Maternal methyl donor and cofactor supplementation in late pregnancy increases ß-cell numbers at 16 days of life in growth-restricted twin lambs.

Restricted growth before birth (IUGR) increases adult risk of Type 2 diabetes by impairing insulin sensitivity and secretion. Altered fetal one-carbon metabolism is implicated in developmental programming of adult health and disease by IUGR. Therefore, we evaluated effects of maternal dietary supplementation with methyl donors and cofactors (MMDS), designed to increase fetal supply, on insulin action in the spontaneously IUGR twin lamb. In vivo glucose-stimulated insulin secretion and insulin sensitivity were measured at days 12-14 in singleton controls (CON, n = 7 lambs from 7 ewes), twins (IUGR, n = 8 lambs from 8 ewes), and twins from ewes that received MMDS (2 g rumen-protected methionine, 300 mg folic acid, 1.2 g sulfur, 0.7 mg cobalt) daily from 120 days after mating (~0.8 of term) until delivery (IUGR+MMDS, n = 8 lambs from 4 ewes). Body composition and pancreas morphometry were assessed in lambs at day 16 IUGR reduced size at birth and increased neonatal fractional growth rate. MMDS normalized long bone lengths but not other body dimensions of IUGR lambs at birth. IUGR did not impair glucose control or insulin action at days 12-14, compared with controls. MMDS increased metabolic clearance rate of insulin and increased ß-cell numerical density and tended to improve insulin sensitivity, compared with untreated IUGR lambs. This demonstrates that effects of late-pregnancy methyl donor supplementation persist until at least the third week of life. Whether these effects of MMDS persist beyond early postnatal life and improve metabolic outcomes after IUGR in adults and the underlying mechanisms remain to be determined.

Neonatal exendin-4 reduces growth, fat deposition and glucose tolerance during treatment in the intrauterine growth-restricted lamb.

BACKGROUND: IUGR increases the risk of type 2 diabetes mellitus (T2DM) in later life, due to reduced insulin sensitivity and impaired adaptation of insulin secretion. In IUGR rats, development of T2DM can be prevented by neonatal administration of the GLP-1 analogue exendin-4. We therefore investigated effects of neonatal exendin-4 administration on insulin action and ß-cell mass and function in the IUGR neonate in the sheep, a species with a more developed pancreas at birth. METHODS: Twin IUGR lambs were injected s.c. daily with vehicle (IUGR+Veh, n = 8) or exendin-4 (1 nmol.kg⁻¹, IUGR+Ex-4, n = 8), and singleton control lambs were injected with vehicle (CON, n = 7), from d 1 to 16 of age. Glucose-stimulated insulin secretion and insulin sensitivity were measured in vivo during treatment (d 12-14). Body composition, ß-cell mass and in vitro insulin secretion of isolated pancreatic islets were measured at d 16. PRINCIPAL FINDINGS: IUGR+Veh did not alter in vivo insulin secretion or insulin sensitivity or ß-cell mass, but increased glucose-stimulated insulin secretion in vitro. Exendin-4 treatment of the IUGR lamb impaired glucose tolerance in vivo, reflecting reduced insulin sensitivity, and normalised glucose-stimulated insulin secretion in vitro. Exendin-4 also reduced neonatal growth and visceral fat accumulation in IUGR lambs, known risk factors for later T2DM. CONCLUSIONS: Neonatal exendin-4 induces changes in IUGR lambs that might improve later insulin action. Whether these effects of exendin-4 lead to improved insulin action in adult life after IUGR in the sheep, as in the PR rat, requires further investigation.

Placental restriction reduces insulin sensitivity and expression of insulin signaling and glucose transporter genes in skeletal muscle, but not liver, in young sheep.

Poor growth before birth is associated with impaired insulin sensitivity later in life, increasing the risk of type 2 diabetes. The tissue sites at which insulin resistance first develops after intrauterine growth restriction (IUGR), and its molecular basis, are unclear. We have therefore characterized the effects of placental restriction (PR), a major cause of IUGR, on whole-body insulin sensitivity and expression of molecular determinants of insulin signaling and glucose uptake in skeletal muscle and liver of young lambs. Whole-body insulin sensitivity was measured at 30 d by hyperinsulinaemic euglycaemic clamp and expression of insulin signaling genes (receptors, pathways, and targets) at 43 d in muscle and liver of control (n = 15) and PR (n = 13) lambs. PR reduced size at birth and increased postnatal growth, fasting plasma glucose (+15%, P = 0.004), and insulin (+115%, P = 0.009). PR reduced whole-body insulin sensitivity (-43%, P < 0.001) and skeletal muscle expression of INSR (-36%), IRS1 (-28%), AKT2 (-44%), GLUT4 (-88%), GSK3α (-35%), and GYS1 (-31%) overall (each P < 0.05) and decreased AMPKγ3 expression in females (P = 0.030). PR did not alter hepatic expression of insulin signaling and related genes but increased GLUT2 expression (P = 0.047) in males. Whole-body insulin sensitivity correlated positively with skeletal muscle expression of IRS1, AKT2, HK, AMPKγ2, and AMPKγ3 in PR lambs only (each P < 0.05) but not with hepatic gene expression in control or PR lambs. Onset of insulin resistance after PR and IUGR is accompanied by, and can be accounted for by, reduced expression of insulin signaling and metabolic genes in skeletal muscle but not liver.

