Ketone-body metabolism after partial hepatectomy in the rat.

Fed or 24 h-starved rats were subjected to two-thirds partial hepatectomy or sham-operation and subsequently starved for 4, 14 or 24 h. Despite high plasma fatty acid concentrations, the partially hepatectomized rats failed to respond to post-operative starvation with increased blood and liver ketone-body concentrations or to maintain the high ketone-body concentrations associated with pre-operative starvation. Hypoglycaemia and hyperlactaemia were observed within 30 min of functional hepatectomy, but not partial hepatectomy, of 24 h-starved rats, and, even after a further 24 h starvation of partially hepatectomized rats, blood glucose concentrations were only slightly decreased. The results are discussed with reference to fat oxidation and gluconeogenesis in the liver remaining after partial hepatectomy.     

Amino acid metabolism in tumour-bearing mice.

Mice bearing the Lewis lung carcinoma showed a high tumour glutaminase activity and significantly higher concentrations of most amino acids than in both the liver and the skeletal muscle of the host. Tumour tissue slices showed a marked preference for glutamine, especially for oxidation of its skeleton to CO2. It is proposed that the metabolism of this particular carcinoma is focused on amino acid degradation, glutamine being its preferred substrate.     

Effect of endurance training upon lipid metabolism in the liver of cachectic tumour-bearing rats

The syndrome of cancer cachexia is accompanied by several alterations in lipid metabolism, and the liver is markedly affected. Previous studies showed that moderate exercise training may prevent liver fat accumulation through diminished delivery of lipids to the liver, increased hepatic oxidation and increased incorporation of triacylglycerol (TAG) into very low density lipoprotein (VLDL). Our aim was to examine the influence of moderate intensity training (8 weeks) upon TAG content, VLDL assembly and secretion, apolipoprotein B (apoB) and microsomal transfer protein (MTP) gene expression in the liver of cachectic tumour-bearing rats. Animals were randomly assigned to a sedentary control (SC), sedentary tumour-bearing (ST) or exercise-trained control (EC) or to an exercise trained tumour-bearing (ET) group. Trained rats ran on a treadmill (60% VO(2max)) for 60 min day(-1), 5 day week(-1), for 8 weeks. TAG content and the rate of VLDL secretion (followed for 3 h), as well as mRNA expression of apoB and MTP, and total cholesterol, VLDL-TAG, VLDL-cholesterol, high density lipoprotein cholesterol (HDL-cholesterol) and tumour weight were evaluated. VLDL-cholesterol showed a decrease in ST (p < 0.05) in relation to SC. Serum TAG, VLDL-TAG and tissue TAG content were all increased in ST (p < 0.01), when compared with SC. ST showed a lower rate of VLDL secretion (p < 0.05) and reduced expression of apoB (p < 0.001) and MTP (p < 0.001), when compared with SC. These parameters were restored to control values (p < 0.05) when the animals were submitted to the exercise training protocol. Tumour weight decreased 10-fold after training (p < 0.001). It is possible to affirm, therefore, that endurance training promoted the re-establishment of lipid metabolism in cachectic tumour-bearing animals, especially in relation to VLDL secretion and assembly.     

Protein kinases from liver mitochondria of tumour-bearing rats.

The mitochondria of liver of Yoshida ascites tumour-bearing rats contained two forms of protein kinase distinguishable on the basis of their kinetic properties, substrate specificity and responses to cyclic adenosine 3',5'-monophosphate (cAMP). One of these (kinase I) was activated 2-3 fold by cAMP while the other form (kinase II) was insensitive to the action of cAMP. Kinase I which was selective towards histone F1 as substrate was obtained as a homogeneous preparation and was observed to have a molecular weight of 170 000 by Sephadex G-150 gel filtration. Protein kinase II appeared to be a smaller protein with molecular weight of 54 000 and was specific towards acidic proteins namely casein and phosvitin. Protein kinases isolated from liver mitochondria of normal rats showed variations in respect to elution profile of DEAE-cellulose and electrophoretic mobility. The preparation corresponding to kinase I did not show stimulatory responses to cAMP.     

