Total pyruvate dehydrogenase complex activity is low in white adipose tissue during the suckling period and increases markedly at weaning on to a high-carbohydrate diet. This is concomitant with an increase in the E1 alpha, E1 beta and E2 subunit protein concentration and their respective mRNAs, suggesting a pretranslational control of this phenomenon. The most marked change is seen for the E1 alpha subunit (17-fold increase in protein concentration). The changes in pyruvate dehydrogenase complex activity and subunit abundance induced by weaning on to a high-carbohydrate diet are precluded if the animals are weaned on to a high-fat diet, suggesting that the nutritional and/or related hormonal changes rather than a developmental stage are responsible for the observed adipose-tissue pyruvate dehydrogenase complex pattern.

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor of the factor VIIa-tissue factor complex of blood coagulation. The primary amino acid sequence of canine TFPI has been deduced from cDNA sequences obtained using the techniques of reverse transcription followed by amplification using PCR and conventional screening of a canine endothelial cell cDNA library. The open reading frame for canine TFPI encodes a signal peptide of 28 amino acids followed by a 40.7 kDa protein of 368 amino acids. Similar to human, rat and rabbit TFPI, canine TFPI contains a negatively-charged cluster of amino acids at its mature amino-terminus, followed by three Kunitz-type proteinase inhibitory domains and a cluster of positively-charged amino acids near its carboxy-terminus. In contrast to other TFPIs, following its second Kunitz-type proteinase inhibitory domain canine TFPI contains an additional amino acid insert which includes a nanomeric peptide-sequence repeated six times. Recombinant canine TFPI was expressed in both bacterial- and insect cell-expression systems for functional analysis and the generation of antibodies. The recombinant canine TFPI inhibits tissue factor-induced coagulation in an in vitro canine system. Immunoprecipitation of TFPI from canine plasma, followed by Western-blot analysis, tentatively identifies canine TFPI as an 80,000 kDa protein. Anti-peptide antibodies raised to the nanomeric peptide repeat immunoprecipitate an identical, cross-reactive, 80,000 kDa protein.

The enzyme activity and the expression (protein and mRNA concentrations) of genes encoding for hepatic carnitine palmitoyl-transferases (CPT) I and II were studied during neonatal development, in response to nutritional state at weaning and during the fed-starved transition in adult rats. The activity, the protein concentration and the level of mRNA encoding CPT I are low in foetal-rat liver and increase 5-fold during the first day of extra-uterine life. The activity and gene expression of CPT I are high during the entire suckling period, in the liver of 30-day-old rats weaned at 20 days on to a high-fat diet and in the liver of 48 h-starved adult rats. The activity and CPT I gene expression are markedly decreased in the liver of rats weaned on to a high-carbohydrate diet. By contrast, the activity, the protein concentration and the level of mRNA encoding CPT II are already high in the liver of term foetuses and remain at this level throughout the suckling period, irrespective of the nutritional state of the animals either at weaning or in the adult.

In newborn-pig hepatocytes, the rate of oleate oxidation is extremely low, despite a very low malonyl-CoA concentration. By contrast, the sensitivity of carnitine palmitoyltransferase (CPT) I to malonyl-CoA inhibition is high, as suggested by the very low concentration of malonyl-CoA required for 50% inhibition of CPT I (IC50). The rates of oleate oxidation and ketogenesis are respectively 70 and 80% lower in mitochondria isolated from newborn-pig liver than from starved-adult-rat liver mitochondria. Using polarographic measurements, we showed that the oxidation of oleoyl-CoA and palmitoyl-L-carnitine is very low when the acetyl-CoA produced is channelled into the hydroxymethylglutaryl-CoA (HMG-CoA) pathway by addition of malonate. In contrast, the oxidation of the same substrates is high when the acetyl-CoA produced is directed towards the citric acid cycle by addition of malate. We demonstrate that the limitation of ketogenesis in newborn-pig liver is due to a very low amount and activity of mitochondrial HMG-CoA synthase as compared with rat liver mitochondria, and suggest that this could promote the accumulation of acetyl-CoA and/or beta-oxidation products that in turn would decrease the overall rate of fatty acid oxidation in newborn- and adult-pig livers.

