Françoise Assimacopoulos-Jeannet

Uncoupling Protein-3 Expression in Rodent Skeletal Muscle Is Modulated by Food Intake but Not by Changes in Environmental Temperature

A new member of the uncoupling protein (UCP) family called UCP3 has recently been cloned and shown to be highly expressed in skeletal muscle of rodents and humans. In the present study, UCP3 was overexpressed in C2C12myoblasts where it acts as an uncoupling protein. Changes in UCP3 mRNA expression were examined in rodent muscles under conditions known to modulate thermogenesis in brown adipose tissue. In skeletal muscle, UCP3 expression did not change in response to 48 h of cold exposure (6 °C), whereas it was decreased by 81% or increased 5.6-fold by 1 week of 50% food restriction or fasting, respectively. It was also decreased by 36% in soleus muscle of obese (fa/fa) as compared with lean Zucker rats. The unexpected rise of UCP3 mRNA level induced by fasting did not change in vitro muscle basal heat production rate but decreased by 31% the capacity to produce heat in response to the uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone. This decrease may reflect underlying uncoupling by UCP3. Up-regulation of UCP3 mRNA after a 24-h fast was still observed in mice exposed at thermoneutrality. These results show that the increase in UCP3 expression induced by fasting is associated with the maintenance of thermogenesis measured in muscle in vitro and is not modulated by environmental temperature. The notion that UCP3 expression is modulated by food intake is of importance to better understand the pathophysiology of obesity in humans.

Modulation of obese gene expression in rat brown and white adipose tissues

The ob gene mRNA expression in rat brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) was measured on Northern blots hybridized with a rat ob gene probe. The level of ob gene mRNA in BAT was about 40% of that in WAT. Fasting (36 h) or semi‐starvation (10 days) decreased the ob gene mRNA level in both tissues by 62–68%, and cold exposure at 6°C (24 h) decreased it in BAT (−84%)_but not in WAT. Acute administration of the β 3‐adrenergic agonist Ro 16‐8714 decreased the ob gene mRNA level in BAT (−51%) and WAT (−28%) of lean Zucker rats and only in BAT (−74%) of obese falfa rats. This study demonstrates that, in the rat, the ob gene is not only expressed in WAT but also in BAT, and suggests that in these two tissues, the modulation of the ob gene expression might be more closely associated with known alterations in cell lipid content than with changes in sympathetic activity.

Evidence for an anaplerotic/malonyl-CoA pathway in pancreatic beta-cell nutrient signaling.

A metabolic model of fuel sensing has been proposed in which malonyl-CoA and long-chain acyl-CoA esters may act as coupling factors in nutrient-induced insulin release (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney J, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802–5810, 1992). To gain further insight into the control of malonyl-CoA content in islet tissue, we have studied the short- and long-term regulation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the β-cell. These enzymes catalyze the formation of malonyl-CoA and its usage for de novo fatty acid biogenesis. ACC mRNA, protein, and enzymatic activity are present at appreciable levels in rat pancreatic islets and clonal β-cells (HIT cells). Glucose addition to HIT cells results in a marked increase in ACC activity that precedes the initiation of insulin release. Fasting does not modify the ACC content of islets, whereas it markedly downregulates that of lipogenic tissues. This indicates differential regulation of the ACC gene in lipogenic tissues and the islets of Langerhans. FAS is very poorly expressed in islet tissue, yet ACC is abundant. This demonstrates that the primary function of malonyl-CoA in the β-cells is to regulate fatty acid oxidation, not to serve as a substrate for fatty acid biosynthesis. The anaplerotic enzyme pyruvate carboxylase, which allows the replenishment of citric acid cycle intermediates needed for malonyl-CoA production via citrate, is abundant in islet tissue. Glucose causes an elevation in β (HIT)-cell citrate that precedes secretion, and only those nutrients that can elevate citrate induce effective insulin release. The results provide new evidence in support of the model and explain why malonyl-CoA rises markedly and rapidly in islets upon glucose stimulation: 1) glucose elevates citrate, the precursor of malonyl-CoA; 2) glucose enhances ACC enzymatic activity; and 3) malonyl-CoA is not diverted to lipids. The data suggest that ACC is a key enzyme in metabolic signal transduction of the β-cell and provide evidence for the concept that an anaplerotic/malonyl-CoA pathway is implicated in insulin secretion.

