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.
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MC Sugden and MJ Holness
ML Langdown, MJ Holness, and MC Sugden
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.
LG Fryer, MJ Holness, and MC Sugden
Adipose-tissue lipolysis (assessed from glycerol release) and glucose uptake were examined in parametrial and mesenteric adipocytes prepared from control or hyperthyroid rats in relation to changes in insulin sensitivity. Basal rates of lipolysis did not differ significantly between adipose-tissue depots. Lipolysis was maximally stimulated by noradrenaline at 1 microM, half-maximal anti-lipolytic effects of insulin were observed at approximately 11 microU/ ml insulin, and half-maximal stimulation of glucose uptake was observed at approximately 16 microU/ml insulin in adipocytes from both depots. Wortmannin caused a dose-dependent inhibition of the anti-lipolytic effect of insulin (150 microU/ml) on noradrenaline-stimulated lipolysis. Half-maximal effects of wortmannin were observed at 20-40 nM. The p70S6K inhibitor rapamycin and the mitogen-activated protein kinase kinase inhibitor PD098059 had no effects on noradrenaline-stimulated lipolysis. Hyperthyroidism increased basal rates of lipolysis and the maximal response of lipolysis to noradrenaline stimulation (3.1-fold, P < 0.001 and 2.1-fold, P < 0.05 respectively) in parametrial adipocytes. Hyperthyroidism markedly blunted the sensitivity of noradrenaline-stimulated lipolysis to half-maximal suppression by insulin in both parametrial and mesenteric adipocyte depots, and noradrenaline-stimulated lipolysis at a maximal insulin concentration remained significantly higher in adipocytes prepared from hyperthyroid rats compared with controls. Hyperthyroidism had no effect on basal and little effect on insulin-stimulated glucose uptake. Tri-iodothyronine administered at a low dose selectively influenced the anti-lipolytic action of insulin in parametrial adipocytes, and led to significantly less marked elevation in plasma non-esterified fatty acid concentrations in vivo. The results demonstrate a selective effect of hyperthyroidism to impair insulin's anti-lipolytic action, and are consistent with the operation of different downstream signalling mechanisms for the effects of insulin on adipocyte glucose transport and lipolysis.
MC Sugden, HS Lall, RA Harris, and MJ Holness
The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.
LG Fryer, MJ Holness, JB Decock, and MC Sugden
There is evidence for a role of protein kinase C (PKC) in the development of cardiac hypertrophy. We examined the expression of individual PKC isoforms in the adult rat heart in two distinct, well-characterised in vivo models of cardiac hypertrophy associated with an activated cardiac renin-angiotensin system, namely experimental hyperthyroidism and the TGR(mRen2)27 rat. The cardiac expression of a range of PKC isoforms (PKC-alpha, PKC-omega, PKC-epsilon, PKC-gamma, and PKC-tau) was examined by immuno-blotting. Our work demonstrates that the expression of total cardiac nPKC-omega and nPKC-epsilon relative to protein is selectively and differentially modified in these models. A consistent up-regulation of nPKC-omega in conjunction with overall down-regulation of nPKC-epsilon was observed in both models. The expression of other PKC isoforms was unaffected. The divergent responses of the expression of the two nPKC isoforms to an activated cardiac renin-angiotensin system in vivo in adulthood suggest that these individual nPKC isoforms subserve specific roles in the response.
MJ Holness, ND Smith, GK Greenwood, and MC Sugden
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.