The glucose transporter GLUT2 is expressed predominantly in the liver. Previous studies have shown that glucose increases GLUT2 mRNA concentration in primary cultures of rat hepatocytes. Since insulin controls the glucose metabolism in the liver, it could be involved in the regulation of GLUT2 gene expression. In vivo, hyperinsulinaemia induced a transient inhibitory effect on liver GLUT2 gene expression, the maximal inhibition of GLUT2 mRNA concentration (93 +/- 6%) being observed after 6 h. When hyperglycaemia was associated with hyperinsulinaemia, the decrease in liver GLUT2 mRNA concentration was partially prevented. The respective effects of glucose and insulin were studied in vitro by primary culture of rat hepatocytes. Insulin alone exerted a transient inhibitory effect on GLUT2 mRNA concentration. When insulin and glucose (10-20 mM) were associated, the stimulatory effect of glucose on GLUT2 gene expression was predominant. In conclusion, the present study shows that GLUT2 mRNA concentration was conversely regulated by insulin and glucose, both in vitro and in vivo.

The tissue-specific expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene was studied in 15-day-old suckling rats. The mRNA and protein were present in liver, intestine and kidney, but were absent from brain, heart, skeletal muscles, brown and white adipose tissues. Kidney-cortex mitochondria from suckling rats were able to produce low amounts of ketone bodies from oleate. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels increased slowly during foetal life and markedly after birth. The postnatal increase in liver HMG-CoA synthase mRNA could be due to the increase in plasma glucagon levels, since it rapidly induced the accumulation of HMG-CoA synthase mRNA in cultured foetal hepatocytes. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels remained elevated throughout the suckling period or in rats weaned on to a high-fat carbohydrate-free diet (HF), but decreased by 50% in the liver and totally disappeared from the intestine and the kidney of rats weaned on to a high-carbohydrate low-fat diet (HC). When HC-weaned rats were fed on a HF-diet for a week, HMG-CoA synthase mRNA was re-induced in the intestine and the kidney. The role of hormones and nutrients in the regulation of HMG-CoA synthase gene expression is discussed.

We have studied the time course and relative effects of hypoinsulinaemia and hyperglycaemia on concentrations of uncoupling protein (UCP) and glucose transporter (GLUT4) and their mRNAs in brown adipose tissue (BAT) during the early phase of diabetes induced by streptozotocin. Two days after intravenous injection of streptozotocin, plasma insulin concentration was at its lowest and glycaemia was higher than 22 mmol/l. After 3 days, a 60% decrease in BAT UCP mRNA concentration and a 36% decrease in UCP was observed. Concomitantly, there was an 80% decrease in GLUT4 mRNA and a 44% decrease in GLUT4 levels. When hyperglycaemia was prevented by infusing phlorizin into diabetic rats, BAT UCP mRNA and protein levels were further decreased (respectively 90% and 60% lower than in control rats). In contrast, the marked decreases in GLUT4 mRNA and protein concentrations in BAT were similar in hyperglycaemic and normoglycaemic diabetic rats. Infusion of physiological amounts of insulin restored normoglycaemia in diabetic rats, and BAT UCP and GLUT4 mRNA and protein concentrations were maintained at the level of control rats. When insulin infusion was stopped, a 75% decrease in BAT UCP mRNA level and a 75% decrease in GLUT4 mRNA level were observed after 24 h, but UCP and GLUT4 concentrations did not decrease. This study shows that insulin plays an important role in the regulation of UCP and GLUT4 mRNA and protein concentrations in BAT. Hyperglycaemia partially prevents the rapid decrease in concentration of UCP and its mRNA observed in insulinopenic diabetes whereas it did not affect the decrease in GLUT4 mRNA and protein concentration. It is suggested that UCP is produced by a glucose-dependent gene.