Evidence for a Role of the Microtubular System in the Secretion of Newly Synthesized Albumin and Other Proteins by the Liver

Livers of normal mice were prefused in situ and the secretion of newly synthesized (i.e. labeled) proteins into the perfusate were measured. In control livers, the secretion of newly synthesized proteins was found to be linear with time. In marked contrast, when livers were perfused with vinblastine, vincristine, or colchicine, drugs known to interfere with the hepatic microtubular system, the release of newly synthesized proteins was either strongly inhibited or completely suppressed although total hepatic protein synthesis (estimated by the incorporation of labeled amino acids into hepatic plus perfusate proteins) remained unaltered. Chromatographic separation of the various secreted proteins showed that the release of albumin, globulins, and small polypeptides was decreased to a similar extent by vincristine or colchicine. In the particular case of albumin, it was further observed that total (i.e. liver plus perfusate) labeled amino acid incorporation into albumin was not altered by either vincristine or colchicine, whereas the incorporation of these amino acids into liver albumin was markedly increased but incorporation into perfusate albumin was decreased, suggesting that the translocation of this particular protein from the liver to the perfusate had been affected by the presence of these drugs. It is proposed that the functional integrity of microtubules is necessary for the intracellular movement and eventual release of albumin and other proteins by the liver, and suggested that microtubules might possibly be a site of regulation of hepatic protein secretion.

Lipid infusion lowers sympathetic nervous activity and leads to increased β-cell responsiveness to glucose

We investigated the possible involvement of the autonomic nervous system in the effect of a long-term elevation of plasma free fatty acid (FFA) concentration on glucose-induced insulin secretion (GIIS) in rats. Rats were infused with an emulsion of triglycerides (Intralipid) for 48 hours (IL rats). This resulted in a twofold increase in plasma FFA concentration. At the end of infusion, GIIS as reflected in the insulinogenic index (DeltaI/DeltaG) was 2.5-fold greater in IL rats compared with control saline-infused rats. The ratio of sympathetic to parasympathetic nervous activities was sharply decreased in IL rats relative to controls. GIIS was studied in the presence of increasing amounts of alpha- and beta-adrenoreceptor agonists and antagonists. The lowest concentrations of the alpha2A-adrenoreceptor agonist oxymetazoline, which were ineffective in control rats, reduced GIIS in IL rats. At the dose of 0.3 pmol/kg, GIIS became similar in IL and control rats. The use of beta-adrenoreceptor agonist (isoproterenol) or antagonist (propranolol) did not result in a significant alteration in GIIS in both groups. GIIS remained as high in IL vagotomized rats as in intact IL rats, indicating that changes in parasympathetic tone were of minor importance. Altogether, the data show that lipid infusion provokes beta-cell hyperresponsiveness in vivo, at least in part through changes in alpha2-adrenergic innervation.

Involvement of non-esterified fatty acid oxidation in glucocorticoid-induced peripheral insulin resistance in vivo in rats.