Testing the plasticity of insulin secretion and ß-cell function in vivo: responses to chronic hyperglycaemia in the sheep.

Plasticity of insulin secretion is essential to maintain the action of insulin during insulin resistance and to prevent diabetes. Investigation of the plasticity of insulin secretion and its regulation is challenging, and the objective of this study was to develop a novel large-animal-based model. The effect of chronic moderate hyperglycaemia on the plasticity of insulin secretion, ß-cell mass and function was determined in sheep. Adolescent sheep (120 days old) were infused with 25% glucose for 16 days to increase blood glucose by 50% (n = 10), and control animals (n = 9) were infused with saline. Glucose- and arginine-stimulated insulin secretion, insulin sensitivity and glucose effectiveness were measured in vivo before and during treatment (days 10-14), and ß-cell mass was measured at the end of treatment. Hyperglycaemia increased blood glucose (+53%) and plasma insulin (+403%; each P < 0.003) and did not alter whole-body insulin sensitivity. Hyperglycaemia increased glucose-stimulated insulin secretion (particularly second phase; five-fold) and arginine-stimulated insulin secretion (particularly first phase; four-fold). Hyperglycaemia reduced ß-cell mass (∼50%, P = 0.038) and increased glucose- and arginine-stimulated insulin secretion relative to ß-cell mass five-fold (P = 0.060) and 20-fold (P = 0.007), respectively. Chronic hyperglycaemia therefore induces marked adaptation and upregulation of glucose- and arginine-stimulated insulin secretion by enhancing ß-cell function rather than increasing ß-cell mass in the sheep, consistent with long-term adaptations seen in humans. This marked plasticity of insulin secretion in response to moderate hyperglycaemia provides a novel model for the investigation of factors affecting its capacity and underlying determinants.

Impaired beta-cell function and inadequate compensatory increases in beta-cell mass after intrauterine growth restriction in sheep.

Poor growth before birth increases the risk of non-insulin-dependent diabetes mellitus (NIDDM) and impairs insulin secretion relative to sensitivity. We investigated the effects of intrauterine growth restriction in sheep on insulin secretion, beta-cell mass, and function from before birth to young adulthood and its molecular basis. Pancreas was collected from control and placentally restricted sheep as fetuses (d 143 gestation), lambs (aged 42 d), and young adults (aged 556 d), following independent measures of in vivo insulin secretion and sensitivity. beta-Cells and islets were counted after immunohistochemical staining for insulin. In lambs, gene expression was measured by RT-PCR and expressed relative to 18S. beta-Cell mass correlated positively with fetal weight but negatively with birth weight in adult males. Glucose-stimulated insulin disposition and beta-cell function correlated negatively with fetal weight but positively with birth weight in adult males. Placental restriction increased pancreatic expression of IGF-II and IGF-I but decreased that of voltage-gated calcium channel, alpha1D subunit (CACNA1D) in lambs. In male lambs, pancreatic IGF-II and insulin receptor expression correlated strongly and positively with beta-cell mass and CACNA1D expression with glucose-stimulated insulin disposition. Restricted growth before birth in the sheep does not impair insulin secretion, relative to sensitivity, before birth or in young offspring. IGF-II and insulin receptor are implicated as key molecular regulators of beta-cell mass compensation, whereas impaired expression of the voltage-gated calcium channel may underlie impaired beta-cell function after intrauterine growth restriction. With aging, the insulin secretory capacity of the beta-cell is impaired in males, and their increases in beta-cell mass are inadequate to maintain adequate insulin secretion relative to sensitivity.

A novel basal lamina matrix of the stratified epithelium of the ovarian follicle.