GLUT-2, glucokinase (GK) and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was studied in the liver of chronically catheterized diabetic rats during the 3 days after an intravenous injection of 65 mg of streptozotocin (STZ)/kg. At 6 h after the STZ injection, portal plasma insulin levels were 270 +/- 32 mu-units/ml and blood glucose was 1.4 +/- 0.4 mmol/l, owing to pancreatic beta-cell destruction. GLUT-2 and PEPCK mRNA concentrations were rapidly and dramatically decreased (> 90%), whereas GK mRNA was increased. After 30 h, plasma insulin concentrations were lower than 5 mu-units/ml and blood glucose was > 20 mmol/l. GLUT-2 and PEPCK mRNA concentrations increased 2-fold and GK mRNA disappeared progressively. In order to assess the relative roles of hyperglycaemia and insulinopenia, blood glucose was clamped at 6.4 +/- 0.5 mmol/l from 18 to 72 h after STZ injection by phlorizin infusion (0.5-2 g/day per kg) or at 6.6 +/- 0.3 mmol/l from 18 to 48 h after STZ injection by insulin infusion (0.25 unit/min per kg). GLUT-2 mRNA concentrations were 50% lower in phlorizin-infused than in untreated diabetic rats. The low levels of GK mRNA and the high levels of PEPCK mRNA were unaffected by normalization of hyperglycaemia in phlorizin-infused diabetic rats. In insulin-infused rats (portal plasma insulin levels of 40 mu-units/ml) GLUT-2 mRNA levels were 25% of those in untreated diabetic rats, and they increased rapidly 6 h after insulin infusion was stopped. Liver GLUT-2 protein concentration showed similar changes in response to STZ injection and to phlorizin or insulin treatment, but after a delay of several hours. From this work we conclude that GLUT-2 gene expression is dramatically and rapidly (< 6 h) decreased by portal hyperinsulinaemia and increased by hyperglycaemia.

The presence of type I insulin-like growth factor (IGF-I) receptors on placental membranes led to the hypothesis that these receptors might play a critical role in the rapid growth of this organ. Diabetes induces feto-placental overgrowth, but it is not known whether it modifies IGF-I receptor activity in fetal and/or placental tissues. To answer this question, we have partially purified and characterized placental receptors from normal and streptozotocin-induced diabetic rats. In normal rats, binding of 125I-IGF-I to a 140 kDa protein corresponding to the alpha subunit of the receptor was observed in cross-linking experiments performed under reducing conditions. Stimulation by IGF-I induces the autophosphorylation of a 105 kDa phosphoprotein representing the beta subunit of the receptor. In rats made hyperglycaemic and insulinopenic by streptozotocin injection on day 1 of pregnancy, placental IGF-I receptor-binding parameters were not different from controls on day 20 of pregnancy. In contrast, the autophosphorylation and kinase activity of IGF-I receptors of diabetic rats were increased 2-3-fold in the basal state and after IGF-I stimulation. The present study indicates that the rat placental IGF-I receptor possesses structural characteristics similar to that reported for fetal-rat muscle, and suggests that the high-molecular-mass beta subunit could represent a type of receptor specifically expressed during prenatal development. In addition, it clearly demonstrates that diabetes induces functional alterations in IGF-I receptor kinase activity that may play a major role in the placental overgrowth in diabetic pregnancy.