SummaryThe mechanism by which glucocorticoids induce insulin resistance was studied in normal rats administered for 2 days with corticosterone then tested by euglycaemic hyperinsulinaemic clamps. Corticosterone administration induced a slight hyperglycaemia, hyperinsulinaemia and increased non-esterified fatty acid levels. It impaired insulin-stimulated total glucose utilization (corticosterone 15.7±0.7; controls 24.6±0.8 mg·kg−1·min−1), as well as residual hepatic glucose production (corticosterone 4.9±1.0; controls 2.0±0.7 mg·kg−1·min−1). During the clamps, insulin did not decrease the elevated non-esterified fatty acid levels in corticosterone-administered rats (corticosterone 1.38±0.15, controls 0.22±0.04 mmol/l). Corticosterone administration decreased the in vivo insulin-stimulated glucose utilization index by individual muscles by 62±6%, and the de novo glycogen synthesis by 78±2% (n=8–9 muscles). GLUT4 protein and mRNA levels were either unchanged or slightly increased by corticosterone administration. Inhibition of lipid oxidation by etomoxir prevented corticosterone-induced muscle but not hepatic insulin resistance. In conclusion, glucocorticoid-induced muscle insulin resistance is due to excessive nonesterified fatty acid oxidation, possibly via increased glucose fatty-acid cycle ultimately inhibiting glucose transport, or via decreased glycogen synthesis, or by a direct effect on glucose transporter translocation or activity or both.

Lipid rather than glucose metabolism is implicated in altered insulin secretion caused by oleate in INS-1 cells

A comprehensive metabolic study was carried out to understand how chronic exposure of pancreatic β-cells to fatty acids causes high basal secretion and impairs glucose-induced insulin release. INS-1 β-cells were exposed to 0.4 mM oleate for 3 days and subsequently incubated at 5 or 25 mM glucose, after which various parameters were measured. Chronic oleate promoted triglyceride deposition, increased fatty acid oxidation and esterification, and reduced malonyl-CoA at low glucose in association with elevated basal O2 consumption and redox state. Oleate caused a modest (25%) reduction in glucose oxidation but did not affect glucose usage, the glucose 6-phosphate and citrate contents, and the activity of pyruvate dehydrogenase of INS-1 cells. Thus changes in glucose metabolism and a Randle-glucose/fatty acid cycle do not explain the altered secretory properties of β-cells exposed to fatty acids. The main response of INS-1 cells to chronic oleate, which is to increase the oxidation and esterification of fatty acids, may contribute to cause high basal insulin secretion via increased production of reducing equivalents and/or the generation of complex lipid messenger molecule(s).A comprehensive metabolic study was carried out to understand how chronic exposure of pancreatic beta-cells to fatty acids causes high basal secretion and impairs glucose-induced insulin release. INS-1 beta-cells were exposed to 0.4 mM oleate for 3 days and subsequently incubated at 5 or 25 mM glucose, after which various parameters were measured. Chronic oleate promoted triglyceride deposition, increased fatty acid oxidation and esterification, and reduced malonyl-CoA at low glucose in association with elevated basal O(2) consumption and redox state. Oleate caused a modest (25%) reduction in glucose oxidation but did not affect glucose usage, the glucose 6-phosphate and citrate contents, and the activity of pyruvate dehydrogenase of INS-1 cells. Thus changes in glucose metabolism and a Randle-glucose/fatty acid cycle do not explain the altered secretory properties of beta-cells exposed to fatty acids. The main response of INS-1 cells to chronic oleate, which is to increase the oxidation and esterification of fatty acids, may contribute to cause high basal insulin secretion via increased production of reducing equivalents and/or the generation of complex lipid messenger molecule(s).

In-vivo Effects of Hyperinsulinemia On Lipogenic Enzymes and Glucose-transporter Expression in Rat-liver and Adipose Tissues

Chronic hyperinsulinemia with maintenance of euglycemia was imposed on normal rats for 4 days. In white adipose tissue, hyperinsulinemia resulted in a twofold increase in GLUT4 protein and mRNA and a sixfold to 15-fold increase in fatty acid synthase (FAS) and acetyl coenzyme A (CoA) carboxylase (ACC) activity, respectively. Lipogenic enzyme mRNA was also markedly increased (20- to 30-fold). This was specific for white adipose tissue and was not observed in brown adipose tissue. In the liver, hyperinsulinemia was accompanied by a threefold increase in glucokinase (GK) activity and mRNA and by a threefold to fivefold increase in lipogenic enzyme activities and mRNA. In agreement with the changes in lipogenic activities, lipogenesis was markedly increased in white adipose tissue and liver of hyperinsulinemic rats. The data strongly suggest that in the rat, insulin is a driving force leading to increased lipid synthesis in liver and white adipose tissue.

