Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes.

The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing V(MAX) and K(M) for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1.

Adrenergic receptor density in brown adipose tissue of active and hibernating hamsters and ground squirrels.

The ligand-binding characteristics (B(max) and K(D)) of alpha(1)- and beta(1)/beta(2)-adrenoceptors were investigated in membranes prepared from brown adipose tissue (BAT) of warm-acclimated, cold-acclimated, hibernating and arousing ground squirrels (Spermophillus undulatus) and hamsters (Mesocricetus auratus) by specific binding of [(3)H]prazosin and [(3)H]CGP-12177, respectively. The physiological state did not change the affinity for the adrenoceptors in the BAT of ground squirrels and hamsters. There was a significant decrease in alpha(1)-receptor density in arousing ground squirrels and a significant decrease in beta(1)/beta(2) density in hibernating ground squirrels. The level of alpha(1)-receptors was in all conditions higher than that of beta(1)/beta(2) receptors. The results indicate a possible change in balance of adrenoceptor density in the processes of cold acclimation, hibernation and arousal. The balance between the various adrenoceptor subtypes may be important for the final effect of catecholamines in BAT in different physiological states.

Inhibitory effects of halothane on the thermogenic pathway in brown adipocytes: localization to adenylyl cyclase and mitochondrial fatty acid oxidation.

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.

Norepinephrine induces slow calcium signalling in murine brown preadipocytes through the beta-adrenoceptor/cAMP/protein kinase A pathway.

The mechanism of adrenergically activated calcium signalling in isolated murine brown preadipocytes (stromal-vascular fraction) was studied with Fura-2. Norepinephrine (NE) generated in preadipocytes a slow Ca(2+)-response ( approximately 10 nM/min) without a burst and a maximum, whereas in mature brown adipocytes, the quick burst reached 1.5 microM [Ca(2+)](i). Thapsigargin, which is known to discharge Ca(2+) ions from the IP(3)-sensitive stores, initiated a huge capacitative calcium entry in mature brown adipocytes but failed to stimulate a response in preadipocytes. The beta-selective antagonist nadolol almost completely prevented the effect of NE on [Ca(2+)](i), while the antagonist of alpha-adrenoceptors phentolamine caused only a approximately 25% reduction of the cellular response. Forskolin or the cell-permeable Br-cAMP caused [Ca(2+)](i) rise, which were even higher than with NE. The protein kinase A (PKA) inhibitor N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) reduced and the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX), N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide (OPC-3911), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) or the protein phosphatase inhibitor okadaic acid enhanced the NE-, isoproterenol- or forskolin-initiated cellular calcium responses. It was concluded that (i) brown preadipocytes lacked a trigger mechanism of initiation of [Ca(2+)](i) rises and (ii) the cAMP- and protein kinase A-mediated phosphorylation played an important role in the beta-adrenoceptor-initiated calcium signalling in these cells. All these features distinguish brown adipocyte precursors from differentiated brown adipocytes, where calcium signalling is initiated exclusively via alpha(1)-adrenoceptors and the trigger mechanism.

Sperm quality as reflected through morphology in salmon alternative life histories.

Male salmon exhibit alternative mating strategies, as both older anadromous adults and precocious juveniles (parr) participate in the spawning of a single female. This study tested the following hypotheses: 1) different intensities of sperm competition may reflect different sperm tail optima; 2) long spermatozoa are superior to short ones, with an associated cost on sperm longevity; and 3) a disfavored role in sperm competition selects for parr investing more in sperm quality. Comparisons included sperm morphological traits, whereas sperm quality was investigated by motility duration observations, measurement of the sperm adenylate system, and fertilization experiments. No evidence of different adaptive sperm dimensions between the male types was found. Positive association between spermatocrit and energy charge was, however, detected. Sperm length parameters correlated positively with ATP, energy charge, and fertilization success, whereas no evidence for an effect of sperm morphology on longevity was found. Male parr had greater spermatocrit than adults and fertilized equal proportions of eggs as adults despite a pronounced numerical subordinance in the fertilization experiments. It is concluded that a long sperm tail and midpiece may be selected to optimize energetic demands under conditions of increased sperm competition intensity.