Previous experiments have shown that insulin-induced glucose utilization is increased in white adipose tissue of young obese Zucker rats. We have investigated the possible role of over-expression of the muscle/fat glucose transporter (Glut 4) and key lipogenic enzymes in this increased insulin-responsiveness. The amount or activity and the mRNA concentrations of Glut 4, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were measured before and after weaning in white adipose tissue of obese and lean Zucker rats. Comparison of the levels of Glut 4 and lipogenic-enzyme expression in 15-day-old suckling and 30-day-old weaned rats on a high-carbohydrate diet shows a marked increase in the latter group. The increase was, in lean and obese rats respectively, 6- and 7-fold for the amount of Glut 4 and 2- and 3-fold for its mRNA concentrations, 40- and 100-fold for the activity of lipogenic enzymes (FAS and ACC) and 30- and 10-fold for their mRNA concentrations. Furthermore, all these parameters, except the amount of Glut 4, were 2-5-fold higher in obese rats, both before and after weaning. Changes at weaning were largely blunted when rats were weaned on to a high-fat diet, although the differences between lean and obese rats persisted, and even became significant for the amount of Glut 4. Whatever the experimental conditions, plasma insulin levels were significantly higher in obese than in lean rats. These results indicate the existence of an enhanced expression of Glut 4, FAS and ACC in white adipose tissue of young obese fa/fa rats which could be related to the increased plasma insulin levels.

Glucokinase (EC 2.7.1.2) first appears in the liver of the rat 2 weeks after birth and increases after weaning on to a high-carbohydrate diet. We investigated the hormonal regulation of glucokinase (GK) mRNA in primary cultures of hepatocytes from 10-12-day-old suckling rats. GK mRNA was undetectable in such cells after 48 h of culture in serum-free medium devoid of hormones. Addition of insulin or tri-iodothyronine (T3) to the medium resulted in induction of GK mRNA. The effects of insulin and T3 were dose-dependent and additive. Dexamethasone alone did not induce GK mRNA, but enhanced the response to insulin and decreased the response to T3. Induction of GK mRNA by insulin was not affected when the medium glucose concentration was varied between 5 and 15 mM, nor when culture was conducted in glucose-free medium supplemented with lactate and pyruvate or galactose. The time course of initial accumulation of GK mRNA in response to insulin was characterized by a lag of 12 h and an induction plateau reached after 36 h. If hepatocytes were then withdrawn from insulin for 24 h and subsequently subjected to a secondary stimulation by insulin, GK mRNA re-accumulated with much faster kinetics and reached the fully induced level within 8 h. Both primary and secondary responses to insulin were abolished by actinomycin D. These results provide insight into the role of hormonal stimuli in the ontogenic development of hepatic glucokinase.

Lipoprotein-associated coagulation inhibitor (LACI) inhibits activated Factor X (Xa) directly and, in an Xa-dependent fashion, inhibits Factor VIIa-tissue factor (TF), presumably by forming a quaternary Xa-LACI-VIIa-TF complex. LACI isolated from the conditioned media of HepG2 cells grown in the presence of [32P]orthophosphate was observed to be covalently phosphorylated. Dephosphorylation of 32P-LACI with phosphatase resulted in an almost complete removal of the radiolabel. Phosphoamino acid analysis of the purified 32P-LACI established that the phosphorylation occurred on (a) serine residue(s). At its N-terminus, LACI contains a cluster of acidic residues C-terminal to the serine-2 residue. Such a site is characteristic of the sites phosphorylated by casein kinase II (CKII) in protein substrates. Edman degradation of endogenously labelled 32P-LACI revealed that the serine-2 residue was a major site of phosphorylation. Phosphorylation of purified LACI by bovine CKII was observed to occur in vitro; amino acid sequence analysis demonstrated that CKII phosphorylated LACI at the serine-2 residue. Recombinant LACI expressed from mouse C127 fibroblasts transfected using a bovine-papilloma-virus expression vector was found to be endogenously phosphorylated. By using site-directed mutagenesis, an altered form of LACI was produced in which the serine-2 residue had been changed to alanine. This altered LACI, although expressed in similar quantity to the wild-type LACI, was not detectably phosphorylated. Using the altered LACI in functional studies demonstrated that a serine residue at position 2, and thus the phosphorylation of this site, was not essential for LACI‘s inhibition of Xa and VIIa-TF activities.

