Suzanne G. Laychock

Neonatal Nutrition: Metabolic Programming of Pancreatic Islets and Obesity

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.

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A2 activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from E. coli membranes. Phospholipase A2 activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A2 activity which was assayable in islet homogenates. Glucose stimulated phospholipase A2 in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A2 activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A2 activity measured in the presence of high calcium concentrations. Phospholipase A2 activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A2 activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.

Metabolic programming: Role of nutrition in the immediate postnatal life

SummaryAlthough genes and dietary habits are generally implicated in the aetiology of the prevailing obesity epidemic, the steep increase in the incidence of obesity within a relatively short span of time suggests that other contributing factors may be at play. The role of nutritional experience during the very early periods of life is increasingly being recognized as contributing to growth and metabolic changes in later life. Epidemiological data and studies from animal models have established a strong correlation between an aberrant intrauterine environment and adult-onset disorders in offspring. The nutritional experience in the immediate postnatal life is another independent factor contributing to the development of metabolic diseases in adulthood. Although studies on the small-litter rat model have shown that overnourishment during the suckling period results in adult-onset metabolic disorders, our studies have shown that a change in the quality of calories—specifically, increased carbohydrate intake by newborn rat pups in the immediate postnatal period—results in chronic hyperinsulinaemia and adult-onset obesity. Several functional alterations in islets and in the hypothalamic energy homeostatic mechanism appear to support this phenotype. Remarkably, female rats that underwent the high-carbohydrate dietary modification as neonates spontaneously transmitted the obesity phenotype to their offspring, thus establishing a vicious generational effect. The high-carbohydrate diet-fed rat model has particular relevance in the context of the current human infant feeding practices: reduction in breast feeding and increase in formula feeding for infants, accompanied by early introduction of carbohydrate-enriched baby foods.

Prostaglandin synthesis and metabolism in isolated pancreatic islets of the rat

Isolated pancreatic islets of Langerhans of the rat which were sonicated and incubated with radiolabeled arachidonic acid for 1 hr synthesized several species of prostaglandins (PGs). Both thin-layer and high-performance liquid (HPLC) chromatographic techniques demonstrated the synthesis by islet sonicates of PGF2 alpha and PGE2 equivalents, in addition to the 15-keto-13, 14-dihydro metabolites of these primary PGs. In addition, HPLC allowed the identification of 6-keto-PGF1 alpha (the metabolite of prostacyclin) as a major PG synthesized from arachidonate by this tissue. Islet vascular elements, as well as endocrine cells, may contribute to the synthesis of the latter compound. Lesser amounts of arachidonate were incorporated into PG-like compounds eluting as thromboxane. The synthesis of PGs was sensitive to the protein concentration of islet sonicate, and a five-fold dilution of protein resulted in a comparable reduction in arachidonate incorporation into PGs. Labeled arachidonate was also incorporated into compounds which elute as hydroxy or hydroperoxyeicosatetraenoic acids on HPLC. Thus, isolated pancreatic islets synthesize a variety of PGs which may have a physiological role in hormone secretion from this endocrine organ.

Insulin Secretion, myo-Inositol Transport, and Na+-K+-ATPase in Glucose-Desensitized Rat Islets

Glucose-induced insulin secretion is desensitized during long-term exposure of pancreatic islet beta-cells to elevated glucose levels. This study characterizes an in vitro model of glucose-induced desensitization in cultured isolated islets of the rat. Insulin secretion in desensitized islets cultured with 11 mM glucose for 4–7 days was progressively reduced compared with the normal freshly isolated (fresh) islets. When desensitized islets were returned to a basal concentration of glucose (5.5 mM) for up to 2 h, the glucose sensitivity of insulin secretion was restored to normal (recovered islets). Carbachol and L-arginine also reversed the effects of desensitization. However, basal insulin release was elevated in desensitized and recovered islets. Sodium-dependent myo-inositol uptake was reduced during desensitization by up to 49% within 4 days. myo-Inositol uptake was restored to normal in a time-dependent manner during recovery of islets at 5.5 mM glucose. The recovery of myo-inositol uptake paralleled that of insulin release. The apparent transport constant for myo-inositol uptake was significantly increased during desensitization, whereas the maximum uptake was not changed. myo-Inositol supplementation (35 or 250 µM) during islet culture did not alter myo-inositol uptake or insulin secretion in desensitized islets. Na+)-K+)-ATPase activity, but not 5′-nucleotidase activity, in desensitized islets was also inhibited by 65 and 47% when compared with fresh islet and recovered islet Na+)-K+-ATPase activity, respectively. Thus, cultured islets represent an appropriate model to study biochemical parameters associated with the onset and reversibility of glucose desensitization of insulin secretion. associated with the onset and reversibility of glucose desensitization of insulin secretion. Alterations in Na+-K+-ATPase activity and coupled myo-inositol uptake during desensitization may contribute to the defective pattern of insulin release in this model.