The profile of melatonin production in tumour-bearing rats

The pineal gland is involved in the regulation of tumour growth through the anticancer activity of melatonin, which presents immunomodulatory, anti-proliferative and anti-oxidant effects. In this study we measured melatonin content directly in the pineal gland, in an attempt to clarify the modulation of pineal melatonin secretory activity during tumour growth. Different groups of Walker 256 carcinosarcoma bearing rats were sacrificed at 12 different time points during 24h (12h:12h light/dark cycle) on different days during the tumour development (on the first, seventh and fourteenth day after tumour inoculation). Melatonin content in the pineal gland was determined by high-performance liquid chromatography with electrochemical detection. During tumour development the amount of melatonin secreted increased from 310.9 ng/mg of protein per day from control animals, to 918.1 ng/mg of protein per day 14 days after tumour implantation, and there were changes in the pineal production profile of melatonin. Cultured pineal glands obtained from tumour-bearing rats turned out to be less responsive to noradrenaline, suggesting the existence, in vivo, of putative factor(s) modulating pineal melatonin production. The results demonstrated that during tumour development there is a modification of pineal melatonin production daily profile, possibly contributing to cachexia, associated to changes in pineal gland response to noradrenaline stimulation.     

Effect of a moderate intensity exercise training protocol on the metabolism of macrophages and lymphocytes of tumour-bearing rats

It is commonly accepted that moderate intensity exercise is beneficial to the immune system. We tested the influence of a moderate intensity training protocol (8 weeks) upon immune system function in Wistar tumour-bearing (TB) rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, together with some functional parameters (hydrogen peroxide production and lymphocyte proliferative response). These substrates were chosen since they represent the most important energetic and synthetic metabolites for these cellular types. The training protocol caused a decrease of 17.4 per cent in the production of H(2)O(2) by macrophages, as well as a decrease in glucose consumption (25 per cent) and lactate production (47.1 per cent), and an increase in the production of labelled CO(2) from the oxidation of [U-(14)C]-glucose, in TB rats. The training protocol was also able to induce changes in the maximal activity of some key enzymes in the metabolism of glucose and glutamine, a reduction of hexokinase (68.8 per cent) activity and an increase in the activity of citrate synthase (10.1 per cent) in TB rats. The training protocol increased the proliferative response of lymphocytes cultivated in the absence of mitogens (75 per cent), of those cultivated in the presence of ConA (38.2 per cent) and in the presence of LPS (45.0 per cent). These cells also showed an increase in the maximal activity of some key enzymes of the glycolytic and glutaminolytic pathways. Our data demonstrated that the training protocol was able to induce an increase in aerobic utilisation of both substrates in lymphocytes and macrophages. The training protocol was also able to prevent several changes in glucose and glutamine metabolism that are normally present in sedentary TB rats. These changes in immune cell metabolism induced by the training protocol were able to increase TB rat survival.     

Increased liver alanine in the tumour-bearing host. Altered levels of some enzymes involved in alanine metabolism

A 2-fold increase in hepatic alanine concentration was observed in rats bearing a Walker 256 carcinoma growing sub-cutaneously. Decreases were observed in the activities of both cytosolic and mitochondrial isozyme forms of L-alanine-2-oxoglutarate aminotransferase. Activities of two enzymes involved in a secondary pathway of haem synthesis involving alanine, L-alanine-4,5-dioxovalerate aminotransferase and the NADP-requiring isozyme form of 4-oxo-5-hydroxyvalerate dehydrogenase were also reduced but there was no change in liver porphyrin concentration. L-alanine-glyoxalate aminotransferase activity was unaffected. The results are discussed in relation to the utilisation of alanine as a gluconeogenic substrate in the tumor-bearing host.     

Hyaluronidase in sera of tumour-bearing nude mice.

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l(-1) (mean+/-SD 432+/-70 mU l(-1), median 440 mU l(-1)). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.     

Ketone-body metabolism after surgical stress or partial hepatectomy. Evidence for decreased ketogenesis and a site of control distal to carnitine palmitoyltransferase I.

Rats were subjected to laparotomy, or laparotomy and partial hepatectomy, at 0-48 h before administration of water or medium-chain-length triacylglycerol, having been starved post-operatively. Functional hepatectomies were performed at intervals after the intragastric load. Blood ketone-body concentrations after medium-chain triacylglycerol administration and/or functional hepatectomy of these rats were compared with values obtained in starved control rats. Decreased ketonaemia in response to medium-chain triacylglycerol was observed for up to 48 h after partial hepatectomy and at 1 and 2 h after laparotomy, but not at 24 or 48 h after laparotomy. Rates of ketone-body clearance after functional hepatectomy were unaffected by prior laparotomy or partial hepatectomy. Ketonaemia after medium-chain-triacylglycerol administration was only partially blocked by inhibition of CPT I (carnitine palmitoyltransferase I). The results demonstrate sustained effects of partial hepatectomy and short-term effects of surgical stress to decrease ketonaemia via inhibition of ketogenesis at site(s) distal to CPT I.     