A gastric [U-14C]glucose load (4.8 mg/g body wt.) was delivered to unrestrained post-absorptive or 30 h-starved rats bearing peripheral and portal vein catheters and continuously perfused with [3-3H]glucose, in order to compare their metabolic and hormonal responses. In the basal state, portal and peripheral glycaemia were less in starved rats than in rats in the post-absorptive period (P less than 0.01), whereas blood lactate was similar. Portal insulinaemia (P less than 0.05) and protal glucagonaemia (P less than 0.005) were lower in starved rats, but insulin/glucagon ratio was higher in post-absorptive rats (P less than 0.005). The glucose turnover rate was decreased by starvation (P less than 0.005). After glucose ingestion, blood glucose was similar in post-absorptive and starved rats. A large portoperipheral gradient of lactate appeared in starved rats. Portal insulinaemia reached a peak at 9 min, and was respectively 454 +/- 68 and 740 +/- 65 mu-units/ml in starved and post-absorptive rats. Portal glucagonaemia remained stable, but was higher in post-absorptive rats (P less than 0.05). At 60 min after the gastric glucose load, 30% of the glucose was delivered at the periphery in both groups. The total glucose appearance rate was higher in starved rats (P less than 0.05), as was the glucose utilization rate (P less than 0.05), whereas the rate of appearance of exogenous glucose was similar. This was due to a non-suppressed hepatic glucose production in the starved rats, whereas it was totally suppressed in post-absorptive rats. At 1 h after the glucose load, the increase in both liver and muscle glycogen concentration was greater in starved rats. Thus short-term fasting induces an increased portal lactate concentration after a glucose load, and produces a state of liver insulin unresponsiveness for glucose production, whereas the sensitivity of peripheral tissues for glucose utilization is unchanged or even increased. This might allow preferential replenishment of the peripheral stores of glycogen.

The expression of different glucose transporter isoforms was measured during the development and differentiation of the rat mammary gland. Before conception, when the mammary gland is mainly composed of adipocytes, Glut 4 and Glut 1 mRNAs and proteins were present. During pregnancy, the expression of Glut 4 decreased progressively, whereas that of Glut 1 increased. In the lactating mammary gland only Glut 1 was present, and was expressed at a high level. The absence of Glut 4 suggests that glucose transport is not regulated by insulin in the lactating rat mammary gland.

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.

The changes of insulin responsiveness of white adipose tissue during the suckling-weaning transition in the rat were investigated in vitro on isolated adipocytes. Insulin binding, glucose transport and glucose metabolism in adipocytes from suckling rats and from rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet were compared. Despite similar insulin binding, insulin-stimulated glucose transport rate is lower in adipocytes from suckling rats and HF-weaned rats than in adipocytes from HC-weaned rats. Moreover, whereas insulin markedly stimulates glucose metabolism in adipocytes from HC-weaned rats, glucose metabolism is totally unresponsive to insulin in adipocytes from suckling and HF-weaned rats. This insulin resistance is associated with a very low rate of lipogenesis and low activities of acetyl-CoA carboxylase, fatty acid synthase and pyruvate dehydrogenase.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.

The insulin-resistant obese fa/fa rat is a convenient model in which to study a potential effect of metformin, a biguanide used in the treatment of non-insulin-dependent diabetes, on insulin-mediated glucose utilization. Female fa/fa rats were given metformin orally for 8 days. Studies were performed on anaesthetized post-absorptive rats 5 h after the last dose of metformin. Glucose production and utilization were enhanced 1.5-fold in metformin-treated rats. The enhanced glucose production was almost entirely due to increased glucose recycling. The digestive tract was the only tissue responsible for the enhanced glucose utilization.

