A key role for interferon regulatory factors in mediating early-life metabolic defects in male offspring of maternal protein restricted rats.

An adverse intra-uterine environment, induced by maternal consumption of diets high in saturated fat or low in protein have been implicated as a potential trigger for development of metabolic disease in later life. However, the underlying mechanisms responsible for this programming of obesity have yet to be described. Recent studies have demonstrated that interferon regulatory factors 3 (IRF3) and 4 (IRF4) function to repress adipogenesis. We investigated whether impaired IRF3 and IRF4 function may predispose to development of metabolic disease in a model of programmed obesity. Changes in IRF3 and IRF4 levels, adipogenic gene expression, and adiponectin signalling were measured in white adipose tissue from programmed male offspring of rat dams fed a low-protein diet (MLP), which are predisposed to obesity. 3T3L1 adipocytes were used to determine novel regulatory mechanisms governing IRF expression. IRF3 and IRF4 levels were suppressed in MLP rats, together with raised lipogenic and adipogenic gene expression. Adiponectin and adiponectin receptor 1 and 2 mRNA levels were reduced in MLP rats, along with levels of PPARα and activity of AMP-activated protein kinase (AMPK), 2 downstream targets of adiponectin. Further studies determined that both IRF3 and IRF4 are induced by adiponectin, with adiponectin-AMPK and adiponectin-PPARα signalling regulating IRF3 and IRF4, respectively. We have demonstrated that impaired ability to repress adipogenesis and lipogenesis, through dysregulated adiponectin-PPARα-AMPK-IRF signalling, may play a causal role in predisposing MLP offspring to development of obesity and metabolic disease in later life.

Sirtuin 3 regulates mouse pancreatic beta cell function and is suppressed in pancreatic islets isolated from human type 2 diabetic patients.

AIMS/HYPOTHESIS: Sirtuin (SIRT)3 is a mitochondrial protein deacetylase that regulates reactive oxygen species (ROS) production and exerts anti-inflammatory effects. As chronic inflammation and mitochondrial dysfunction are key factors mediating pancreatic beta cell impairment in type 2 diabetes, we investigated the role of SIRT3 in the maintenance of beta cell function and mass in type 2 diabetes. METHODS: We analysed changes in SIRT3 expression in experimental models of type 2 diabetes and in human islets isolated from type 2 diabetic patients. We also determined the effects of SIRT3 knockdown on beta cell function and mass in INS1 cells. RESULTS: SIRT3 expression was markedly decreased in islets isolated from type 2 diabetes patients, as well as in mouse islets or INS1 cells incubated with IL1ß and TNFα. SIRT3 knockdown in INS1 cells resulted in lowered insulin secretion, increased beta cell apoptosis and reduced expression of key beta cell genes. SIRT3 knockdown also blocked the protective effects of nicotinamide mononucleotide on pro-inflammatory cytokines in beta cells. The deleterious effects of SIRT3 knockdown were mediated by increased levels of cellular ROS and IL1ß. CONCLUSIONS/INTERPRETATION: Decreased beta cell SIRT3 levels could be a key step in the onset of beta cell dysfunction, occurring via abnormal elevation of ROS levels and amplification of beta cell IL1ß synthesis. Strategies to increase the activity or levels of SIRT3 could generate attractive therapies for type 2 diabetes.

The involvement of PPARs in the causes, consequences and mechanisms for correction of cardiac lipotoxicity and oxidative stress.

Chronically-elevated plasma lipid concentrations, particularly when combined with high glucose, elicit a plethora of effects that cause the progressive deterioration of insulin sensitivity and ultimately cellular malfunction or death. This review addresses how metabolic abnormalities in white adipose tissue leading to excessive lipid or abnormal adipokine release can be modified by PPARγ activation. It also discusses the etiology of cardiac lipotoxicity and oxidative stress, in relation to imbalanced lipid delivery and clearance and how PPARα activation can be used to correct some of these effects.

Nicotinamide mononucleotide protects against pro-inflammatory cytokine-mediated impairment of mouse islet function.

