Regulation of lipogenesis in mitogen-stimulated human lymphocytes by thyroid hormones

The hormonal regulation of precursor incorporation into cellular lipids has been investigated in human lymphocytes stimulated with phytohemeagglutinine. Addition of thyroxine (5 micrograms/ml) for 72 h increased incorporation of [14C]acetate into the triacylglycerol fraction to 290% above the hormone-free control values. Incorporation into the cholesterol fraction was elevated up to 188% under the same conditions. Triiodothyronine was less effective than thyroxine: maximal effects were 153% of the control for triacylglycerols and 142% for cholesterol. Similar results were obtained when [14C]palmitic acid was used as a precursor for triacylglycerol synthesis. Effects of insulin on the parameters described were less pronounced than those obtained with thyroid hormones. Cellular triacylglycerol and protein contents were not elevated significantly by thyroid hormone addition. Further, incorporation of labelled thymidine, uridine, and leucine into acid-precipitable products was not elevated by triiodothyronine above mitogen-stimulated levels. It is concluded, that rapidly dividing lymphocytes provide a suitable system for studies concerning human lipid metabolism.     

Independent influence of insulin, catecholamines, and thyroid hormones on metabolic syndrome

The objective of this study was to examine whether metabolic syndrome, defined according to adult treatment panel III criteria, is associated with insulin, catecholamines, and thyroid hormones, independently of age and gender. A cohort of 651 euthyroid overweight and obese patients, 440 women and 211 men, aged 18-68 years, were examined. Central fat accumulation (indirectly measured by waist circumference), fasting thyroid-stimulating hormone (TSH), FT(3), FT(4), insulin, glucose, and lipid (cholesterol, HDL-cholesterol, and triglyceride) serum concentrations, 24-h urinary catecholamines, and the level of insulin resistance (estimated by homeostasis model assessment for insulin resistance (HOMA(IR))) were measured. Patients with metabolic syndrome showed higher insulin (P < 0.001) and FT(3) (P < 0.001) serum levels and higher 24-h urinary noradrenaline (P < 0.001) than subjects without this syndrome. The number of metabolic syndrome parameters was directly associated with insulin (P < 0.001) and FT(3) (P < 0.05) serum levels, and with 24-h urinary noradrenaline (P < 0.001) in the whole population. When a multiple regression analysis was performed with the metabolic syndrome as the dependent variable, and age, gender, and insulin, and TSH, FT(3), FT(4) serum levels, and 24-h urinary noradrenaline and adrenaline as independent variables, the metabolic syndrome maintained an independent positive association with age (P < 0.001), male sex (P < 0.001), insulin (P < 0.001), and 24-h urinary noradrenaline (P < 0.001). In conclusion, this study suggests that insulin and noradrenaline cooperate independently to the development of the metabolic syndrome.     

Effects of insulin on lipolysis and lipogenesis in hamster white adipocytes with high sensitivity to hormones

A modified procedure for preparation of hamster adipocytes by collagenase digestion under carefully controlled conditions has been developed. The adipocytes were 4- to 8-fold more sensitive to catecholamine stimulation of lipolysis than cells prepared by a commonly used method (Hittelman, K.J., Wu, C.F. and Butcher, R.W. (1973) Biochim. Biophys. Acta 304, 188-196) and also more sensitive to the anti-lipolytic action of insulin. The effects of insulin on lipogenesis, measured as [3H]glucose conversion to cell lipids, and on catecholamine-stimulated lipolysis were compared under identical conditions with the same cell batch. Isoprenaline-stimulated lipolysis was found to be half-maximally inhibited by an insulin concentration 8-fold lower than that stimulating lipogenesis to a corresponding extent (half-maximal effects at insulin concentrations of 40 vs. 300 pM). A similar difference was found when cells had been stimulated with adrenaline instead of isoprenaline.     

Hormone-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. Regulation of insulin- and cAMP-dependent activation by phosphorylation.

