Influence of thyroxine and luteinizing hormone on some enzymes concerned with lipogenesis in adipose tissue, testis and adrenal gland

The activities of hexokinase, citrate-cleavage enzyme, ;malic enzyme' and NADP-linked isocitrate dehydrogenase have been measured in the adipose tissue, testes and adrenals of normal rats, hypophysectomized rats and hypophysectomized rats treated with either thyroxine or thyroxine plus luteinizing hormone. Hypophysectomy reduced the activity of all four enzymes in all three tissues. Thyroxine alone restored the activity of all four enzymes in adipose tissue towards normal but failed to do so in either testes or adrenals. Thyroxine and luteinizing hormone restored the citrate-cleavage enzyme activity of testes and increased the activity of hexokinase from the low value after hypophysectomy. Neither ;malic enzyme' nor isocitrate dehydrogenase was increased by thyroxine or thyroxine and luteinizing hormone in testes. The differential stimulation of enzyme activity by thyroxine in the different tissues suggests thyroxine as having a special significance in adipose-tissue lipogenesis.     

Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver.

The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate mental manipulation, nutritional conditions were ridgidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reductionin activities of fatty acid synthetase and carboxylase in adipose in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissure, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3-H2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitary-regulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at lease in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3-H from 3-H2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led tto a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.     

The effect of diabetes and insulin on adipose lactate dehydrogenase isozymes.

Adipose lactate dehydrogenase (LDH) (EC 1.1.1.27) isozyme distribution was altered in streptozotocin-diabetic and fasting rats resulting from a relative reduction of subunit A. Treatment with insulin for 2 days partially restored the relative content of isozyme 5 to control values in the diabetic rats, and the effect of insulin was not inhibited by simultaneous injection of actinomycin D or puromycin. When the epididymal adipose tissues isolated from control animals were incubated in vitro with dibutyryl adenosine 3',5'-cyclic monophosphate or epinephrine, a relative decrease in subunit A was observed; whereas, either compound caused an increase in subunit A in diabetic tissues. The findings suggest that the redistribution of LDH isozyme under these conditions is to prevent excessive accumulation of lactate in the tissue.     

Expression of lactic dehydrogenase isoenzymes in rabbit muscle during development

• 1.1. Rabbit cDNA probes for H and M lactic dehydrogenase subunits were used to monitor mRNA levels in different muscle types during growth.
• 2.2. At the same time, lactic dehydrogenase activity and relative quantities of H and M protein subunits were measured.
• 3.3. The main results are that mRNA abundance depends on muscle type and age, and mRNA abundance is not correlated with enzymatic activity.

     

Influence of hormones on the nicotinamide nucleotide coenzymes of adipose tissue

The concentrations of the oxidized and reduced forms of the nicotinamide nucleotides were measured in the epididymal fat pads of normal, alloxan-diabetic and hypophysectomized rats. In both alloxan-diabetic rats and hypophysectomized rats the weight of the adipose tissue fell, as did the total content of NADH and NADPH; in addition, NAD⁺ was decreased in the alloxan-diabetic group. Of these changes the most marked was in NADPH and this was the only significant difference when the results were expressed as nicotinamide nucleotides/mg. of tissue protein. The concentration of NADPH in the hypophysectomized rats was not altered by treatment with growth hormone but was restored to normal by treatment with thyroxine. These results are discussed in relation to the known effect of these hormonal conditions on lipid synthesis in adipose tissue.     

Effect of temperature on lipoprotein lipase and lipogenic enzyme activities in brown adipose tissue of hypophysectomized rats