AIMS/HYPOTHESIS: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD(+) biosynthesis, exists as intracellular NAMPT (iNAMPT) and extracellular NAMPT (eNAMPT). eNAMPT, secreted from adipose tissue, promotes insulin secretion. Administration of nicotinamide mononucleotide (NMN), a product of the eNAMPT reaction, corrects impaired islet function in Nampt ( +/- ) mice. One of its potential targets is the NAD(+)-dependent deacetylase sirtuin 1. We hypothesised that altered NAMPT activity might contribute to the suppression of islet function associated with inflammation, and aimed to determine whether NMN could improve cytokine-mediated islet dysfunction. METHODS: Acute effects of NMN on cytokine-mediated islet dysfunction were examined in islets incubated with TNFα and IL1ß, and in mice fed a fructose-rich diet (FRD) for 16 weeks. Changes in iNAMPT, eNAMPT and inflammation levels were determined in FRD-fed mice. RESULTS: FRD-fed mice displayed markedly lower levels of circulating eNAMPT, with impaired insulin secretion and raised islet expression of Il1b. NMN administration lowered Il1b expression and restored suppressed insulin secretion in FRD-fed mice. NMN also restored insulin secretion in islets cultured with pro-inflammatory cytokines. The changes in islet function corresponded with changes in key markers of islet function and differentiation. The anti-inflammatory effects of NMN were partially blocked by inhibition of sirtuin 1. CONCLUSIONS/INTERPRETATION: Chronic fructose feeding causes severe islet dysfunction in mice. Onset of beta cell failure in FRD-fed mice may occur via lowered secretion of eNAMPT, leading to increased islet inflammation and impaired beta cell function. Administration of exogenous NMN to FRD-fed mice corrects inflammation-induced islet dysfunction. Modulation of this pathway may be an attractive target for amelioration of islet dysfunction associated with inflammation.

Metformin opposes impaired AMPK and SIRT1 function and deleterious changes in core clock protein expression in white adipose tissue of genetically-obese db/db mice.

AIM: AMPK activates SIRT1 in liver and skeletal muscle. Impaired circadian function is associated with development of obesity. SIRT1 regulates circadian function and is suppressed in white adipose tissue (WAT) of obese patients. We examined the potential role of AMPK and SIRT1 in regulation of circadian components in WAT of obese db/db mice and in mice fed a high-fat diet (HFD), and investigated whether metformin-mediated activation of AMPK opposed any deleterious changes in the WAT clock mechanism. METHODS: db/+ and db/db mice were administered metformin (250 mg/kg/day; 7 days). Separately, mice were fed HFD for 16-weeks. 3T3-L1 adipocytes were incubated with metformin, EX527 or FK866, inhibitors of SIRT1 and NAMPT, respectively. Gene and protein expression were measured by qRT-PCR and immunoblotting. RESULTS: AMPK activity, NAMPT expression and SIRT1 expression were decreased in WAT of db/db and HFD mice, in association with suppressed expression of the core circadian components CLOCK and BMAL1. Expression of Pparγ and the adipogenic repressors Irf3 and Irf4 were also suppressed. Metformin increased AMPK activity in WAT of db/db mice and in metformin-treated adipocytes, with increased NAMPT, SIRT1 and circadian component expression. Metformin-mediated induction of Clock mRNA in adipocytes was blocked by inhibition of NAMPT and SIRT1. CONCLUSIONS: Decreased AMPK-SIRT1 signalling in db/db and HFD mice impacts WAT circadian function causing dysregulated lipid regulation, favouring an obese phenotype. Metformin mediates a phenotypic shift away from lipid accretion through AMPK-NAMPT-SIRT1 mediated changes in clock components, supporting chronotherapeutic treatment approaches for obesity.

Peroxisome proliferator-activated receptor alpha deficiency modifies glucose handling by isolated mouse adipocytes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a transcription factor that regulates enzymes involved in fatty acid (FA) utilisation. PPARalpha null mice have recently been demonstrated to have increased whole-body glucose turnover in vivo. This has been attributed to increased glucose uptake by adipose tissue, but the impact of PPARalpha deficiency on the characteristics of glucose handling by isolated adipocytes ex vivo is unknown. To determine directly the impact of PPARalpha deficiency on adipocyte glucose handling, thereby excluding any influence of humoral/neuronal factors, we examined total glucose metabolism as well as glucose disposition towards alternative fates in epididymal adipocytes isolated from wild-type and PPARalphanull mice. Total glucose metabolism (oxidation, incorporation into FA and glycerol moieties of triglyceride (TAG) and conversion to lactate) was measured under basal conditions (low glucose) and 'stimulated lipogenic' conditions (high glucose + insulin). Adipocytes from PPARalpha null mice had higher rates of glucose metabolism under both basal and stimulated lipogenic conditions, with increased glucose utilisation both for oxidation and entry into the synthesis of the FA and glycerol components of lipid. In particular, the capacity of adipocytes from PPARalpha-deficient mice to utilise glucose for synthesis of the glycerol backbone of TAG was greatly enhanced under stimulated (high glucose + insulin) conditions. The increased use of glucose for the glycerol moiety of adipocyte TAG may therefore contribute to, and provide explanation for, enhanced glucose turnover in PPARalpha null mice.

