Stimulation of leukotriene synthesis in intact polymorphonuclear cells by the 5-lipoxygenase inhibitor 3-oxo-tirucallic acid.

ABSTRACT Commercially available extracts from Boswellia serrata resin used as anti-inflammatory drugs or phytonutrients show paradoxical concentration-dependent potentiating and inhibitory actions on 5-lipoxygenase (5-LO) product synthesis in stimulated PMNs. In our attempt to characterize the stimulating constituents, we identified the tetracyclic triterpene 3-oxo-tirucallic acid (3-oxo-TA), which, in the range from 2.5 to 15 M, enhanced 5-LO product formation in ionophore-challenged polymorphonuclear cells (PMNs) (e.g., from 1981 Ϯ 177 to 3042 Ϯ 208 pmol at 10 M 3-oxo-TA), and initiated Ca 2ϩ mobilization, MEK-1/2 phosphorylation, 5-LO translocation, and 5-LO product formation in resting cells (534 Ϯ 394 pmol/ 5 ϫ 10 6 PMNs). In cell-free 5-LO assays, 3-oxo-TA acted only inhibitory (IC 50 value of about 3 M), demonstrating the pivotal role of intact cell structure for its activating property. In 3-oxo-TA-challenged PMNs, the mitogen-activated protein kinase kinase (MEK)-1/2 inhibitor PD098059 abolished 5-LO product formation, along with inhibition of MEK-1/2 phosphorylation and 5-LO translocation. The 3-acetoxy derivative of 3-oxo-TA acted like 3-oxo-TA in intact PMNs, whereas 3-hydroxy-TA barely stimulated MEK phosphorylation in resting cells and showed only inhibition on ionophore-induced 5-LO product synthesis. Steroid-type tetracycles neither induced 5-LO activation nor had enhancing or inhibitory effects. In summary, defined natural tetracyclic triterpenes, which act as inhibitors of the 5-LO in the cell-free assay, initiate 5-LO activation by a MEK-inhibitor sensitive mechanism and potentiate stimulated product synthesis in intact cells. Because TAs contribute significantly to the overall biological effects of B. serrata resin extracts, special precaution for standardization is recommended when using B. serrata preparations as drugs or dietary supplements. 5-Lipoxygenase (5-LO; EC 1.13.11.34) catalyzes the first two steps in the biosynthesis of leukotrienes and 5(S)-HETE from arachidonic acid. Leukotrienes and 5-oxo-eicosa-tetraenoic acid, a final metabolite from 5(S)-HETE The enzymatic activity of 5-LO, as well as its binding to other macromolecules, is regulated in a highly complex manner (for concise reviews on many aspects of 5-LO, products, and receptors, se

Methyl pyruvate stimulates pancreatic beta-cells by a direct effect on KATP channels, and not as a mitochondrial substrate.

In pancreatic beta-cells, methyl pyruvate is a potent secretagogue and is widely used to study stimulus-secretion coupling. In contrast with pyruvate, which barely stimulates insulin secretion, methyl pyruvate was suggested to act as an effective mitochondrial substrate. We show that methyl pyruvate elicited electrical activity in the presence of 0.5 mM glucose, in contrast with pyruvate. Accordingly, methyl pyruvate increased the cytosolic free Ca(2+) concentration after an initial decrease, similar to glucose. The initial decrease was inhibited by thapsigargin, suggesting that methyl pyruvate stimulates ATP production. This assumption is supported by the observation that methyl pyruvate hyperpolarized the mitochondrial membrane potential, similar to glucose. However, in contrast with glucose, methyl pyruvate even slightly decreased NAD(P)H autofluorescence and did not influence ATP production or the ATP/ADP ratio. This observation questions the suggestion that methyl pyruvate acts as a powerful mitochondrial substrate. The finding that methyl pyruvate directly inhibited a cation current across the inner membrane of Jurkat T-lymphocyte mitochondria suggests that this metabolite may increase ATP production in beta-cells by activating the respiratory chains without providing reduction equivalents. We conclude that this mechanism may account for a slight and transient increase in ATP production. We further show that methyl pyruvate inhibited the K(ATP) current measured in the standard whole-cell configuration, an effect that was at least partly antagonized by diazoxide. Accordingly, single-channel currents in inside-out patches were blocked by methyl pyruvate. We conclude that inhibition of K(ATP) channels, and not activation of metabolism, mediates the induction of electrical activity in pancreatic beta-cells by methyl pyruvate

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane–associated ATP-sensitive K+ (KATP) channels of pancreatic β cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of KATP channels on loss of mouse β cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of KATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1–/– islets was less susceptible to oxidative stress induced by the oxidant H2O2. This was likely, at least in part, a result of the reduced ability of H2O2 to hyperpolarize plasma membrane potential and reduce cytosolic free Ca2+ concentration ([Ca2+]c) in the Sur1–/– β cells. Remarkably, Sur1–/– β cells were less prone to apoptosis induced by H2O2 or an NO donor than WT β cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1–/– cells to H2O2-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca2+]c. Sur1–/– mice were less susceptible than WT mice to streptozotocin-induced β cell destruction and subsequent hyperglycemia and death, which suggests that loss of KATP channel activity may protect against streptozotocin-induced diabetes in vivo

Lupanine Improves Glucose Homeostasis by Influencing KATP Channels and Insulin Gene Expression

The glucose-lowering effects of lupin seeds involve the combined action of several components. The present study investigates the influence of one of the main quinolizidine alkaloids, lupanine, on pancreatic beta cells and in an animal model of type-2 diabetes mellitus. In vitro studies were performed with insulin-secreting INS-1E cells or islets of C57BL/6 mice. In the in vivo experiments, hyperglycemia was induced in rats by injecting streptozotocin (65 mg/kg body weight). In the presence of 15 mmol/L glucose, insulin secretion was significantly elevated by 0.5 mmol/L lupanine, whereas the alkaloid did not stimulate insulin release with lower glucose concentrations. In islets treated with l-arginine, the potentiating effect of lupanine already occurred at 8 mmol/L glucose. Lupanine increased the expression of the Ins-1 gene. The potentiating effect on secretion was correlated to membrane depolarization and an increase in the frequency of Ca2+ action potentials. Determination of the current through ATP-dependent K+ channels (KATP channels) revealed that lupanine directly inhibited the channel. The effect was dose-dependent but, even with a high lupanine concentration of 1 mmol/L or after a prolonged exposure time (12 h), the KATP channel block was incomplete. Oral administration of lupanine did not induce hypoglycemia. By contrast, lupanine improved glycemic control in response to an oral glucose tolerance test in streptozotocin-diabetic rats. In summary, lupanine acts as a positive modulator of insulin release obviously without a risk for hypoglycemic episodes