It has been shown that the same modifications on the composition of brown adipose tissue (BAT) which are normally induced following cold stimulation are also observed in hypophysectomized rats acclimated either at 28 degrees C or 15 degrees C. To test the possibility of BAT stimulation in hypophysectomized rats, we have determined some enzymatic activities known to modulate the energy supply to that organ. Seven week old Long-Evans rats were hypophysectomized. Three weeks later, they were exposed to either 28 degrees C or 15 degrees C ambient temperature for five or six weeks. Hypophysectomized rats were compared to age matched or weight matched controls. Total lipoprotein lipase activity (LPL) (triglyceride uptake) was enhanced in BAT of 28 degrees C hypophysectomized rats compared to controls. Cold acclimation led to a large increased activity. Total LPL activity was comparable in BAT of hypophysectomized and control rats. Total malic enzyme and glucose-6-phosphate dehydrogenase activities (in situ lipogenesis) were doubled in BAT of 28 degrees C hypophysectomized compared to controls. A large enhancement was observed in BAT of either 15 degrees C control or 15 degrees C hypophysectomized rats. Among the studied organs (liver, white adipose tissue, heart, BAT) hypophysectomy promotes the three enzyme activities only in BAT. These variations were discussed with relation to the effect of hypophysectomy on brown adipose tissue at 15 degrees C and 28 degrees C.     

Occurrence and properties of lactic dehydrogenases of fermentative mycoplasmas

Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(-)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(-)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.     

Some biochemical effects of the growth hormone analogue produced by plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate metabolism of adipose tissue from normal, diabetic, and hypophysectomized rats

Plerocercoids of Spirometra mansonoides produce a functional analogue of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but has not been shown to duplicate all of the actions reported for GH. The purpose of this study was to determine the effects of plerocercoid infection (chronic PGF treatment) on glucose metabolism of adipose tissue and to compare the effects to those elicited by insulin and GH in intact, diabetic, and hypophysectomized male rats. Groups of rats were constantly exposed to PGF (via plerocercoid infection) or injected twice daily with bovine GH, insulin, or saline for 10 days. Basal oxidation rates of [U-14C]glucose to 14CO2 in adipose tissue segments were measured in vitro immediately after tissue removal. Other aliquots of adipose tissue were preincubated in hormone-free medium for 3 hr prior to testing the ability of the tissue to respond to insulin or human GH (hGH) added in vitro. Adipose tissue from PGF-treated intact and hypophysectomized rats had significantly elevated basal glucose oxidation rates, and the tissue was sensitive to further stimulation by insulin or hGH. The results obtained with intact and hypophysectomized rats were essentially the same, indicating that the effects of PGF were not due to suppression of endogenous GH. The basal glucose oxidation rate in adipose tissue from diabetic rats was stimulated (P less than 0.01) by PGF, but the tissue was not sensitive to insulin added in vitro. Furthermore, PGF had no effect on body growth or blood glucose concentrations of diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP-dependent protein kinase activity and lipolysis in adipose tissue. Effect of fasting, oligomycin and iodoacetamide

The release of glycerol into the medium, the concentration of cAMP, and the cAMP-dependent protein-kinase activity were studied in adipocytes and in fat-pads obtained from epididymal adipose tissue of rats under different conditions of feeding. An increase in the tissue concentration of cAMP and in the protein-kinase activity was observed in vivo at 48 and 96 h of fasting. A diminished release of glycerol was found in adipocytes from rats fasted for 48 h, in the absence of glucose, and the maximum concentration of cAMP was inferior to that of fed rats. Oligomycin and iodoacetamide, in the presence of epinephrine and glucose, produce a diminution in the values of the parameters studied. No significant differences were observed, however, in the responses of tissue obtained from fed and fasting rats to these compounds. The present results confirm previous observations and show the dependence of the lipolytic process on carbohydrate metabolism.     

Effects of hypophysectomy and acute growth hormone treatment upon glucose metabolism in adipose tissues and isolated adipocytes of rats.

