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.

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.

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.

Early alterations in the brown adipose tissue adenylate cyclase system of pre-obese Zucker rat fa/fa pups: decreased G-proteins and beta 3-adrenoceptor activities.

This study was undertaken to determine whether receptor and non-receptor components of the adenylate cyclase (AC) cascade were altered in brown adipose tissue (BAT) of 14-day-old pre-obese (fa/fa) rats, before endocrine status is strongly modified by fa gene expression. Activity of the AC catalytic subunit did not differ between the two genotypes. In fa/fa rats compared with control Fa/fa rats, there was a 50% decrease in the activity of alpha Gs (stimulated by NaF or guanosine 5'-[gamma-thio]triphosphate) but no change in protein content (Western blotting). alpha Gi function, assessed by the inhibitory action of low concentrations of guanosine 5'-[beta gamma-imido]triphosphate upon 10(-4) M forskolin-stimulated AC activity, was equally low in both genotypes. Analysis of dose-response curves for different beta-agonists revealed that (i) both the basal and the maximally stimulated activity of AC were 2-fold lower in fa/fa rats than in Fa/fa rats; (ii) BRL37344 and CGP12177 (beta 3 agonists) were less potent in fa/fa than in Fa/fa rats (Kact. multiplied by 2); (iii) noradrenaline and isoprenaline (Iso), at the low-affinity site (beta 3-AR), were less potent in fa/fa than in Fa/fa pups (Kact. increased by 30 and 20% respectively). At the high-affinity site (mainly beta 1) these two agonists were more potent in fa/fa than in Fa/fa rats (Kact. decreased by 40 and 80% respectively). In good agreement with the latter result, the beta 1-adrenergic receptor (beta 1-AR)-selective antagonist CGP20712A had more effect on the Iso-stimulated AC activity in pre-obese than in lean pups (2-fold decreased in IC50). Binding experiments with [3H]CGP12177 show that in BAT of suckling rats, beta 3-ARs represent 80% of the total beta-ARs. Bmax values for the two sites were not affected by the genotype, although the beta 3-AR mRNA concentration in BAT (quantitative reverse-transcriptase PCR) was 3-fold lower in fa/fa rats than in Fa/fa pups. In conclusion, these results provide evidence for alterations in beta 1- and beta 3-AR signalling in BAT of 14-day-old suckling pre-obese Zucker rats with a decreased activity of alpha Gs. The impaired AC responsiveness to catecholamines might be a primary contributor to the development of this genetic obesity.