Fasting-induced increases in aquaporin 7 and adipose triglyceride lipase mRNA expression in adipose tissue are attenuated by peroxisome proliferator-activated receptor alpha deficiency.

OBJECTIVE: To investigate the impact of peroxisome proliferator-activated receptor alpha deficiency on gene expression of adipose triglyceride lipase and the glycerol transporter aquaglyceroporin 7 in white adipose tissue in the fed and fasted states in relation to glycerol release by isolated adipocytes. MEASUREMENTS: Studies using wild-type and peroxisome proliferator-activated receptor alpha null mice. Hormone and metabolite concentrations, real-time polymerase chain reaction (PCR), basal and stimulated adipocyte lipolysis, estimated by glycerol release. RESULTS: Peroxisome proliferator-activated receptor alpha deficiency blocked the increase in aquaglyceroporin 7 transcript level and attenuated the increase in adipose triglyceride lipase transcript level in white adipose tissue elicited by fasting. Fasting glycerol levels were lower in peroxisome proliferator-activated receptor alpha null than wild-type mice, despite increased mobilization of adipocyte fat reserves in vivo as indicated by reduced adipose tissue masses (three distinct depots) and a significantly lower epididymal adipocyte diameter. Basal net glycerol release was unchanged but beta-adrenergic-stimulated net glycerol release was higher with isolated adipocytes from fasted peroxisome proliferator-activated receptor alpha null mice compared with those of fasted wild-type mice. CONCLUSION: Peroxisome proliferator-activated receptor alpha deficiency prevents effects of fasting to increase adipocyte aquaglyceroporin 7 gene expression, and influences the regulation of inter-tissue glycerol flux after fasting via lowered adipocyte aquaglyceroporin 7 expression. Lowered gene expression of adipose triglyceride lipase and aquaglyceroporin 7 in peroxisome proliferator-activated receptor alpha null mice is not limiting for adipose triglyceride breakdown in vivo during fasting.

Hyperthyroidism impairs pancreatic beta cell adaptations to late pregnancy and maternal liporegulation in the rat.

AIMS/HYPOTHESIS: Hyperthyroidism modifies lipid dynamics (increased oxidation), impairs insulin action and can suppress insulin secretion. We therefore examined the impact of hyperthyroidism on the relationship between glucose-stimulated insulin secretion (GSIS) and insulin action, using late pregnancy as a model of physiological insulin resistance that is associated with compensatory insulin hypersecretion to maintain glucose tolerance. Our aim was to examine whether hyperthyroidism compromises the regulation of insulin secretion and the ability of insulin to modulate circulating lipid concentrations in late pregnancy. MATERIALS AND METHODS: Hyperthyroidism was induced by tri-iodothyronine (T(3)) administration from day 17 to 19 of pregnancy. GSIS was assessed during an IVGTT and during hyperglycaemic clamps in vivo and in vitro, using step-up and -down islet perifusions. RESULTS: Hyperthyroidism in pregnancy elevated the glucose threshold for GSIS and impaired GSIS at low and high glucose concentrations in islet perifusions. In the intact animal, insulin secretion (after bolus glucose) was more rapidly curtailed following removal of the glucose stimulus to secretion. In contrast, GSIS was maintained during protracted hyperglycaemia (hyperglycaemic clamps) in the hyperthyroid pregnant state in vivo. CONCLUSIONS/INTERPRETATION: Hyperthyroidism in vivo during late pregnancy blunts GSIS in subsequently isolated and perifused islets at low and high glucose concentrations. It also adversely affects GSIS under conditions of an acute glucose challenge in vivo. In contrast, GSIS is maintained during sustained hyperglycaemia in vivo, suggesting that in vivo factors can rescue GSIS. The ability of insulin to suppress systemic lipid levels during hyperglycaemic clamps was impaired. We therefore suggest that higher circulating lipids may preserve GSIS under conditions of sustained hyperglycaemia in the hyperthyroid pregnancy.

Interactive influences of peroxisome proliferator-activated receptor alpha activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat.

