous fluctuations also in plasma levels of pancreatic polypeptide (PP). Mean +/- SE basal plasma levels of PP were 236 +/- 15 pg/ml in 11 monkeys, 64 +/- 12 in nine normal-weight human subjects, and 74 +/- 10 in nine obese human subjects. 1) PP levels fluctuated with periods of 6-26 min. The fluctuations in PP were less regular than and did not temporally correlate with the fluctuations in plasma levels of insulin, glucagon, or glucose (usual periods 8-12 min). 2) In human subjects the concentration but not the periodicity of PP was related to obesity. 3) Comparisons of simultaneously determined levels of PP in portal and central venous plasma of monkeys suggested that PP may be extracted to varying degrees by the liver, even under basal well-controlled conditions, and that neither the size of the PP portal-central gradient nor the period and amplitude of fluctuations was associated with PP concentration. We conclude that plasma PP levels fluctuate with such a large amplitude that these fluctuations must be considered in the interpretation of experimental results based on limited numbers of samples.

Possible role of endogenous arachidonic acid metabolites in stimulated release of insulin and glucagon from the isolated, perfused rat pancreas.

Enhancement of arachidonic acid metabolism results in increased insulin secretion. To determine which pathways of arachidonic acid metabolism were involved in this stimulation, we studied the effects of various inhibitors of arachidonate metabolism on arginine-induced insulin and glucagon secretion in the isolated, perfused rat pancreas. The release of PGE2 from the pancreas was monitored to document the efficacy of the inhibitory drugs. p-Bromophenacyl bromide, a phospholipase A2 inhibitor, diminished PGE2 release and significantly inhibited both the early and late phases of insulin and glucagon release in response to arginine. Flurbiprofen, a specific cyclooxygenase inhibitor, decreased the early phase of insulin release and inhibited both phases of arginine-stimulated glucagon secretion; these decreases were concurrent with a large inhibition of PGE2 release. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, at a dose of 10(-5) M did not affect PGE2 release, inhibited the early phase of insulin release, and did not modify glucagon secretion. The combination of flurbiprofen and nordihydroguaiaretic acid, although the most potent in inhibiting PGE2, lowered only the early phase of insulin and had no effect on glucagon secretion. We conclude that: (1) endogenous cyclooxygenase-derived metabolites of arachidonic acid promote insulin and glucagon release, (2) endogenous lipoxygenase products preferentially stimulate insulin release, and (3) phospholipase A2 activity has an intrinsic modulatory effect on insulin and glucagon secretion.

Role of arachidonate lipoxygenase and cyclooxygenase products in insulin and glucagon secretion from rat pancreatic islets.

Rat pancreatic islets incubated in nutrient medium were used to study the role of endogenous arachidonic acid metabolism in pancreatic hormone secretion. Both glucose and fetal calf serum stimulated radioimmunoassayable PGE2 production and insulin secretion from islets. These effects were abolished by the phospholipase inhibitor p-bromophenacyl bromide or by concurrent inhibition of cyclooxygenase and lipoxygenase by flurbiprofen plus nordihydroguaiaretic acid (NDGA), respectively. Bromophenacyl bromide also inhibited glucagon secretion. When used alone, flurbiprofen caused a significant enhancement of glucose-induced insulin secretion that was attributed to reactive stimulation of lipoxygenase-product formation rather than to selective cyclooxygenase inhibition. NDGA given alone in the presence of stimulatory concentrations of glucose suppressed the normal eight-fold rise in insulin secretion, but caused a marked enhancement in glucagon secretion that could be overcome by simultaneous inclusion of flurbiprofen. We concluded that: (1) Increased metabolism of arachidonic acid in pancreatic islets amplifies the secretion of insulin and glucagon. (2) The lipoxygenase as well as the cyclooxygenase pathways of arachidonate metabolism participate in the amplification of insulin secretion. (3) The observations made in this study are inconclusive with respect to the involvement of the lipoxygenase and cyclooxygenase pathways in glucagon secretion; an inhibitory role for lipoxygenase pathway products is suggested.

