Adipocyte functions are modulated by cell size change: potential involvement of an integrin/ERK signalling pathway.

OBJECTIVES: Adipocyte is the only cell whose size may vary dramatically in physiological conditions. We hypothesized that increase in fat cell size per se could modulate several signalling pathways by changing the relationships between the cell and the extracellular matrix. The aim of the current study was (i). to examine whether within the same fat depot, metabolic functions of adipocyte were modified by cell size and (ii). if such an adaptation exists, to look for an integrin/extracellular-signal-regulated kinases (ERKs) signalling pathway. RESULTS: We isolated two populations of adipocytes with different volumes (67 and 22 x 10(3) microm(3)) within the same adipose location. In large compared to small fat cells, fatty acid synthase and lipoprotein lipase activities were increased two- and seven-fold, respectively; GLUT4 protein concentration and leptin expression were increased three-fold; lipolytic capacity was increased four-fold. The integrin/ERK signalling pathway could be the one responsible for the adaptation of adipose functions to cell size. In large compared with small adipocytes, we showed that beta(1)-integrins are present in adipose membranes and at a higher concentration in large than in small cells. In isolated adipocytes, stimulation of beta(1)-integrins with a specific monoclonal antibody results in ERK(1) and ERK(2) activation. In large compared to small cells, cytoplasmic concentrations of these two mitogen-activated protein kinases were increased two-fold, whereas their activities were increased 10-fold. CONCLUSION: A beta(1)-integrin/ERKs signalling pathway is present in mature adipocyte. Increase in cell size, by modifying the relationships between cell and extracellular matrix, could turn on this pathway. Since ERKs can modulate transcription factors and subsequently modulate gene expression important for adipose function, this pathway could play an important role in the adaptation of adipose functions to cell size.

Impaired beta-adrenergic signaling pathway in white adipocytes of suckling fa/fa Zucker rats: a defect in receptor coupling.

BACKGROUND: In fa/fa Zucker rats, leptin receptor deficiency is responsible for both a deficit of energy expenditure and hyperphagia which lead to massive obesity and insulin resistance in adulthood. This obesity is also characterised by alterations of the beta-adrenergic signaling pathway. OBJECTIVE: To determine whether alterations in beta-adrenergic pathway could occur at the onset of obesity when fa/fa rats are not yet hyperinsulinemic. ANIMALS: Fourteen-day-old suckling fa/fa and Fa/fa littermates (from heterozygous lean (Fa/fa) female and homozygous obese (fa/fa) male mating). MEASUREMENTS: Membranes were prepared from isolated adipocytes after collagenase treatment of inguinal adipose tissue. The response of adenylyl-cyclase activity to stimulation by isoprenaline, GTPgamma-S or forskolin was studied. Bmax and Kd of (beta1+beta2) and of beta3 adrenoceptors were measured using 3H-CGP saturation binding experiments. mRNA concentration of beta1- and beta3-AR was determined by semi-quantitative RT-PCR. G(s)alpha protein was quantified by Western blotting and Gi protein by ADP-ribosylation. RESULTS: Despite an almost normal body weight, inguinal fat pad weight was increased two-fold by the expression of fa mutation. This increase was entirely accounted for by fat cell hypertrophy (x2.5 in volume). In fa/fa compared to Fa/fa pups, response of adenylyl cyclase to isoprenaline was decreased two-fold but responses to GTPgammaS or forskolin were unchanged. Density of (beta1+beta2) and beta3-AR was not affected by the fa/fa genotype, as well as G(s)alpha and Gi concentration. CONCLUSION: Response of inguinal fat cells to catecholamines was decreased without any quantitative modifications of the different elements of the adenylyl cyclase cascade. This suggests an alteration in the coupling between beta-AR and G proteins. Due to the important increase in fat cell volume we hypothesize that changes in the physical properties of plasma membranes and/or changes in cytoskeleton-extracellular-matrix interactions could disturb the beta-adrenergic pathway responsiveness. In addition to the excess of lipid storage, which occurs very early at the onset of obesity, the impairment of the responsiveness to catecholamines reported in this study might worsen the obesity syndrome.

Evidence for the presence of several phosphodiesterase isoforms in brown adipose tissue of Zucker rats: modulation of PDE2 by the fa gene expression.

The present study was undertaken to characterise the phosphodiesterases (PDEs) present in brown adipose tissue (BAT) of Zucker rat pups and to determine whether the capacity for degradation of cyclic nucleotides was affected by the fatty genotype. Regardless of the genotype, PDE2-4 contributed to total PDE activity, the PDE3 activity equalling the sum of PDE2 and 4 activities. In fa/fa compared to Fa/fa rats, (a) PDE2 activity was significantly increased, (b) Western blot analysis of PDE2 revealed two signals at 71 and 105 kDa, with changes in protein being in good parallelism with changes in activity, (c) the PDE2 mRNA concentration was also significantly increased. In good agreement, the cGMP concentration was decreased in BAT from fa/fa pups.