In 32PO4-labeled adipocytes, isoproterenol (ISO) or physiologically relevant concentrations of insulin rapidly increased phosphorylation of a particulate 135-kDa protein which has been identified as a cGMP-inhibited "low Km" cAMP phosphodiesterase (CGI-PDE) by several criteria, including selective immunoprecipitation with anti-CGI-PDE IgG (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 533-537). The time courses and concentration dependences for phosphorylation of CGI-PDE by ISO and insulin correlated with CGI-PDE activation in the presence of these agents; effects of ISO were somewhat more rapid than those of insulin. Adenosine deaminase, which metabolizes the adenylate cyclase inhibitor adenosine, also rapidly induced phosphorylation and activation of CGI-PDE. Phenylisopropyladenosine (an adenosine deaminase-resistant adenosine analog) prevented or reversed both adenosine deaminase-stimulated phosphorylation and activation of CGI-PDE (IC50 approximately 0.2 nM). Incubation of adipocytes with 0.1 nM insulin in the presence of ISO rapidly produced 30-200% greater activation and phosphorylation of CGI-PDE than the expected added effects of insulin and ISO individually; both effects preceded the insulin-induced decreases in protein kinase A activity and inhibition of lipolysis. These and other results indicate that CGI-PDE can be phosphorylated at distinct sites and activated by cAMP-dependent and insulin-dependent serine kinase(s), that the activation state of CGI-PDE reflects its relative phosphorylation state, and that synergistic phosphorylation/activation of CGI-PDE may be important in the antilipolytic action of insulin.     

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T₃) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T₃ (1·10^?6) and cyclic AMP (2·10^?4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[¹⁴C]isobutyric acid as well as net alanine uptake and urea production were elevated by T₃ and by cyclic AMP. T₃ increased the oligomycin-sensitive O₂ consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T₃ on hepatic gluconeogenesis was independent of exogenous Ca²⁺ concentration. T₃ by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese (v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phosphoenolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phosphoenolpyruvate. Cyclic AMP action was not affected by the further addition of T₃. Glucagon (1·10^?8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T₃ stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.     

Inhibitory effect of thyroid hormones on pituitary cyclic AMP phosphodiesterase activity in vitro

Thyroid hormones effectively inhibited partially purified cyclic AMP phosphodiesterase from the anterior and posterior lobes of bovine pituitary gland competitively with the substrate, but thyronine, diiodotyrosine, monoiodotyrosine, thyrotropin, thyrotropin releasing hormone, dexamethasone, and luteinizing hormone releasing hormone did not inhibit this enzyme activity.K _i values were found to be 1.0 and 2.5 M for thyroxine, and 8.0 and 13.5 M for triiodothyronine in the anterior and posterior lobes, respectively.     

Regulation of cyclic AMP levels in canine thyroid slices by alpha-adrenergic action.

In an attempt to clarify the role of adrenergic receptors in metabolic responses, interaction of norepinephrine with TSH was studied in canine thyroid slices with regard to cyclic AMP levels. Norepinephrine caused a very rapid (within 1 min), but quite transient increase in cyclic AMP levels. The elevation of cyclic AMP levels induced by TSH was markedly inhibited by norepinephrine. Phentolamine, an α-adrenergic blocker, not only prevented the decline of cyclic AMP levels that followed the rise by norepinephrine, but also abolished the norepinephrine effect on the TSH-induced elevation of cyclic AMP levels. Propranolol, a β-adrenergic blocker, exhibited no such effects. These results indicate that the α-adrenergic receptors control cyclic AMP levels in the thyroid gland.     