The effect of insulin on glucose metabolism in mammary gland was studied by the euglycaemic/hyperinsulinaemic-clamp technique. Measurement of metabolite concentrations and enzyme activities in the mammary gland suggests two sites of action of insulin: phosphofructokinase-1 and acetyl-coA carboxylase. The increase in phosphofructokinase-1 activity could be linked to the 2-fold increase in fructose 2,6-bisphosphate concentration, since no change in maximal activity and in sensitivity of the enzyme toward fructose 6-phosphate was detected in vitro.

This study was undertaken to determine the factors involved in the development of insulin-sensitivity at weaning. Glucose kinetics were studied in suckling rats and in rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet, in the basal state and during euglycaemic-hyperinsulinaemic-clamp studies. These studies were coupled with the 2-deoxyglucose technique, allowing a measure of glucose utilization by individual tissues. In the basal state, the glycaemia was higher in HF-weaned rats (124 +/- 4 mg/dl) than in suckling (109 +/- 1 mg/dl) and HC-weaned rats (101 +/- 3 mg/dl). Glucose turnover rates were similar in the three groups of animals (14 mg/min per kg). Nevertheless, basal metabolic glucose clearance rate was 20% lower in HF-weaned rats than in the other groups. During the euglycaemic-hyperinsulinaemic experiments, hepatic glucose production was suppressed by 90% in HC-weaned rats, whereas it remained at 40% of basal value in suckling and HF-weaned rats, indicating an insulin resistance of liver of these animals. Glucose clearance rate during the clamp was 18.3 +/- 0.9 ml/min per kg in suckling rats, whereas it was 35.3 +/- 1.2 ml/min per kg in HC-weaned rats and 27.8 +/- 1.1 ml/min per kg in HF-weaned rats, indicating an insulin resistance of glucose utilization in suckling, and to a lower extent, in HF-weaned rats. The deoxyglucose technique showed that peripheral insulin resistance was localized in muscles and white adipose tissue of suckling and HF-weaned rats. These results indicate that the switch from milk to a HC diet is an important determinant of the development of insulin-sensitivity at weaning in the rat.

Fatty acid oxidation and synthesis were studied in isolated hepatocytes from adult rats adapted for 44 days on low-fat, high-carbohydrate (LF), diet or high-fat diets, composed of long-chain (LCT) or medium-chain (MCT) triacylglycerols. The rates of [1-14C]octanoate oxidation were almost similar in each group studied, whereas the oxidation of [1-14C]oleate was 50% lower in the LF group than in animals adapted to high-fat diets. The rates of oleate oxidation are inversely correlated with the rates of lipogenesis. However, it seems unlikely that [malonyl-CoA] itself represents the sole mechanism involved in the regulation of oleate oxidation during long-term LCT or MCT feeding, since: (1) despite a 3-fold higher concentration of malonyl-CoA in MCT-fed rats than in LCT-fed ones, the rates of oleate oxidation are similar; (2) when malonyl-CoA concentration is increased after stimulation of lipogenesis (by adding lactate + pyruvate) in MCT-fed rats, to a level comparable with that of the LF group, the rate of oleate oxidation remains 55% higher than that measured under similar conditions in the LF-fed rats; (3) in the LF group, the 90% decrease in malonyl-CoA concentration [by 5-(tetradecyloxy)-2-furoic acid] is not associated with a stimulation of oleate oxidation. By contrast, the sensitivity of carnitine palmitoyltransferase I (CPT I) to malonyl-CoA is markedly decreased in the LCT- and MCT-fed rats, by 90% and 70% respectively. The relevance of this decrease in the sensitivity of CPT I is discussed.

The effects of fasting on glucose metabolism in the conscious resting rat were studied. Fasting decreased whole-body glucose utilization by 40%. The fast induced a decrease in glucose utilization in muscles which are constantly working even in the resting state, i.e. heart, diaphragm and postural muscles. No modification was observed in other tissues.