Noradrenergic control of ascorbic acid and glutathione concentrations in brown fat of cold-exposed rats.

Ascorbic acid and glutathione concentrations increase in brown fat of cold-exposed rats. This phenomenon can be reproduced by noradrenaline or isoproterenol administration, and thus seems to be under sympathetic control. Histological study shows ascorbic acid storage in brown adipocyte nuclei.

Effect of noradrenaline chronic administration on brown fat phospholipids.

Chronic cold exposure of rats (9 days at 5 degrees C) induces an alteration of the fatty acid composition of phospholipids in brown adipose tissue. The alteration is due to an increase of the unsaturation degree of these lipids. The phenomenon can be reproduced by 10(-7) mole. h-1 administration of noradrenaline for 9 days in rats kept at 25 degrees C. Thus, phospholipid alteration in brown fat of cold exposed rats is most probably a consequence of the increase of sympathetic tone which occurs in this tissue during exposure to cold.

Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue. Evidence for a rapid beta-adrenoreceptor-mediated and transcriptionally regulated step during activation of thermogenesis.

A cloned cDNA sequence for the unique mitochondrial uncoupling protein of rat brown adipose tissue has been used to assay the corresponding mRNA in several situations. When thermogenesis in brown adipose tissue is stimulated (exposure of adult rats to the cold, birth) a rapid and prolonged increase in the level of uncoupling protein mRNA is observed. Such an increase can be mimicked by injection of animals with a new beta-adrenoreceptor agonist BRL 26830A. Conversely it is known that mice and rats with genetic or surgical obesity have a weakly thermogenic brown adipose tissue with a reduced norepinephrine turnover. A reduced level of uncoupling protein mRNA was measured in obese fa/fa rats 10 days or 10 weeks old and in obese rats with a lesion of the ventromedial hypothalamic area but not in obese ob/ob mice. Moreover, exposure of obese animals to cold or dosing with BRL 26830A strikingly increased the level of uncoupling protein mRNA. Measurement of the relative concentration of nascent Ucp transcripts in nuclei isolated from brown adipose tissue indicates that Ucp gene is acutely (within 15 min) regulated at the level of transcription and is controlled via activation of beta-adrenoreceptors of plasma membrane. Ucp gene transcription is decreased in obese fa/fa rats but can be fully and rapidly turned on after injection of BRL 26830A.

Increase of uncoupling protein and its mRNA in brown adipose tissue of rats fed on 'cafeteria diet'.

The effect of 'cafeteria diet' on mitochondrial uncoupling protein in brown adipose tissue of rats was examined. 'Cafeteria diet' induced an increase of the 32 kDa uncoupling protein in electrophoresed proteins of brown-fat mitochondria. Use of a cDNA probe corresponding to uncoupling-protein mRNA indicated that this mRNA was increased in rats fed on the 'cafeteria diet'. Nevertheless, this effect was weak compared with that observed in rats adapted to cold.

Cold exposure or chronic noradrenaline treatment induces an increase in the calmodulin-like immunoreactivity of brown adipose tissue of rats.

Cell proliferation is often associated with an increase in calmodulin, the ubiquitous intracellular calcium receptor of non-muscle cells. A long lasting increase in the proliferative activity of brown adipose tissue is induced by cold exposure in the rat. The present work showed that this phenomenon is also associated with a rapid and long lasting increase in the calmodulin content of this tissue. It was equally shown that this increase can be reproduced by noradrenaline administration.

Factors controlling brown adipose tissue development.

Brown adipose tissue (BAT) is a site of non-shivering thermogenesis in mammals. Thermogenesis in brown adipocytes is related to the presence of a specific mitochondrial component called "uncoupling protein". When animals are chronically exposed to cold, their BAT is enlarged and exhibits several changes such as cellular differentiation, hyperplasia, mitochondriogenesis and marked synthesis of uncoupling protein. The hormonal and neural factors controlling this adaptive response have been studied. It is concluded that sympathetic innervation of BAT and released noradrenaline play an essential role in the development of BAT.

Effects of cold exposure on calcitonin secretion in the young rat.

The effects of cold exposure on calcitonin (CT) secretion were evaluated in young rats. Acute cold exposure (5 h to 5 degrees C) induced a rise in plasma CT concentrations and a decrease in thyroidal CT stores without change in total and ionized plasma calcium levels. The cold activation of sympathico-adrenomedullary axis and the inhibition of CT response to cold after beta-antagonist treatment might suggest that endogenous catecholamines can enhance CT secretion in young rats. Cold adaptation (3 weeks to 5 degrees C) induced a fall in plasma calcium concentration and a rise in thyroidal CT stores without change in plasma CT levels. The high plasma glucocorticoid levels which are known to occur during chronic cold exposure could be involved in the rise of thyroidal CT content in cold adapted rats.

Rapid increase of mitochondrial uncoupling protein and its mRNA in stimulated brown adipose tissue. Use of a cDNA probe.

