L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of the rat and RINm5F insulinoma cells were determined. Both L-Arg and glucose stimulation increased islet cGMP levels, and glucose potentiated the response to L-Arg alone. A competitive inhibitor of L-Arg metabolism to nitric oxide, NG-monomethyl-L-arginine, reduced glucose- and L-Arg-stimulated insulin release and glucose-induced increases in cGMP; however, basal insulin release was slightly increased. D-Arg and L-ornithine did not affect islet cGMP levels, although insulin release was stimulated. RINm5F cell cGMP levels and insulin release increased in response to L-Arg in a concentration- and time-related manner, whereas glucose and L-histidine were without effect. 8-Bromo-cGMP also slightly increased RINm5F cell insulin release. Sodium nitroprusside as a source of nitric oxide increased RINm5F cell cGMP production. Methylene blue and LY83583, inhibitors of soluble guanylyl cyclase activation, reduced RINm5F cell cGMP levels in the presence and absence of L-Arg; LY83583 also reduced glucose-stimulated cGMP levels in islets. Insulin release by glucose and L-Arg was also inhibited by methylene blue and LY83583 in islets. We conclude that glucose and L-Arg stimulate guanylyl cyclase activity and cGMP formation in beta-cells at least in part through metabolism to the reactive intermediate nitric oxide. However, neither nitric oxide nor cGMP synthesis is obligatory for insulin secretion.

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Pancreatic islet β‐cells undergo apoptosis (APO) following culture with cytokines (CTK) in an in vitro model for type 1 diabetes mellitus. The aim was to determine if sphingosine 1‐phosphate (S1P) modulates the biochemical pathways that mediate the β‐cell APO response. Isolated rat islets or cells were pretreated with S1P (400 nM) for 5 h prior to and during CTK stimulation for 24‐48 h. APO was determined by TUNEL staining. CTK (1 ng/ml interleukin‐1β and 5 ng/ml interferon‐γ ) induced 28±2% APO in islet cells after 48 h. S1P alone did not affect islet cell APO, however, S1P together with CTK reduced APO to 13±3% (P<0.001 vs. CTK) of total cells. Caspase 3 activity in INS‐1e cells was also a measure of APO. CTK increased caspase 3 activity after 24 h to 193±33% of control (C) cells without CTK treatment (P<0.001). The presence of S1P or dihydro‐S1P (400 nM) with CTK reduced INS‐1e cell caspase 3 activity to 127±15% C (P>0.05) and 105±14% C (P>0.05), respectively; the occurrence of APO cells with S1P alone was 94±7% C (P>0.05). Forskolin (50 nM) reduced CTK‐induced caspase 3 activity to 121±5% C (P>0.05). Analysis by real‐time PCR showed that the mRNA expression for the anti‐APO gene Bcl‐xL increased by 337±34% C (P<0.001) in CTK‐treated islets, and that S1P augmented the expression to 570±63% C (P<0.01); with S1P alone Bcl‐xL mRNA levels were 108±15% C. In conclusion, S1P protects the islet β‐cell from cytokine‐induced APO to a similar extent as forskolin, and increased expression of Bcl‐xL and reduced activity of caspase 3 play a role in the antiapoptotic effects. (Supported by Juvenile Diabetes Research Foundation grant 1‐2002‐613)

Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [3H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [3H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [3H]CP-55,940 to sperm, defined as total binding displaced by (-)delta 9THC, was saturable: KD 5.16 +/- 1.02 nM; Hill coefficient 0.98 +/- 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 +/- 122 cannabinoid receptors per cell. Binding of [3H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (-)delta 9THC, and (+)delta 9THC. The rank order of potency to inhibit binding of [3H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (-)delta 9THC > (+)delta 9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [3H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation.

Obese individuals are more likely to suffer from diseases termed the "metabolic syndrome," which includes type 2 diabetes. It is now recognized that early life dietary experiences play an important role in the etiology of such diseases. In this context, the consequences of a high carbohydrate (HC) dietary intervention in neonatal rats is being studied in our laboratory. Artificial rearing of 4-day-old rat pups on a HC milk formula up to Day 24 results in the immediate onset of hyperinsulinemia, which persists throughout the period of dietary intervention. Several adaptations at the biochemical, cellular, and molecular levels in the islets of these HC rats support the onset and persistence of the hyperinsulinemic condition during this period. Some of these adaptations include a distinct leftward shift in the insulin secretory capacity, increased hexokinase activity, increased gene expression of preproinsulin and related transcription factors and specific kinases in 12-day-old HC islets, and alterations in the number and size of islets. These adaptations are programmed and expressed in adulthood thereby sustain the hyperinsulinemic condition in the postweaning period and form the basis for adult-onset obesity. HC females spontaneously transmit the HC phenotype (chronic hyperinsulinemia and adult-onset obesity) to their progeny. Collectively, our results indicate that even a mere switch in the nature of the source of calories (from fat rich in rat milk to carbohydrate rich in the HC milk formula) during critical phases of early development in the rat results in metabolic programming of islet functions leading to chronic hyperinsulinemia (throughout life) and adult-onset obesity. This metabolic programming, once established, forms a vicious cycle because HC female rats spontaneously transmit the HC phenotype to their progeny. The results from our laboratory in the context of metabolic programming due to neonatal nutritional experiences are discussed in this review.