Comparative effects of forskolin and isoproterenol on the cyclic AMP content of human adipocytes

Alterations in adipocyte cyclic AMP concentrations in response to 100 microM forskolin and 10 microM isoproterenol over a 4 hour period were found to be similar; with each agent, a peak response was noted within 30 minutes. In general, the greater the magnitude of peak response, the more rapid the decline of cyclic AMP concentration during the ensuing 3 1/2 hours. Alpha-2 adrenergic activation, achieved with 10 microM clonidine or 10 microM epinephrine, substantially reduced the cyclic AMP concentrations in cells stimulated by 100 microM forskolin or 10 microM isoproterenol. Isoproterenol-stimulated cells appeared to be more sensitive to alpha adrenergic inhibition than did forskolin-stimulated cells. Cells preincubated for 3 hours with 100 microM forskolin were markedly less responsive to a second exposure to the diterpine. Cells exposed to forskolin for 3 hours also had a reduced response when incubated with isoproterenol; thus, desensitization to forskolin appears to be heterologous. Forskolin desensitization did not appear to be dependent on cellular ATP depletion since cells mildly stimulated during preincubation were as severely desensitized as those adipocytes strongly stimulated. Maximum desensitization required a preincubation time of 1-2 hours with either isoproterenol or forskolin.     

Regulation of enzymes of glucose metabolism and lipogenesis in diabetic rat liver by thyroid hormones

Experimental diabetes in rats is associated with a degree of hypothyroidism. Hepatic enzymes involved in carbohydrate and lipid metabolism were estimated in control (untreated), control +T3 treated, alloxan diabetic and alloxan diabetic + T3-treated rats. The key glycolytic enzymes, phosphofructokinase and pyruvate kinase, were decreased in activity in diabetes and unchanged by further treatment with T3. In contrast, certain enzymes involve in lipogenesis, ATP-citrate lyase 'malic' enzyme and 6-phosphogluconate dehydrogenase, which were decreased in activity in diabetes, were increased to, or above, control values when diabetic rats were treated with T3. It is suggested that T3 deficiency may play a role in the decrease in enzyme activities observed in experimental diabetes, in particular, some enzymes associated with lipogenesis and the provision of NADPH.     

The action of adenosine in relation to that of insulin on the low-Km cyclic AMP phosphodiesterase in rat adipocytes.

The adenosine-sensitive cyclic AMP phosphodiesterase of rat adipocytes was found to reside in the same subcellular fraction as the enzyme sensitive to insulin. There were several similarities between the action of adenosine and that of insulin on the enzyme. The action of adenosine on the phosphodiesterase is probably like that of insulin, both being receptor-mediated, although different sites or different receptors could be involved. Adenosine analogues with intact ribose but a modified purine moiety elicited a response similar to that of adenosine. Added Ca2+ was also not a requirement for the action of adenosine. The action of adenosine was not synergistic with that of insulin, neither was adenosine essential for insulin action. Insulin stimulated the enzyme even at low cell concentrations and in the presence of adenosine deaminase. Adenosine, however, enhanced the effect of insulin, but only at insulin concentrations that produced submaximal effects. Thus the mechanisms of action could be similar or related. The time-course effect of a suboptimal concentration of insulin was transitory, like that of adenosine, and was influenced by the presence of adenosine, whereas that of a maximally effective concentration of insulin was sustained for at least 20 min and was not affected by the presence of adenosine. Isoprenaline enhanced phosphodiesterase activity stimulated by optimal concentrations of either adenosine or insulin, suggesting that their effects were mediated through different mechanisms of action.     

The effects of lipolytic hormones on calcium uptake in endoplasmic reticulum of adipocytes