The increase in mitochondrial uncoupling protein in brown adipose tissue during acute stimulation by exposure of animals to cold was examined. Uncoupling protein level increased during the first hours of tissue stimulation. Use of a cDNA probe shows that synthesis of uncoupling protein mRNA was quickly stimulated. Animals treated with propranolol exhibited neither increase in uncoupling protein mRNA nor increase in the protein itself.

Increased level of mRNA for the uncoupling protein in brown adipose tissue of rats during thermogenesis induced by cold exposure or norepinephrine infusion.

We have studied by in vitro translation the mRNA population from rat brown fat in two situations where the thermogenic activity of the tissue is stimulated. Both cold exposure of rats and the presence of pheochromocytoma result in marked alterations among the polypeptides synthesized. Notably, in these two situations there was an increased labeling of polypeptides of the size of the heat-shock proteins and an increased labeling of the 32,000-dalton uncoupling protein of brown fat mitochondria, which is responsible for energy dissipation as heat. Stimulation of the thermogenic activity of brown fat thus results in a high level of mRNA encoding the uncoupling protein. Chronic delivery of norepinephrine mimicked the increase of mRNA for the uncoupling protein observed in animals exposed to cold or bearing pheochromocytoma. Thus, a neuromediator such as norepinephrine is able to trigger the induction of the mRNA encoding the mitochondrial uncoupling protein and is able to stimulate mitochondriogenesis.

Noradrenaline controls the concentration of the uncoupling protein in brown adipose tissue.

The importance of noradrenaline in the control of the level of the uncoupling protein responsible for the high thermogenic capacity of brown adipose tissue mitochondria was examined. It was observed that chronic infusion of noradrenaline through mini-osmotic pumps increased the mitochondrial concentration of this uncoupling protein to the same extent as chronic exposure to cold.

Development and activation of brown fat in rats with pheochromocytoma PC 12 tumors.

To study the neurohormonal determinism of cellular growth and differentiation and mitochondrial development in brown adipose tissue (BAT), this organ was analyzed in rats bearing uncloned or cloned (PC 12) pheochromocytoma; comparison was made with cold adaptation. Both uncloned and PC 12 tumors induced an enlargement of tissue weight and DNA total content, although smaller than during cold adaptation. The following striking modifications were observed in rats bearing PC 12 tumors: strong vasodilation, increase in protein and phospholipid percentage, alteration of the fatty acid composition of phospholipids, increase in mitochondrial protein, large increase of the GDP binding to isolated mitochondria, and marked rise in specific amount of 32,000-dalton uncoupling protein (ascertained using immunological approach). It is concluded that secretions of PC 12 tumors can induce the same alterations in BAT as does the sympathetic system during cold adaptation of animals. An important contribution of norepinephrine to these effects is evidenced, but a specific function of other trophic factors secreted by PC 12 cells and by sympathetic nerves can be postulated.

Parallel increase of ascorbic acid and glutathione contents in brown adipose tissue during chronic cold exposure.

Spontaneous lipid peroxidation rate was found unchanged in the brown adipose tissue of rats chronically exposed to cold, although oxidative metabolism, ascorbic acid and poly-unsaturated phospholipid amounts increased. It is suggested that the concomitant increase in glutathione concentration may protect the tissue from a possible peroxidative process.

Localization of serotonin and dopamine in the brown adipose tissue of the rat and their variations during cold exposure.

The variations of several biogenic amines in brown adipose tissue (BAT) during cold exposure were studied and their localization investigated with histological methods. The study of serotonin and its metabolite 5-HIAA suggests that BAT serotonin is mobilized during acute and chronic cold exposure. This amine was found to be principally stored, together with histamine, in mast cells. The mast cell number in BAT was doubled during cold adaptation, as was the histamine content of the tissue. Using radio-enzymatic assay and high pressure liquid chromatography, only small amounts of dopamine were found in BAT. Since no specific dopamine-storing structure was detected (for example SIF cells), this low amount of dopamine is probably the precursor pool for noradrenaline synthesis and is most likely stored in the noradrenergic innervation of the tissue. BAT is known to be sensitive to both exogenous serotonin and exogenous dopamine; according to our results serotonin could play a role in BAT regulation while the role of dopamine remains hypothetical.

Ultrastructural and biochemical characterization of human brown adipose tissue in pheochromocytoma.

The amount and distribution of brown adipose tissue (BAT) in healthy adult human subjects remain uncertain. BAT has been described in patients with pheochromocytoma. This pathological situation was chosen to try to characterize human BAT using both morphological and biochemical criteria. Typical multilocular brown adipocytes were observed; the cytoplasm of these adipocytes was filled with mitochondria with numerous tightly packed cristae, as in rat or hamster BAT. Isolated mitochondria exhibited a loose respiratory coupling sensitive to guanosine diphosphate; the chloride conductance of these mitochondria was reduced by the addition of this nucleotide. It is concluded that in patients with pheochromocytoma, there is a large amount of a tissue around the adrenal glands and kidneys which fullfills the ultrastructural and biochemical criteria for a BAT.