AIMS/HYPOTHESIS: We sought to elucidate whether excess glucocorticoids and increased dietary lipids act synergistically to impair glucose tolerance and, if so, whether activation of peroxisome proliferator-activated receptor alpha (PPARalpha) has an adverse or beneficial effect on glucose tolerance. METHODS: Dexamethasone (100 microg kg(-1) body weight day(-1); 5 days) was administered to insulin-resistant rats fed a high-saturated-fat (HF) diet for 4 weeks. The PPARalpha agonist WY14643 was administered (50 mg kg(-1) body weight intraperitoneally) 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed in vivo after an acute glucose bolus injection, and in vitro using step-up and step-down islet perifusions. RESULTS: Although neither PPARalpha activation nor dexamethasone alone affected fasting glycaemia in the HF group, dexamethasone in combination with PPARalpha activation elicited marked postabsorptive hyperglycaemia. Dexamethasone treatment of HF rats had little effect on GSIS after an acute glucose challenge in vivo, but induced glucose intolerance. PPARalpha activation augmented GSIS in dexamethasone-treated HF rats in vivo, restoring glucose tolerance. Contrasting with data obtained in vivo, greatly enhanced peak rates of GSIS were observed ex vivo in perifusions of islets from dexamethasone-treated HF rats compared with those from untreated HF rats, an effect attenuated by antecedent PPARalpha activation. CONCLUSIONS/INTERPRETATION: The study demonstrates that glucocorticoid excess precipitates the development of glucose intolerance in rats maintained on a high-saturated-fat diet. It does this by interrupting the negative feedback loop between insulin sensitivity and secretion in vivo, such that further enhancement of compensatory insulin secretion is not possible. PPARalpha activation restores the coupling between insulin secretion and action.

Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation.

PDC (pyruvate dehydrogenase complex) catalyses the oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle. Regulation of PDC determines and reflects substrate preference and is critical to the 'glucose-fatty acid cycle', a concept of reciprocal regulation of lipid and glucose oxidation to maintain glucose homoeostasis developed by Philip Randle. Mammalian PDC activity is inactivated by phosphorylation by the PDKs (pyruvate dehydrogenase kinases). PDK inhibition by pyruvate facilitates PDC activation, favouring glucose oxidation and malonyl-CoA formation: the latter suppresses LCFA (long-chain fatty acid) oxidation. PDK activation by the high mitochondrial acetyl-CoA/CoA and NADH/NAD(+) concentration ratios that reflect high rates of LCFA oxidation causes blockade of glucose oxidation. Complementing glucose homoeostasis in health, fuel allostasis, i.e. adaptation to maintain homoeostasis, is an essential component of the response to chronic changes in glycaemia and lipidaemia in insulin resistance. We develop the concept that the PDKs act as tissue homoeostats and suggest that long-term modulation of expression of individual PDKs, particularly PDK4, is an essential component of allostasis to maintain homoeostasis. We also describe the intracellular signals that govern the expression of the various PDK isoforms, including the roles of the peroxisome proliferator-activated receptors and lipids, as effectors within the context of allostasis.

Acute (24 h) activation of peroxisome proliferator-activated receptor-alpha (PPARalpha) reverses high-fat feeding-induced insulin hypersecretion in vivo and in perifused pancreatic islets.

Abnormal depletion or accumulation of islet lipid may be important for the development of pancreatic beta cell failure. Long-term lipid sensing by beta cells may be co-ordinated via peroxisome proliferator-activated receptors (PPARs). We investigated whether PPARalpha activation in vivo for 24 h affects basal and glucose-stimulated insulin secretion in vivo after intravenous glucose administration and ex vivo in isolated perifused islets. Insulin secretion after intravenous glucose challenge was greatly increased by high-fat feeding (4 weeks) but glucose tolerance was minimally perturbed, demonstrating insulin hypersecretion compensated for insulin resistance. The effect of high-fat feeding to enhance glucose-stimulated insulin secretion was retained in perifused islets demonstrating a stable, long-term effect of high-fat feeding to potentiate islet glucose stimulus-secretion coupling. Treatment of high-fat-fed rats with WY14,643 for 24 h reversed insulin hypersecretion in vivo without impairing glucose tolerance, suggesting improved insulin action, and ex vivo in perfused islets. PPARalpha activation only affected hypersecretion of insulin since glucose-stimulated insulin secretion was unaffected by WY14,643 treatment in vivo in control rats or in perifused islets from control rats. Our data demonstrate that activation of PPARalpha for 24 h can oppose insulin hypersecretion elicited by high-fat feeding via stable long-term effects exerted on islet function. PPARalpha could, therefore, participate in ameliorating abnormal glucose homeostasis and hyperinsulinaemia in dietary insulin resistance via modulation of islet function, extending the established requirement for PPARalpha for normal islet lipid homeostasis.

Therapeutic potential of the mammalian pyruvate dehydrogenase kinases in the prevention of hyperglycaemia.