Glucose oxidation and incorporation into lipid were measured in epididymal adipose tissues and isolated adipose cells of normal and hypophysectomized rats in an effort to determine whether the acute hypoglycemic effect of a systemic growth hormone (GH) injection was related to alterations in the glucose metabolism of adipose tissue. The rats were fed rat chow or a high sucrose diet and received 100 mug GH intraperitoneally 30 minutes or three and one-half hours before sacrifice. Hypophysectomized rats showed a lower plasma glucose as compared with normal rats on both diets. Thirty minutes after a GH injection there was a further decrease of the plasma glucose which, however, was not present in those rats receiving GH three and one-half hours before sacrifice. Adipose tissues from hypophysectomized rats fed the high sucrose diet showed a blunted insulin sensitivity as compared with normal rats on a similar diet. The insulin sensitivity of these tissues was further decreased 30 minutes after a GH injection. Basal glucose metabolism of isolated adipocytes from hypophysectomized rats, as compared with normal rats, was depressed if they were fed rat chow, was at normal levels if they were fed the high sucrose diet and was increased if they were fed the sucrose diet and received triiodothyronine and cortisone supplements. No manipulations of diet or hormonal treatments made the isolated adipocyte from hypophysectomized rats sensitive to insulin either 30 minutes or three and one-half hours after a GH injection. Since basal glucose utilization is not enhanced by GH injection and both the blunted insulin sensitivity of adipose tissue and the absent insulin sensitivity of adipopocytes would be expected to produce hyperglycemia rather than hypoglycemia, it is concluded that immediate systemic effects of a GH injection on carbohydrate metabolism are not related to changes in glucose metabolism of the peripheral adipose tissues.     

The histochemical localization of lactic dehydrogenase isoenzymes in the rat nephron by means of an improved polyvinyl alcohol method

Summary The histochemical localization of lactic dehydrogenase (LDH) activity and the prevailing type of isoenzyme in different segments of the nephron in male and female rats are described. Polyvinyl alcohol was added to the incubation medium in order to reduce enzyme diffusion. Localization of the reaction product was further improved by the use of a high concentration of Nitro BT (and of PMS and NAD).The three segments of the proximal tubules exhibited clearly different staining patterns. In glomeruli, terminal parts of the third proximal segments, thin limbs of Henle, and collecting ducts M subunits of LDH predominated, whereas in the remaining tubular segments H subunits prevailed. Sex differences were observed in respect to the LDH reaction in the proximal tubules, but not in the rest of the nephron. The localization of -hydroxy acid oxidase was also investigated, as this enzyme oxidizes lactate and therefore contributes to the reaction. Under certain conditions, i.e. high substrate concentration, the activity of -hydroxy acid oxidase was negligible in comparison with that of LDH.The Abbreviations used are PVA polyvinyl alcohol - PMS phenazine methosulfate - NAD nicotinamide adenine dinucleotide - Nitro BT nitroblue tetrazolium - Tris Tris (hydroxymethyl) aminomethane - PCMB p-chloromercuribenzoate Supported by grants from Fonden til Lægevidenskabens Fremme, and the Danish Medical Research Council.The terms H isoenzyme and M isoenzyme denote isoenzymes of LDH containing predominantly H subunits and M subunits, respectively.     

Acute effects in vivo of anti-insulin serum on rates of fatty acid synthesis and activities of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats.

Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.     

Adaptive changes in enzyme activity and metabolic pathways in adipose tissue from meal-fed rats

A number of metabolic factors and the activity of a number of enzymes were determined in meal-fed (animals fed a single daily 2 hr meal) and nibbling (ad libitum-fed) rats. The dependency of the observed adaptive changes on the ingestion of carbohydrate was studied by feeding diets high in carbohydrate or fat. Glucose-6-phosphate dehydrogenase and NADP-malic dehydrogenase were more active in adipose tissue from high carbohydrate meal-fed rats than in tissue from ad libitum-fed rats. The activity in adipose tissue of isocitric dehydrogenase, 6-phosphogluconate dehydrogenase, and NAD-malic dehydrogenase did not increase significantly in response to meal-feeding the high carbohydrate diet. No increase in lipogenesis or enzyme activity could be demonstrated in adipose tissue from rats meal-fed a high fat diet. Lipase activity of adipose tissue was increased by high carbohydrate meal-feeding and decreased by feeding a high fat diet. The in vitro uptake of palmitate-1-(14)C by adipose tissue was depressed by a high fat diet and enhanced in rats meal-fed a high carbohydrate diet. Diaphragm or slices of liver from high fat-fed rats oxidized palmitate-1-(14)C more rapidly than did tissue from ad libitum-fed animals. Evidence is presented for the quantitative importance of citrate as a source of extramitochondrial acetyl CoA in adipose tissue of meal-eating and ad libitum-fed rats. The relationship of extramitochondrially formed citrate to the NAD-malic dehydrogenase-malic enzyme system in adipose tissue is discussed.     