Leucine and tryptophan metabolism in rats.

The rate of tryptophan metabolism in isolated liver cells from animals fed on a high-leucine diet was greater than for cells from control animals. Leucine inhibited tryptophan metabolism and tryptophan uptake in isolated liver cells, probably by competing for membrane transport. Leucine had no effect on tryptophan 2,3-dioxygenase in vitro. 4-Methyl-2-oxovalerate increased tryptophan oxidation in incubations containing albumin, by displacing bound tryptophan and increasing the availability of the amino acid to the cell. The results suggest that, under extreme conditions, when the availability of tryptophan is low, leucine may be pellagragenic.     

Ketone-body metabolism in hyperthyroid rats: reduced activity of D-3-hydroxybutyrate dehydrogenase in both liver and heart and of succinyl-coenzyme A: 3-oxoacid coenzyme A-transferase in heart

The specific activity of D-3-hydroxybutyrate dehydrogenase is reduced by about a third in liver and heart mitochondria of hyperthyroid rats. State 3 respiration is also reduced in isolated mitochondria from the same animals when DL-3-hydroxybutyrate is the substrate. Determination of the kinetic parameters of the membrane-bound D-3-hydroxybutyrate dehydrogenase in liver of hyperthyroid rats reveals a decreased in maximal velocity (Vmax). The Michaelis and dissociation constants of NAD+ and D-3-hydroxybutyrate are also significantly influenced, thus indicating that both the affinity and the binding of this enzyme toward its substrates are affected. In hyperthyroid rats a significant ketone-body increase is found in both liver and heart: in blood, an almost doubled concentration can be measured. At the same time, in heart mitochondria of these animals the activity of succinyl-coenzyme A: 3-oxoacid coenzyme A-transferase is significantly reduced. The decrease in both D-3-hydroxybutyrate dehydrogenase and 3-oxoacid coenzyme A-transferase associated with the increase in ketone bodies supports the suggestion that there is a lower utilization of these compounds by peripheral tissues. In the blood of hyperthyroid rats a higher D-3-hydroxybutyrate/acteoacetate ratio is also found, probably resulting from a selective utilization of the two compounds in this pathological state.     

Protein synthesis, myosin ATPase activity and myofibrillar protein composition in hearts from tumour-bearing rats and mice.

Growing rats and adult weight-stable mice bearing a transplantable methylcholanthrene-induced sarcoma were compared with animals with various states of malnutrition. Heart protein synthesis was measured in vivo. Myocardial RNA, myofibrillar protein composition and the Ca2+-activated ATPase activity in heavy chains of native myosin were measured. 'Fingerprints' were made from myosin by trypsin treatment to evaluate possible structural changes in the protein. Cardiac protein-synthesis rate was decreased by 20% in growing tumour-bearing rats, by 35% in protein-malnourished (rats) and by 47% in starved rats, compared with freely fed controls (P less than 0.05). Adult tumour-bearing mice showed no significant decrease in myocardial protein synthesis. Pair-weighed control mice had significantly depressed heart protein synthesis. Protein translational efficiency was maintained in both tumour-bearing rats and mice, but was decreased in several groups of malnourished control animals. The Ca2+-activated myosin ATPase activity was decreased in all groups of malnourished animals, including tumour-bearing mice and rats, without any evidence of a change in cardiac isomyosin composition. We conclude that loss of cardiac muscle mass in tumour disease is communicated by both depressed synthesis and increased degradation largely owing to anorexia and host malnutrition. Increased adrenergic sensitivity in hearts from tumour-bearing and malnourished animals is not communicated by increased Ca2+-activated ATPase activity. This may be down-regulated in all groups with malnutrition, without any observable alterations in the isomyosin profile.     

Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats

Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer-induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4-dinitrophenylhydrazine assay), both malondialdehyde- (MDA-) and 2-hydroxy-4-nonenal- (HNE-) protein adducts, Mn-superoxide dismutase (Mn-SOD), catalase, heme oxygenase-1 (HO-1) and 3-nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH-130 hepatoma. In the muscles of the tumour-bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA-protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn-SOD, catalase, and HO-1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer-induced cachexia.     

Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats

 Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression. Received: 16 October 1997 / Accepted: 8 January 1998