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyses the oxidative decarboxylation of pyruvate, and links glycolysis to the tricarboxylic acid cycle and ATP production. Adequate flux through PDC is important in tissues with a high ATP requirement, in lipogenic tissues (since it provides cytosolic acetyl-CoA for fatty acid (FA) synthesis), and in generating cytosolic malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase (CPT I). Conversely, suppression of PDC activity is crucial for glucose conservation when glucose is scarce. This review describes recent advances relating to the control of mammalian PDC activity by phosphorylation (inactivation) and dephosphorylation (activation, reactivation), in particular regulation of PDC by pyruvate dehydrogenase kinase (PDK) which phosphorylates and inactivates PDC. PDK activity is that of a family of four proteins (PDK1-4). PDK2 and PDK4 appear to be expressed in most major tissues and organs of the body, PDK1 appears to be limited to the heart and pancreatic islets, and PDK3 is limited to the kidney, brain and testis. PDK4 is selectively upregulated in the longer term in most tissues and organs in response to starvation and hormonal imbalances such as insulin resistance, diabetes mellitus and hyperthyroidism. Parallel increases in PDK2 and PDK4 expression appear to be restricted to gluconceogenesic tissues, liver and kidney, which take up as well as generate pyruvate. Factors that regulate PDK4 expression include FA oxidation and adequate insulin action. PDK4 is also either a direct or indirect target of peroxisome proliferator-activated receptor (PPAR) alpha. PPAR alpha deficiency in liver and kidney restricts starvation-induced upregulation of PDK4; however, the role of PPAR alpha in heart and skeletal muscle appears to be more complex. These observations may have important implications for the pharmacological modulation of PDK activity (e.g. use of PPAR alpha activators) for the control of whole-body glucose, lipid and lactate homeostasis in disease states and suggest that therapeutic interventions must be tissue targeted so that whole-body fuel homeostasis is not adversely perturbed.

Gender-specific programming of insulin secretion and action.

Insulin secretion and glucose tolerance were studied in 20-week-old male and female offspring of rat dams maintained on an isocaloric 20% or 8% protein diet during pregnancy and lactation after transfer to the same diet at weaning. Protein-restricted male and female offspring were also weaned onto a 20% protein diet. In males, post-absorptive insulin concentrations were suppressed by protein restriction from conception to adulthood (by 41%; P<0.001); however, basal insulin levels were 2.6-fold higher (P<0.001) if protein restriction was limited to gestation and lactation. Post-absorptive insulinaemia in females was unaffected by early or sustained protein restriction, but was lower than for males in the control group and the group exposed to protein restriction during early life alone (by 40% (P<0.001) and 52% (P<0.001) respectively). Plasma insulin/blood glucose ratios were higher in males compared with females in both control and early protein-restricted groups (1.6-fold (P<0.05) and 2.3-fold (P<0.001) respectively). A positive linear relationship existed between mean ambient insulin and glucose concentrations in males (r=1.0) and females (r=0.9), but the gradient was 12.4-fold greater (P<0.01) in males. beta-Cell function was evaluated after intravenous glucose challenge. In males, the acute insulin response and the suprabasal 30-min area under the insulin curve were dramatically higher in rats exposed to protein restriction during gestation and lactation alone (2.6- and 2.8-fold respectively; P<0.001). In contrast, these parameters were lowered by extending the exposure to protein restriction to adulthood in males, and by either early or prolonged exposure to protein restriction in females. The insulin resistance index was increased (2.5-fold; P<0.001) in male, but not female, rats exposed to protein restriction during gestation and lactation alone, and was not increased by extending the period of protein restriction to adulthood in either sex. Thus the data have demonstrated gender-specific lowering of insulin sensitivity due to protein restriction during early life only. The insulinogenic index (insulin response in relation to prevailing glycaemia) was increased in male, but not female, rats exposed to protein restriction during gestation and lactation alone (3.0-fold; P<0.001). A modest decline in insulin secretion in the female groups exposed to protein restriction until either the end of lactation or adulthood was compensated by increased insulin sensitivity, as demonstrated by significant decreases in the insulin resistance index in both groups (by 48% and 52% respectively; P<0.05). Glucose disappearance rates did not differ between the male and female control or early protein-restricted groups but were higher in both male (31%; P<0.05) and female groups (46%; P<0.001) exposed to protein restriction from conception to adulthood. Marked gender differences in glucose-stimulated insulin secretion were not associated with gender differences with respect to glucose tolerance. Our data therefore demonstrated that exposure to protein restriction during early life alone leads to relative insulin resistance and hyperinsulinaemia in adulthood, but this relationship is gender specific, observed only in males, and glucose tolerance is maintained.

Selective modification of pyruvate dehydrogenase kinase isoform expression in rat pancreatic islets elicited by starvation and activation of peroxisome proliferator-activated receptor-alpha: implications for glucose-stimulated insulin secretion.