Brown-adipose-tissue glucose utilization rate and its insulin-sensitivity were measured in vivo in the anaesthetized rat by a 2-deoxy[1-3H]glucose technique. Glucose utilization can be increased 60-fold by insulin, to reach extremely high rates. Glucose utilization and its insulin-sensitivity are modulated in accordance with physiological or pathological conditions.

A quantitative method allowing determination of glucose metabolism in vivo in muscles and white adipose tissue of the anaesthetized rat is presented. A tracer dose of 2-deoxy[3H]glucose was injected intravenously in an anaesthetized rat and the concentration of 2-deoxy[3H]glucose was monitored in arterial blood. After 30-80 min, three muscles, the soleus, the extensor digitorum longus and the epitrochlearis, periovarian white adipose tissue and brain were sampled and analysed for their content of 2-deoxy[3H]glucose 6-phosphate. This content could be related to glucose utilization during the same time period, since (1) the integral of the decrease of 2-deoxy[3H]glucose in arterial blood was known and (2) correction factors for the analogue effect of 2-deoxyglucose compared with glucose in the transport and phosphorylation steps were determined from experiments in vitro. Glucose utilization was then measured by this technique in the tissues of post-absorptive rats in the basal state (0.1 munit of insulin/ml of plasma) or during euglycaemic-hyperinsulinaemic glucose clamp (8 munits of insulin/ml of plasma) and of 48 h-starved rats. Results corresponded qualitatively and quantitatively to the known physiological characteristics of the tissues studied.

Insulin action on glucose transport and metabolism was studied in paraovarian adipocytes from 3-month-old female rats and compared with insulin action in epididymal adipocytes from closely age-matched males. At maximal insulin concentrations the stimulations of 2-deoxyglucose uptake (4-fold the basal value) and of [U-14C]glucose incorporation into CO2 and total lipids (3- and 2-fold the basal values respectively) were similar in adipocytes from rats of both sexes. At submaximal insulin concentrations (less than 0.2 nM) the ability of paraovarian adipocytes to transport and to metabolize glucose was higher than that of epididymal adipocytes; accordingly an increase in insulin binding was observed in paraovarian adipocytes as compared with epididymal adipocytes. These results show that paraovarian adipocytes from mature female rats were highly responsive to insulin, and exhibited a higher sensitivity to the hormone than did epididymal adipocytes from male rats of the same age.

Under basal conditions (zero insulin), paraovarian adipocytes from 19-day-pregnant rats exhibited the same rates of [U-14C]glucose conversion into CO2 and total lipids as did those from age-matched virgin rats. The dose-response curves for insulin stimulation of glucose metabolism were similar in both groups: maximal response (+100% over basal values) and high sensitivity (half-maximal effect at 0.05 nM-insulin). The present results suggest that the insulin resistance in vivo that occurs during late pregnancy may involve circulating factors lost in vitro.

The development of fatty acid metabolism was studied in isolated hepatocytes from newborn rats. Ketone-body production from oleate is increased 6-fold between 0 and 16 h after birth. This increase is related to an enhanced beta-oxidation rather than to a channeling of acetyl-CoA from the tricarboxylic acid cycle to ketone-body synthesis. The increase in oleate oxidation is not related to a decreased esterification rate, as the latter is already low at birth and does not decrease further. At birth, lipogenic rate is 2-3-fold lower than in fed adult rats and it decreases to undetectable values in 16 h-old rats. A 90% inhibition of lipogenesis in hepatocytes of newborn rats (0 h) by glucagon and 5-(tetradecyloxy)-2-furoic acid does not lead to an increased oxidation of non-esterified fatty acids. This suggests that the inverse relationship between lipogenesis and ketogenesis in the starved newborn rat is not responsible for the switch-on of fatty acid oxidation at birth. Moreover, ketogenesis from octanoate, a medium-chain fatty acid the oxidation of which is independent of carnitine acyltransferase, follows the same developmental pattern at birth as that from oleate.