Brown adipose tissue heat production in heat acclimated and perchlorate treated rats

The noradrenaline-induced energy dissipation rate was measured with a direct microcalorimeter in brown adipose tissue taken from rats acclimated to 34 degrees C (HA), perchlorate treated (PC) and heat acclimated-perchlorate treated (HAPC). The response to 10(-7) M NA was reduced by 45%, 47% and 86% in HA, PC and HAPC groups, respectively, as compared to a control group kept at 24 degrees C. In the same groups, the response to 10(-6) M NA was reduced by 34%, 7% and 64%, respectively. The specific activity of the soluble alpha-glycerophosphate dehydrogenase in brown fat from HA rats was reduced by 50%, whereas it was not altered in the PC animals. It is concluded that the sensitivity to noradrenaline of the brown adipose tissue thermogenic mechanisms is decreased in hypothyroidism, and that the acclimation temperature and the thyroid status per se each have a different influence on brown adipose tissue function.     

NADPH generating enzymes in Leydig cells from diabetic rats.

Impaired testosterone biosynthesis in Leydig cells from streptozotocin treated rats is correlated with the reduced activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase. The results shown demonstrate that in the diabetic state the activity of these enzymes is reduced by almost 50 to 59% from normal levels. Insulin treatment restored their activities to normal levels. The diminished supply of NADPH in diabetic interstitial tissue is not the unique factor in the control of steroidogenesis, since the availability of large amounts of exogenous NADPH in the incubations of Leydig cell did not reduce the differences in testosterone synthesis observed when compared with normal cells.     

Effect of streptozotocin-induced diabetes and insulin treatment on the synthesis of hexokinase II in the skeletal muscle of the rat

The relative rate of synthesis of hexokinase II in the skeletal muscle of the normal, streptozotocin-diabetic, and diabetic insulin-treated rat was determined by the rate of incorporation of [3H]leucine into hexokinase II and the total cytosolic proteins to determine if the rate of hexokinase II synthesis was altered relative to that of the average protein. This relative rate of synthesis of hexokinase II is approximately 1.9 times higher in the normal than in the diabetic rat. The administration of insulin to the diabetic animal increases the rate of hexokinase synthesis to approximately normal levels. An enzyme-linked immunosorbent assay procedure was developed to determine the amount of hexokinase II protein in the skeletal muscle extracts, and immunoprecipitation was utilized to determine the hexokinase II activity. The specific activity of hexokinase II was determined from these analyses. The specific activity of hexokinase II was the same in the skeletal muscle extracts from normal, streptozotocin-diabetic, and diabetic insulin-treated rats. These results suggest that the decrease in muscle hexokinase activity is not caused by the loss of an activator of the enzyme nor by the increased formation of a hexokinase inhibitor in streptozotocin-induced diabetes; rather the decrease in hexokinase II activity reported in diabetic rats relative to normal animals is a result of decreased synthesis coupled to increased degradation in the diabetic relative to the normal animal.     

Effects of hypophysectomy, growth hormone, and thyroxine on protein turnover in heart.

Cardiac atrophy following hypophysectomy was accompanied by decreased heart content of RNA and polysomes and increased levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in vitro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower RNA content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, heart RNA content and rates of protein synthesis were equal to or greater than those found in normal hearts. When plasma levels of amino acids, glucose, fatty acids, and insulin, and rates of beating and ventricular pressure development observed in normal and hypophysectomized rats were simulated during in vitro perfusion, hearts from hormone-deficient rats had reduced rates of protein synthesis but unaltered rates of degradation. Cathepsin D activity in heart homogenates (+ Triton X-100) was elevated during cardiac atrophy when expressed per g of tissue but not when expressed per heart.