The pyruvate dehydrogenase complex (PDC) has a pivotal role in islet metabolism. The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of PDC. Starvation increases islet PDK activity (Am J Physiol Endocrinol Metab 270:E988-E994, 1996). In this study, using antibodies against PDK1, PDK2, and PDK4 (no sufficiently specific antibodies are as yet available for PDK3), we identified the PDK isoform profile of the pancreatic islet and delineated the effects of starvation (48 h) on protein expression of individual PDK isoforms. Rat islets were demonstrated to contain all three PDK isoforms, PDK1, PDK2, and PDK4. Using immunoblot analysis with antibodies raised against the individual recombinant PDK isoforms, we demonstrated increased islet protein expression of PDK4 in response to starvation (2.3-fold; P < 0.01). Protein expression of PDK1 and PDK2 was suppressed in response to starvation (by 27% [P < 0.01] and 10% [NS], respectively). We demonstrated that activation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) by the selective agonist WY14,643 for 24 h in vivo leads to specific upregulation of islet PDK4 protein expression by 1.8-fold (P < 0.01), in the absence of change in islet PDK1 and PDK2 protein expression but in conjunction with a 2.2-fold increase (P < 0.01) in islet PPAR-alpha protein expression. Thus, although no changes in islet PPAR-alpha expression were observed after the starvation protocol, activation of PPAR-alpha in vivo may be a potential mechanism underlying upregulation of islet PDK4 protein expression in starvation. We evaluated the effects of antecedent changes in PDK profile and/or PPAR-alpha activation induced by starvation or PPAR-alpha activation in vivo on glucose-stimulated insulin secretion (GSIS) in isolated islets. GSIS at 20 mmol/l glucose was modestly impaired on incubation with exogenous triglyceride (1 mmol/l triolein) ( approximately 20% inhibition; P < 0.05) in islets from fed rats. Starvation (48 h) impaired GSIS in the absence of triolein (by 57%; P < 0.001), but GSIS after the further addition of triolein did not differ significantly between islets from fed or starved rats. GSIS by islets prepared from WY14,643-treated fed rats did not differ significantly from that seen with islets from control fed rats, and the response to triolein addition resembled that of islets prepared from fed rather than starved rats. PPAR-alpha activation in vivo led to increased insulin secretion at low glucose concentrations. Our results are discussed in relation to the potential impact of changes in islet PDK profile on the insulin secretory response to lipid and of PPAR-alpha activation in the cause of fasting hyperinsulinemia.

Role of peroxisome proliferator-activated receptor-alpha in the mechanism underlying changes in renal pyruvate dehydrogenase kinase isoform 4 protein expression in starvation and after refeeding.

The pyruvate dehydrogenase complex (PDC) occupies a strategic role in renal intermediary metabolism, via partitioning of pyruvate flux between oxidation and entry into the gluconeogenic pathway. Inactivation of PDC via activation of pyruvate dehydrogenase kinases (PDKs), which catalyze PDC phosphorylation, occurs secondary to increased fatty acid oxidation (FAO). In kidney, inactivation of PDC after prolonged starvation is mediated by up-regulation of the protein expression of two PDK isoforms, PDK2 and PDK4. The lipid-activated transcription factor, peroxisome proliferator-activated receptor-alpha (PPAR alpha), plays a pivotal role in the cellular metabolic response to fatty acids and is abundant in kidney. In the present study we used PPAR alpha null mice to examine the potential role of PPAR alpha in regulating renal PDK protein expression. In wild-type mice, fasting (24 h) induced marked up-regulation of the protein expression of PDK4, together with modest up-regulation of PDK2 protein expression. In striking contrast, renal protein expression of PDK4 was only marginally induced by fasting in PPAR alpha null mice. The present results define a critical role for PPAR alpha in renal adaptation to fasting, and identify PDK4 as a downstream target of PPAR alpha activation in the kidney. We propose that specific up-regulation of renal PDK4 protein expression in starvation, by maintaining PDC activity relatively low, facilitates pyruvate carboxylation to oxaloacetate and therefore entry of acetyl-CoA derived from FA beta-oxidation into the TCA cycle, allowing adequate ATP production for brisk rates of gluconeogenesis.

Maternal glucocorticoid treatment modulates placental leptin and leptin receptor expression and materno-fetal leptin physiology during late pregnancy, and elicits hypertension associated with hyperleptinaemia in the early-growth-retarded adult offspring.