In hepatocytes isolated from 48 h-old starved of suckling newborn pigs or from 15-day-old starved piglets, the rate of ketogenesis from oleate or from octanoate is very low. This is not due to an inappropriate fatty acid uptake by the isolated liver cells, but results from a limited capacity for fatty acid oxidation. Some 80-95% of oleate taken up is converted into esterified fats, whatever the age or the nutritional conditions. Three lines of indirect evidences suggest that fatty acid oxidation is not controlled primarily by malonyl-CoA concentration in newborn pig liver. Firstly, the addition of glucagon does not increase fatty acid oxidation or ketogenesis. Secondly, the rate of lipogenesis is very low in isolated hepatocytes from newborn pigs. Thirdly, the rates of oxidation and ketogenesis from octanoate are also decreased in isolated hepatocytes from newborn and young piglets. The huge rate of esterification of fatty acids in the liver of the newborn pigs probably represents a species-specific difference in intrahepatic fatty acid metabolism.

In hepatocytes from 1-day-old rats, active gluconeogenesis occurs in parallel with active ketogenesis, although the carbon atoms of non-esterified fatty acids do not participate in glucose synthesis. Once a significant ketogenesis is established, a further increase does not enhance gluconeogenesis. Indeed, octanoate is more ketogenic than oleate, but stimulates gluconeogenesis to a similar extent.

Metabolic interactions between fatty acid oxidation and gluconeogenesis were investigated in vivo in 16h-old newborn rats under various nutritional states. As the newborn rat has no white adipose tissue, starvation from birth induces a low rate of hepatic fatty acid oxidation. Hepatic gluconeogenesis in inhibited in the starved newborn rat when compared with the suckling rat, which receives fatty acids through the milk, at the steps catalysed by pyruvate carboxylase and glyceraldehyde 3-phosphate dehydrogenase. These inhibitions are rapidly reversed by triacylglycerol feeding. Inhibition of fatty acid oxidation by pent-4-enoate in the suckling animal mimics the effect of starvation on the pattern of hepatic gluconeogenic metabolites. It is concluded that, in the newborn rat in vivo, hepatic fatty acids oxidation can increase the gluconeogenic flux by providing the acetyl-CoA necessary for the reaction catalysed by pyruvate carboxylase and the reducing equivalents (NADH) to displace the reversible reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase in the direction of gluconeogenesis.

Inhibition of fatty acid oxidation with pent-4-enoate in suckling newborn rats caused a fall in blood [glucose] and blood [ketone bodies] and inhibition of gluconeogenesis from lactate. Glucose utilization was not increased in newborn rats injected with pent-4-enoate. Active fatty acid oxidation appears to be essential to support gluconeogenesis and to maintain normal blood [glucose] in suckling newborn rats.

1. The injection of L-alanine (50-100 mg/kg) into 35-day-old rats that had been starveed for 48 h increased blood L-alanine concentration to values observed in fed animals and lowered the blood concentration of 3-hydroxybutyrate within 2 min. 2. This hypoketon aemic action of L-alanine was specific for 3-hydroxybutyrate, since the acetoacetate concentrations did not change significantly. 3. The decrease in 3-hydroxybutyrate elicited by L-alanine was not related to changes in the blood concentrations of insulin, glucagon, growth hormone, glucose, unesterified fatty acids, lactate or pyruvate. 4. The injection of L-alanine resulted in a decrease in total ketones that was apparently unrelated to their increased peripheral utilization. These results are interpreted as an anti-ketogenic action of L-alanine. 5. The data suggest that L-alamine lowers ketone-body formation in starved rats, possibly via an alteration in hepatic redox equilibrium.

Inhibition of gluconeogenesis with 3-mercaptopicolinate in suckling newborn rats caused a fall in blood [glucose], but no change in their high plasma [free fatty acid] and blood [ketone bodies]. Active gluconeogenesis seems to be more important than sparing of glucose by high concentrations of fat-derived substrates for the maintenance of normal blood [glucose] in suckling newborn rats.