Functional disconnection of brown adipose tissue in hypothalamic obesity in rats

The metabolic responses to electrical nerve stimulation, norepinephrine or octanoate additions were studied using continuous monitoring of NAD(P)H/NADP redox state by reflexion spectrophotometry of interscapular brown adipose tissues from control and ventromedial hypothalamic (VMH) lesioned rats. The responses to these stimuli were all greatly decreased already 3 days after VMH lesions, indicating a reduced cell capacity to oxidize free fatty acids. Measurements of interscapular brown adipose tissue composition 4–5 weeks after VMH lesions showed a decrease of both DNA concentration and total content, indicating some tissue involution.It is concluded that the involvement of the ventromedial bypothalamus in the activation of brown adipose tissue provided a possible anatomical clue concerning pathways connecting thermal and weight regulations.

Uncoupling protein-3 expression in skeletal muscle and free fatty acids in obesity

The newly identified mitochondrial uncoupling proteins UCP2 and UCP3, by contrast with UCP1, are highly expressed in human beings, UCP2 being ubiquitous and UCP3 specific to skeletal muscle. Because of its tissue distribution, UCP3 might have an important role in wholebody energy homoeostasis in human beings. Variations in its degree of expression might contribute to the interindividual variability in resting energy expenditure and in the energybalance dysregulation found in obesity and type 2 diabetes. We investigated whether mRNA expression of UCP2 and UCP3 in human vastus lateralis muscle was correlated with variables related to fat or glucose metabolism and with resting energy expenditure in a mixed population of obese patients and obese patients with type 2 diabetes. Nine obese, 10 h fasted patients, including three with type 2 diabetes (untreated but dietetically controlled, fasting plasma glucose of 8·1 to 10·5 mmol/L, table), volunteered, with written consent, to participate, which was accepted by the local Human Investigation Committee. We measured lean body mass with bioelectrical impedance. Plasma glucose concentrations were determined with a Beckman Glucose Analyzer II (Beckman Instruments, CA, USA), and plasma free fatty acids and insulin concentrations with commercial kits (Wako Chemicals GmbH, Germany, and Abbott, IL). Glucose uptake was measured with a euglycaemic hyperinsulinaemic clamp, and glucose and lipid oxidation rates, as well as resting energy expenditure, were determined by indirect calorimetry. The concentrations of UCP2 and UCP3 mRNA in vastus lateralis biopsy fragments were measured by northern blot. The expression of UCP2 and UCP3 mRNA normalised to the respective concentrations of 18S rRNA were compared with 12 physical, biological, and metabolic variables and analysed (Simple Pearson ProductMoment correlations). The mRNA expression of UCP3 was positively and linearly correlated with circulating free fatty acids (r=0·83; p=0·005), whereas that of UCP2 was not (r=0·40). When adjustments were made for age, percentage of fat mass, body-mass index, lean body mass and bodyweight, the correlation between UCP3 mRNA and concentrations of free fatty acids was stronger (r=0·99; p 0·05) was seen between UCP2 or UCP3 mRNA expression and age, percentage fat mass, body-mass index, lean body mass, resting energy expenditure per kg lean body mass, bodyweight, fasting plasma glucose, insulin concentrations, insulin-induced glucose uptake, glucose oxidation, and lipid oxidation. Free fatty acids might control muscle UCP3 expression. A 10-day severe calorie restriction increases muscle UCP2 and UCP3 mRNA expression, and in rodents infusion of free fatty acids increases muscle UCP3 mRNA. Our data suggest a role for muscle UCP3 in the metabolic adaptations to increases in fatty-acid supply, and, therefore, the involvement of UCP3 in a compensatory mechanism linking obesity to increased muscle thermogenesis.