Basal laminas are important sheets of specialized extracellular matrix that underlie and surround groups of cells, such as epithelia or endothelia, enabling the cells to orientate their basal/apical polarity and creating a microenvironment for them. Basal laminas can also individually encapsulate whole cells, such as muscle cells, thereby forming a microenvironment but not polarizing the enclosed cells. Other mesenchymal or stromal cells exist with no basal lamina. In the course of studying the bovine follicular basal lamina which underlies the multilayered epithelium of the ovarian follicle, we identified a developmentally regulated novel extracellular matrix (which we call focimatrix for focal intra-epithelial matrix). Focimatrix is composed of basal lamina-like material deposited as plaques or aggregates between the multilayers of the epithelial granulosa cells. The focimatrix does not encapsulate individual or groups of cells and therefore does not form a microenvironment for them. Focimatrix contains collagen type IV subunits alpha1 and alpha2 (but not alpha3-alpha6), and laminin chains alpha1, beta2 and gamma1 (but not alpha2 or beta1), and nidogen-1 and perlecan (but not versican). The amount of focimatrix increases with increasing follicular size, and its appearance precedes the expression by granulosa cells of the enzymes for steroid hormone synthesis, cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), in the days preceding ovulation. The expression in granulosa cells of two components examined, nidogen-1 and perlecan, also increases substantially when follicles enlarge to a sufficient size capable of ovulating. Following ovulation the follicular basal lamina is degraded, and presumably focimatrix is too since it is not detected in corpora lutea that develop from the ovulating follicles. During this development the granulosa cells undergo an epithelial-mesenchymal transition (EMT) into luteal cells following ovulation, and substantially increase their expression of steroidogenic enzymes in the process. During EMT epithelial cells lose polarity. Since focimatrix exists on more than one side of the granulosa cells, we propose that it disrupts the polarity induced by the follicular basal lamina in the lead up to ovulation. Hence focimatrix maybe a key part of the follicular/luteal EMT.

Fast Ca(2+)-dependent inactivation of the store-operated Ca2+ current (ISOC) in liver cells: a role for calmodulin.

Store-operated Ca2+ channels (SOCs) provide a major pathway for Ca2+ entry in non-excitable cells. SOCs in immortalized liver cells are highly selective for Ca2+ over other cations and are similar to well-studied Ca2+ release activated Ca2+ (CRAC) channels in haematopoietic cell lines. In the present work, employing H4IIE liver cells, we investigated fast inactivation of SOC current (ISOC), which occurs at membrane potentials below -60 mV. This inactivation was significantly reduced when BAPTA, a faster Ca2+ buffer, was used instead of EGTA, and was completely abolished if Na+ was used as a charge carrier in the absence of divalent cations in the external medium. These results suggested that fast inactivation of SOCs in H4IIE cells was Ca2+ dependent and was similar to the fast inactivation of CRAC channels. Experiments showing that the fast inactivation of ISOC was not affected by the disruption of actin by latrunculin B indicate that the cytoskeleton is unlikely to be involved. To elucidate the mechanism of Ca2+ dependence, a possible role of calmodulin (CaM) in SOCs' fast inactivation was investigated. The CaM inhibitors Mas-7 and calmidazolium failed to affect ISOC fast inactivation, whereas over-expression of a CaM inhibitor peptide or a mutant CaM lacking functional EF hands significantly altered the inactivation of ISOC. Out of two exponential components normally required to approximate kinetics of ISOC fast inactivation, the faster component was reduced in amplitude by 30%, compared to the control. The results presented suggest that CaM is responsible for at least part of Ca(2+)-dependent fast inactivation of ISOC in liver cells. It is hypothesized that CaM is tethered to the channel itself and therefore protected from chemical inhibitors.

Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2++Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells.

The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.

Evidence for the expression of transient receptor potential proteins in guinea pig airway smooth muscle cells.

OBJECTIVE: The present study investigates the expression of transient receptor potential (TRPC) proteins in airway smooth muscle (ASM) cells in order to determine whether these proteins may be candidate molecular counterparts of plasma membrane Ca2+-permeable channels involved in the contraction of ASM. METHODS: Expression of TRPC mRNA was detected using specific primers and RT-PCR. Expression of the TRPC1, TRPC3 and TRPC6 proteins was detected using antibodies in immunoprecipitation and Western blot. RESULTS: Guinea pig ASM cells exhibited thapsigargin- and acetylcholine-initiated Ca2+ inflow but none by 1-oleoyl-2-acetyl-sn-glycerol. mRNA encoding each of the TRPC1 to TRPC6 proteins was detected in ASM cells. mRNA encoding TRPC1, TRPC3, TRPC4 and TRPC6 was detected in ASM cells at a concentration approximately equivalent to that in guinea pig brain. mRNA encoding TRPC2 and TRPC5 was more abundant in ASM cells than in brain. The TRPC1 protein, but not the TRPC3 or TRPC6 proteins, was detected in extracts of ASM cells, while all three proteins were detected in brain. CONCLUSION: The results provide evidence for a low level of expression of the TRPC1 to TRPC6 proteins in ASM cells. These proteins may function as store-operated Ca2+ and/or second messenger-activated non-selective cation channels in ASM cells.