Fatty Acid Incorporation into Phospholipids of Isolated Pancreatic Islets of the Rat: Relationship to Insulin Release

Fatty acid incorporation into specific phospholipids of isolated islets of the rat was investigated using unsaturated [14C]arachidonic acid. Glucose (25 mM) stimulated the incorporation of arachidonic acid into phosphatidylinositol (PI) and phosphatidylcholine (PC) in a time-related manner correlated with two phases of insulin release. Arachidonate incorporation was inhibited by calcium deprivation. The sulfonylurea tolbutamide stimulated an early monophasic release of insulin that was accompanied by increased [14C]arachidonate incorporation into PI and PC. The cholinergic agonist and insulin secretagogue, carbamylcholine, also promoted the incorporation of [14C]arachidonate into PI/phosphatidylserine (PS) and PC fractions. 2-Deoxy-D-glucose, which does not support insulin release, did not enhance arachidonate incorporation into phospholipids. However, phenylephrine, an inhibitor of glucose-induced insulin secretion, stimulated arachidonate turnover in PI. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, markedly depressed both glucose-stimulated arachidonate incorporation into phospholipids and insulin release. The stimulated release of arachidonate from endogenous radiolabeled phospholipids provided additional evidence that phospholipase A2 mediates glucose stimulation. However, since glucose also promoted the incorporation of saturated [14C]palmitic acid into PE (phosphatidylethanolamine) and PI/PS fractions, a phospholipase A1 may also mediate the glucose response. Thus, fatty acid incorporation into islet phospholipids mediates the effects of various secretagogues on insulin release. However, the ability of phenylephrine to stimulate arachidonyl PI turnover suggests that fatty acid turnover is not a sufficient stimulus for release. Augmented levels of unsaturated fatty acids in islet cell membranes may promote fusion or activate enzymes important for hormone release.

A dietary intervention (high carbohydrate) during the neonatal period causes islet dysfunction in rats.

Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats and compared with age-matched mother-fed (MF) pups. The plasma insulin and glucagon contents were increased sixfold and twofold, respectively, in HC rats compared with MF rats. There was a distinct leftward shift in the glucose-stimulated insulin secretory pattern for HC islets. HC islets secreted insulin in the absence of any added glucose and in the presence of Ca2+ channel inhibitors. The activities of glucokinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase complex were significantly increased in HC islets compared with MF islets. The protein contents of GLUT-2 and hexokinase were significantly increased in HC islets. These findings indicate that a nutritional intervention in the form of a HC formula only during the suckling period has a profound influence on pancreatic function, causing the onset of hyperinsulinemia.Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats and compared with age-matched mother-fed (MF) pups. The plasma insulin and glucagon contents were increased sixfold and twofold, respectively, in HC rats compared with MF rats. There was a distinct leftward shift in the glucose-stimulated insulin secretory pattern for HC islets. HC islets secreted insulin in the absence of any added glucose and in the presence of Ca(2+) channel inhibitors. The activities of glucokinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase complex were significantly increased in HC islets compared with MF islets. The protein contents of GLUT-2 and hexokinase were significantly increased in HC islets. These findings indicate that a nutritional intervention in the form of a HC formula only during the suckling period has a profound influence on pancreatic function, causing the onset of hyperinsulinemia.

Nitric oxide reduces depolarization-induced calcium influx in PC12 cells by a cyclic GMP-mediated mechanism

The present study was undertaken to determine whether nitric oxide (NO) alters voltage-dependent changes in intracellular calcium levels ([Ca2+]i) using PC12 cells as a neuronal model. The addition to PC12 cells of sodium nitroprusside (SNP), which spontaneously releases NO in aqueous solution, significantly inhibited the KCl-stimulated increase in [Ca2+]i. The inhibitory action of SNP was concentration-dependent and was mimicked by hydroxylamine which also generates NO. Both L-type (nifedipine sensitive) and N-type (omega-conotoxin sensitive) voltage-dependent Ca2+ channels are present in PC12 cells and may be affected by NO-generating agents. In contrast, SNP did not alter [Ca2+]i in response to purinergic receptor stimulation. Preincubation of PC12 cells with 8-bromo-cyclic GMP also inhibited the KCl-stimulated increase in [Ca2+]i. In addition, inclusion of the guanylyl cyclase inhibitor, LY83583, blocked the inhibitory action of SNP on the voltage-sensitive changes in [Ca2+]i. The results suggest that NO selectively inhibits voltage-dependent calcium influx in neuronal cells through a cyclic GMP-dependent mechanism.

GLUCOSE METABOLISM, SECOND MESSENGERS AND INSULIN SECRETION

Insulin secretion from beta cells of the islets of Langerhans in the endocrine pancreas is regulated by glucose, glucose metabolites, metabolic intermediates such as ATP, acetyl CoA and reduced pyridine nucleotides, and classical second messengers. Receptor responses transduced by guanine nucleotide binding proteins modulate metabolic activity, the generation of second messengers, and cell depolarization during stimulus-response coupling in the beta cell. This review will consider insulin secretion as regulated by glucose metabolic pathways and second messengers.

Indomethacin-induced alterations in corticosteroid and prostaglandin release by isolated adrenocortical cells of the cat.

1 The effects of purported prostaglandin synthesis inhibitors on steroid and prostaglandin (E and F) release from trypsin‐dispersed cat adrenocortical cells were investigated. 2 Low indomethacin concentrations potentiated adrenocorticotrophin (ACTH)‐evoked prostaglandin and steroid release, whereas higher concentrations depressed both responses to ACTH. The steroidogenic response to exogenous prostaglandin E2 was not markedly altered over a wide range of indomethacin concentrations. 3 Indomethacin enhanced basal steroid release but did not enhance basal prostaglandin E or F release. 4 5, 8, 11, 14‐Eicosatetraynoic acid (ETA) elicited a concentration‐dependent inhibition of ACTH‐induced steroid release, but had little effect on prostaglandin E2‐induced steroid release. A high concentration of ETA inhibited prostaglandin E and F release. 5 These data are discussed in relation to the concept that prostaglandins provide a critical link in ACTH‐induced corticosteroidogenesis.