To explore interrelationship between the roles of cAMP and calcium ion in hormone-stimulated lipolysis, cAMP accumulation in rat adipocytes and calcium binding in the endoplasmic reticulum were investigated with special reference to the effects of lipolytic hormones under various conditions. ACTH, isoproterenol, DBcAMP and aminophylline significantly increased ATP-dependent calcium uptake in adipocyte endoplasmic reticulum, but only after they were incubated with intact cells and not when they were added after homogenization. In vivo dexamethasone treatment and A-23187 accelerated, while 2.4-dinitrophenol blunted ACTH-stimulated lipolysis, cAMP accumulation and microsomal calcium uptake in parallel. Adrenalectomy, Mn2+ and adenosine enhanced ACTH-stimulated cAMP accumulation in adipocytes but lowered the calcium uptake and lipolysis. Thus, there was consistent parallelism between hormone-stimulated lipolysis and microsomal calcium uptake throughout the study. These data suggest that changes in the microsomal calcium uptake plays a crucial role in the regulation of hormone-induced lipolysis, irrespective of whether or not the intracellular cAMP concentration is involved in the lipolytic mechanism.     

Effect of vertebrate hormones on the cyclic AMP level in Tetrahymena.

Epinephrine, insulin, glucagon and theophylline produced a significant increase in the cAMP level of Tetrahymena, while the enhancing effect of TSH was not significant. The experimental results suggest that Tetrahymena possesses receptors for hormones of higher animals, but has no receptor for the nonsense hormone TSH. The cAMP enhancing effect shown by many hormones of higher animals irrespective of their different functions indicates that apart from the general mediator action of cAMP, some special mediation is also taking place.     

Interactions between insulin and thyroid hormone in the control of lipogenesis.

1. The effects of intragastric glucose feeding and L-tri-iodothyronine (T3) administration on rates of hepatic and brown-fat lipogenesis in vivo were examined in fed and 48 h-starved rats. 2. T3 treatment increased hepatic lipogenesis in the fed but not the starved animals. Brown-fat lipogenesis was unaffected or slightly decreased by T3 treatment of fed or starved rats. 3. Intragastric glucose feeding increased hepatic lipogenesis in control or T3-treated fed rats, but did not increase hepatic lipogenesis in starved control rats. Glucose feeding increased hepatic lipogenesis if the starved rats were treated with T3. Glucose feeding increased rates of brown-fat lipogenesis in all experimental groups. The effects of glucose feeding on liver and brown-fat lipogenesis were mimicked by insulin injection. 4. The increase in hepatic lipogenesis in T3-treated 48 h-starved rats after intragastric glucose feeding was prevented by short-term insulin deficiency, but not by (-)-hydroxycitrate, an inhibitor of ATP citrate lyase. The increase in lipogenesis in brown adipose tissue in response to glucose feeding was inhibited by both short-term insulin deficiency and (-)-hydroxycitrate. 5. The results tend to preclude pyruvate kinase and acetyl-CoA carboxylase as the sites of interaction of insulin and T3 in the regulation of hepatic lipogenesis in 48 h-starved rats. Other potential sites of interaction are discussed.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

In vivo hormonal control of L-type pyruvate kinase gene expression. Effects of glucagon, cyclic AMP, insulin, cortisone, and thyroid hormones on the dietary induction of mRNAs in the liver

Using a cDNA probe complementary to rat L-type pyruvate kinase mRNAs, we studied the respective roles of glucocorticoids, thyroid hormones, glucagon, and insulin in the induction of specific mRNAs in the liver of animals refed either a maltose-rich or a fructose-rich diet. Neither adrenalectomized nor thyroidectomized nor diabetic animals could express L-type pyruvate kinase mRNAs in their liver when refed the carbohydrate-rich diets. When the animals were given the missing hormone, the level of hybridizable mRNAs returned to normal values but administration of the hormone alone failed to induce mRNA synthesis in fasted animals. Both glucagon and cyclic AMP abolished the induction of L-type pyruvate kinase mRNAs in refed animals. Exogenous insulin, whatever the dose, could not reverse the inhibitory action of glucagon. Insulin has usually been regarded as the main regulator of L-type pyruvate kinase gene expression. It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level. Consequently, our results show that three conditions are required for the synthesis of liver L-type pyruvate kinase mRNAs: (i) the presence of dietary carbohydrates, (ii) the cessation of glucagon release, and (iii) the presence of permissive hormones, including insulin.