The adrenergic control of hepatic glucose and FFA metabolism in rainbow trout (Oncorhynchus mykiss): increased sensitivity to adrenergic stimulation with fasting.

The adrenergic control of glucose and FFA release was studied in hepatocytes of rainbow trout (Oncorhynchus mykiss), which were either normally fed or fasted for 3 weeks. Isolated hepatocytes were incubated with adrenaline, noradrenaline, or isoprenaline (nonselective beta-agonist). Identification of the hepatic beta-adrenoceptor was combined with quantification of the difference in its affinity for adrenaline and noradrenaline. To identify the beta-adrenoceptor subtype, isoprenaline incubations were combined with atenolol (selective beta(1)-antagonist) or ICI 118,551 (selective beta2-antagonist). Stimulation of the beta-adrenoceptor resulted in mobilisation of glucose, which was inhibited by ICI 118,551 thus pointing to a beta2-subtype. The affinity of the beta2-adrenoceptor for isoprenaline and adrenaline (beta2-values of 8.3 and 7.9) was clearly higher than for noradrenaline (beta2-value of 6.5). This indicates that at physiological concentrations beta2-adrenoceptors in trout are mainly stimulated by adrenaline and not by noradrenaline. A significant effect of beta-adrenoceptor stimulation on the FFA release was also found, although only at high concentrations (i.e., 10(-6) and 10(-5)M). Again the beta2-adrenoceptor appeared to mediate the stimulation of hepatic FFA release. Upon fasting, both the basal glucose and FFA release were strongly decreased. The ratio between glucose and FFA release decreased from 15.4 to 4.3 upon fasting and at this ratio the energy output for both metabolites became equal. The mobilisation of FFA upon adrenergic stimulation was relatively conserved, namely -35% upon fasting, as opposed to -89% in mobilisation of glucose. This indicates that upon fasting FFA gain importance in hepatic metabolism. The hepatic sensitivity to adrenergic stimulation is enhanced upon fasting, as indicated by an increased beta2-value from 8.3 to 8.9.

Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes.

We examined the effects of diet composition and fasting on lipolysis of freshly isolated adipocytes from gilthead seabream (Sparus aurata). We also analyzed the effects of insulin, glucagon, and growth hormone (GH) in adipocytes isolated from fish fed with different diets. Basal lipolysis, measured as glycerol release, increased proportionally with cell concentration and time of incubation, which validates the suitability of these cell preparations for the study of hormonal regulation of this metabolic process. Gilthead seabream were fed two different diets, FM (100% of fish meal) and PP (100% of plant protein supplied by plant sources) for 6 wk. After this period, each diet group was divided into two groups: fed and fasted (for 11 days). Lipolysis was significantly higher in adipocytes from PP-fed fish than in adipocytes from FM-fed fish. Fasting provoked a significant increase in the lipolytic rate, about threefold in isolated adipocytes regardless of nutritional history. Hormone effects were similar in the different groups: glucagon increased the lipolytic rate, whereas insulin had almost no effect. GH was clearly lipolytic, although the relative increase in glycerol over control was lower in isolated adipocytes from fasted fish compared with fed fish. Together, we demonstrate for the first time that lipolysis, measured in isolated seabream adipocytes, is affected by the nutritional state of the fish. Furthermore, our data suggest that glucagon and especially GH play a major role in the control of adipocyte lipolysis.

Differential role of adrenoceptors in control of plasma glucose and fatty acids in carp, Cyprinus carpio (L.).

Carp were cannulated in the dorsal aorta, and after 2 days of recovery they were infused with 1) norepinephrine, 2) yohimbine (alpha(2)-antagonist) plus norepinephrine, 3) clonidine (alpha(2)-agonist), and 4) isoproterenol (nonselective beta-agonist). Norepinephrine lowered the plasma free fatty acid (FFA) level and raised the plasma glucose level for several hours. Norepinephrine in combination with the alpha(2)-antagonist yohimbine resulted in retardation of the FFA decrease, indicating the involvement of alpha(2)-adrenoceptors. Infusion with the partial alpha(2)-agonist clonidine had a smaller effect. Infusion with isoproterenol caused a marked increase of glucose levels, and unexpectedly a decline of plasma FFA levels, indicating a direct involvement of beta-adrenoceptors. Combination of isoproterenol with either atenolol (beta(1)-antagonist) or ICI-118,551 (beta(2)-antagonist) showed that both beta(1)- and beta(2)-adrenoceptors were involved in the glucose release by isoproterenol. Remarkably, the decline of FFA levels was augmented in the presence of ICI-118,551, whereas with atenolol present plasma FFA levels were increased by isoproterenol. Thus it is concluded that in carp both beta(1)- and beta(2)-adrenoceptors mediate glucose release, whereas lipolysis is controlled by inhibitory beta(1)-adrenoceptors and stimulatory beta(2)-adrenoceptors, as well as by inhibitory alpha(2)-adrenoceptors.

Purification of rat liver mitochondrial phospholipase A2.

Rat liver mitochondrial phospholipase A2 was purified to near homogeneity by a combination of gel-filtration, hydroxyapatite and Matrex gel Blue A column chromatography. The absolute positional specificity of the enzyme for acylester bonds at the sn-2-position was established in experiments with 1-[9,10-3H2]palmitoyl-2-[1-14C]linoleoylphosphatidylethanolamine. Molecular weight estimations revealed Mr values of 15000 by SDS-polyacrylamide gel electrophoresis and of 9700 gel by gel-filtration over Ultrogel AcA 54 columns. The enzyme is unaffected by diisopropylfluorophosphate and thiol reagents such as 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide and iodoacetamide, but is completely inhibited by the alkylating reagent p-bromophenacylbromide.