BACKGROUND: Leptin concentrations are increased during late pregnancy, and leptin receptors are expressed in placental and fetal tissues, suggesting a role for leptin in placental and/or fetal growth, or both. In humans, leptin concentrations in adulthood are inversely related to body weight at birth, independent of adult adiposity, and correlate with fasting insulin. Glucocorticoids and insulin regulate leptin secretion. Excessive exposure to glucocorticoids during late fetal development in the rat causes intrauterine growth retardation (IUGR), together with hypertension and hyperinsulinaemia in adulthood. Leptin may have a role in the development of some forms of hypertension. OBJECTIVE: To determine whether IUGR induced by maternal glucocorticoid treatment during the last third of pregnancy in the rat is associated with modulation of either maternal or fetal leptin concentrations, the placental expression of leptin or the short form of the leptin receptor (ObR-S), or combinations thereof, and to evaluate whether hypertension or hyperinsulinaemia in the early-growth-retarded adult progeny of dexamethasone-treated dams is associated with altered leptin concentrations. DESIGN AND METHODS: Dexamethasone was administered to pregnant rats from day 15 to day 21 of gestation via a chronically implanted subcutaneous osmotic minipump. Protein expression of leptin and ObR-S in the placenta at day 21 of pregnancy was measured by western blotting. Plasma leptin and insulin concentrations were determined by radioimmunoassay and ELISA respectively. Systolic hypertension was measured by tail cuff plethysmography. RESULTS: Dexamethasone administration during the last third of pregnancy decreased placental mass and fetal body weight at day 21 of gestation, caused maternal hyperleptinaemia but fetal hypoleptinaemia, and suppressed placental leptin protein expression whilst up-regulating placental protein expression of ObR-S. The male and female offspring of dexamethasone-treated dams were hypertensive from 12 weeks of age. One-year-old offspring of dexamethasone-treated dams exhibited significant hyperleptinaemia compared with age-matched controls, an effect associated with hyperinsulinaemia in the male, but not female, offspring. CONCLUSIONS: The rat model of maternal dexamethasone treatment is established as a paradigm of 'programmed' hypertension in man. Our data show modification of placental leptin and leptin receptor protein expression by dexamethasone treatment during the last third of pregnancy. We also show that leptin concentrations are suppressed during fetal life but increased in adulthood in this rat model of programmed hypertension. Our data do not necessarily establish a causal relationship between fetal hypoleptinaemia and impaired fetal growth during early life, or between hyperleptinaemia and hypertension in adulthood. Nevertheless, they suggest that hyperleptinaemia may be a component of the cluster of metabolic abnormalities seen in the insulin resistance syndrome in man. They also suggest that excessive fetal exposure to glucocorticoids could be a common early-life stimulus to the association between hyperinsulinaemia, hypertension and hyperleptinaemia often seen in individuals of low birthweight.

Dexamethasone during late gestation exacerbates peripheral insulin resistance and selectively targets glucose-sensitive functions in beta cell and liver.

We examined whether low-dose dexamethasone administration during late pregnancy modifies hepatic and/or peripheral insulin action or glucose-stimulated insulin secretion. Dexamethasone (100 microg/kg maternal body weight/d) was administered via an osmotic minipump from d 14--19 of gestation. Maternal glucose-insulin homeostasis was assessed on d 19 of pregnancy in the postabsorptive state. Insulin secretion and glucose tolerance was assessed after iv glucose, and insulin action examined during insulin infusion at euglycemia. Dexamethasone treatment during late pregnancy elicited fasting hyperinsulinaemia (by 88%; P < 0.001) and hyperglycaemia (by 20%; P < 0.05), and enhanced endogenous glucose production (by 29%; P < 0.001). Insulin secretion and rates of glucose disappearance after iv glucose were greatly impaired (by 44% and 39% respectively; P < 0.05). Suppression of endogenous glucose production by insulin was enhanced by dexamethasone treatment, but insulin's ability to promote glucose clearance was diminished. We demonstrate that excess maternal glucocorticoids during late pregnancy impairs glucose-stimulated insulin secretion and insulin-simulated glucose clearance but enhances insulin's ability to suppress endogenous glucose production. The data also indicate that elevated maternal glucocorticoids impair adaptations of the endocrine pancreas to pregnancy in vivo in that insulin hypersecretion in response to deteriorating peripheral insulin action is no longer apparent, leading to impaired glucose tolerance.

Excessive glucocorticoid exposure during late intrauterine development modulates the expression of cardiac uncoupling proteins in adult hypertensive male offspring.