Arachidonic acid induced release of insulin and glucagon: role of endogenous prostaglandins in pancreatic hormone secretion.

The involvement of prostaglandins in the effects of arachidonic acid (20:4n-6) on insulin and glucagon release was investigated, using the isolated, perfused rat pancreas model. 20:4n-6, the substrate for dienoic prostaglandins, or 20:3n-3, a fatty acid that cannot be metabolized to prostaglandins were perfused over 55 min. 20: 4n-6 evoked triphasic insulin release: early and late phase during, and "off-response" following the perfusion. With 20:3n-3 the early phase of insulin release was 57% of that with 20:4n-6. 20:4n-6 stimulated only an early phase release of glucagon; 20:3n-3 had no effect. Indomethacin (10 microM, a cyclooxygenase inhibitor) inhibited by 50% the early phase of insulin and glucagon release induced by 20:4n-6, but did not modify insulin release during the early phase with 20:3n-3, or the late phase or off-response with either 20:4n-6 or 20:3n-3. We conclude that 1) the early phase release of insulin and glucagon which occurs with arachidonic acid is due in part to pancreatic biosynthesis of prostaglandins; and 2) in the other phases of insulin release evoked by the fatty acids, alternate "nonspecific" mechanisms may be involved.

Effects of lipoxygenase and cyclooxygenase inhibitors on glucose-stimulated insulin secretion from the isolated perfused rat pancreas.

Some of the metabolites of arachidonic acid formed in the lipoxygenase and cyclooxygenase pathways stimulate insulin release. We studied the relative importance of each of these pathways in the modulation of glucose-induced insulin release by using inhibitors of arachidonate metabolism. Perfusion of the isolated rat pancreas with two chemically different inhibitors of cyclooxygenase, flurbiprofen and sodium salicylate, markedly inhibited prostaglandin E2 release, but had little effect on glucose-induced insulin release or on potentiation of insulin release caused by prior exposure to glucose. On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, not only inhibited both phases of glucose-induced insulin release but also abolished the potentiation effect. These effects of NDGA prevailed, when it was administered together with flurbiprofen, which caused profound inhibition of prostaglandin E2 release. We conclude that 1) lipoxygenase pathways play a dominant role in glucose-stimulated insulin release, and 2) endogenous lipoxygenase metabolites influence the potentiating effect of glucose on the release of insulin in response to a subsequent stimulation.

The effect of flurbiprofen, a potent inhibitor of prostaglandin synthesis, on insulin and glucagon release from isolated rat pancreas.

The influence of different levels of inhibition of prostaglandin (PG) synthesis on the release of insulin and glucagon was investigated in the basal state (5.6 mM glucose) and in response to 30-min perfusion of 16.7 mM glucose using the isolated perfused rat pancreas model. Flurbiprofen (FLR), a potent and selective inhibitor of PG synthesis, was present in the perfusate during the entire experimental period at a concentration of 10(-8), 5 X 10(-8), or 10(-6) M; control experiments were performed without the drug. Levels of immunoreactive PGE2, PGF2 alpha, insulin, and glucagon were measured in the portal venous effluent. FLR inhibited PG synthesis in a dose-related manner; PGE2 was inhibited more than PGF2 alpha. Basal and glucose-induced secretion of insulin was augmented by FLR at 5 X 10(-8) M, but was inhibited at 10(-6) M. At 10(-6) M FLR, basal glucagon secretion was inhibited; glucose-induced suppression still occurred without any potentiation. We conclude that 1) endogenous PGs modulate the secretion of insulin and glucagon; 2) divergence of the effects of low and high levels of inhibition of PG biosynthesis on insulin release may be due to altered tissue proportions of various PGs and related autacoids; and 3) the predominant effect of endogenous PGs on glucagon release is tonic stimulation.