We investigated the impact of intrauterine growth retardation and fetal programming of hypertension by maternal dexamethasone treatment on cardiac uncoupling protein (UCP) expression during development and in adulthood in the rat. Dexamethasone administered via an indwelling osmotic pump (100 micrograms/kg body mass per day from day 15 of gestation) decreased fetal body mass at day 21 of gestation (by 13%; P < 0.05), elicited significant (+24%, P < 0.01) systolic hypertension and elevated corticosterone levels (+15%; P < 0.05) in adult (24-week-old) male offspring. Cardiac UCP-2 and UCP-3 protein expression was significantly upregulated during early postnatal development, reaching 1.7-fold and 2.7-fold the respective fetal day-21 levels by postnatal day 7 and reaching plateaus at postnatal days 15-21 (2.5-fold and 3.5-fold of respective fetal levels). Cardiac UCP protein expression at fetal day 21 and the ontogeny of cardiac UCP expression during early postnatal life were unaffected by prenatal dexamethasone treatment. Prenatal dexamethasone treatment did not abrogate the postnatal surge in corticosterone levels or modify plasma non-esterified fatty acid (NEFA) levels over this period. However, UCP-2 and UCP-3 protein expression was significantly downregulated in the hearts of adult hypertensive male offspring of dexamethasone-treated mothers (to 27% and 65% of control values respectively). We propose that changes in cardiac UCP protein expression are linked with changes in cardiac metabolic fuel selection (from glucose-->fatty acids at birth and from fatty acids-->glucose during hypertension).

Fuel-sensing mechanisms integrating lipid and carbohydrate utilization.

Fuel metabolism is highly regulated to ensure adequate energy for cellular function. The contribution of the major metabolic fuels--glucose, lactate and fatty acids (FAs)--often reflects their circulating levels. In addition, regulatory cross-talk and fuel-induced hormone secretion ensures appropriate and co-ordinate fuel utilization. Because its activity can either determine or reflect fuel preference (carbohydrate versus fat), the pyruvate dehydrogenase complex (PDC) occupies a pivotal position in fuel cross-talk. Active PDC permits glucose oxidation and allows the formation of mitochondrially derived intermediates (e.g. malonyl-CoA and citrate) that reflect fuel abundance. FA oxidation suppresses PDC activity. PDC inactivation by phosphorylation is catalysed by pyruvate dehydrogenase kinases (PDKs) 1-4, which are regulated differentially by metabolite effectors. Most tissues contain at least two and often three of the PDK isoforms. We develop the hypothesis that PDK4 is a "lipid status"-responsive PDK isoform facilitating FA oxidation and signalling through citrate formation. Substrate interactions at the level of gene transcription extend glucose-FA interactions to the longer term. We discuss potential targets for substrate-mediated transcriptional regulation in relation to selective PDK isoform expression and the influence of altered PDK isoform expression in fuel sensing, selection and utilization.

Early growth retardation induced by excessive exposure to glucocorticoids in utero selectively increases cardiac GLUT1 protein expression and Akt/protein kinase B activity in adulthood.

In the rat, dexamethasone treatment during late pregnancy leads to intrauterine growth retardation and is used as a model of early programming of adult onset disease. The present study investigated whether pre-natal dexamethasone treatment modifies cardiac glucose transporter (GLUT) protein expression in adulthood and identified signalling pathways involved in the response. Dexamethasone (100 microg/kg body wt per day) administered via an osmotic pump to pregnant rats (day 15 to day 21; term=22 to 23 days) reduced fetal weight at day 21 and caused hypertension, hyperinsulinaemia and elevated corticosterone levels in the adult (24-week-old) male offspring. Cardiac GLUT1 protein expression was selectively up-regulated (2.5-fold; P<0.001), in the absence of altered cardiac GLUT4 protein expression, in adult male offspring of dexamethasone-treated dams. Maternal dexamethasone treatment did not influence cardiac GLUT1 protein expression during fetal or early post-natal life. We examined potential regulatory signalling proteins that might mediate up-regulation of cardiac GLUT1 protein expression in adulthood. We observed marked (2.2-fold; P<0.01) activation of Akt/protein kinase B (PKB), together with modest activation of the anti-apoptotic protein kinase C (PKC) isoforms PKC alpha (88%, P<0.05) and PKC epsilon (56%, P<0.05) in hearts of the early-growth-retarded male offspring. These effects were, however, observed in conjunction with up-regulation of cardiac protein expression of PKC beta(1) (191%, P<0.01), PKC beta(2) (49%, P<0.05) and PKC delta (35%; P<0.01), effects that may have adverse consequences. Maternal dexamethasone treatment was without effect on cardiac extracellular signal-related kinase (ERK) 1 or ERK2 activity in adulthood. In conclusion, our data demonstrate an effect of maternal dexamethasone treatment to up-regulate cardiac GLUT1 protein expression in early-growth-retarded, hypertensive, hyperinsulinaemic adult male offspring, an effect observed in conjunction with activation of Akt/PKB.