Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples.

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 microg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 microg/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYP1A2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYP1A2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, W/C, and C/C were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations.

Changes in zinc and copper homeostasis in human livers and kidneys associated with exposure to environmental cadmium.

This study was undertaken to assess changes in zinc and copper homeostasis in human tissues that could be attributed to human exposure to environmental cadmium, using samples of lung, liver and kidney cortex of 61 Queensland residents, aged 2 to 89 years, who had died of accidental causes. None of the subjects were exposed to cadmium in the workplace. Levels of zinc in liver and kidney cortex samples showed inverse associations with donor age whereas zinc in lung only showed inverse association with gender. Lung zinc levels in females were 14% lower than in males. Zinc in liver and kidney cortex samples were found to exist in at least two pools; one was associated with cadmium that bound to metallothionein (MT) and the other was associated with non-MT bound copper. In liver, the amounts of zinc in the MT pool were smaller compared to those in non-MT pool given that only 7% of zinc variations were explained by cadmium whereas 22% of the liver zinc variations were accounted for by non-MT bound copper. In sharp contrast, larger amounts of zinc in kidney cortex samples were in the MT pool, compared to those in the non-MT pool given that cadmium was found to explain 69% of total zinc variation whereas copper explained only 17% of kidney zinc variations. The levels of copper in liver were found to be increased by 45-50% in subjects with high cadmium exposure level, compared to subjects of similar ages with medium exposure level. The levels of zinc and copper in kidney cortex samples in the subjects with high cadmium exposure were both found to be significantly elevated compared to those found in the medium-exposure group whereas copper contents were about 19-23% greater than in medium- as well as low-exposure groups. Taken together these results indicate increased sequestration of zinc and copper in liver and kidney cortex samples. The increases in metal sequestrations were observed in liver samples having cadmium contents of greater than 1 microg/g wet weight and in kidney cortex having cadmium contents of greater than 26 microg/g wet weight. Zinc and copper contents in lung of this sample group, however, were not associated with cadmium due probably to lower exposure levels compared to those of liver and kidney.

Evidence for a synergistic interaction between cadmium and endotoxin toxicity and for nitric oxide and cadmium displacement of metals in the kidney.

This study was undertaken to examine changes in Zn and Cu homeostasis in the liver and kidney of rats caused by cadmium (Cd) or lipopolysaccharide (LPS) administration. Twenty-five male, 7- to 8-week-old Wistar rats were divided into five groups: saline only treatment, saline treatment and food deprivation, exposure to a single dose of Cd, exposure to LPS alone, and exposure to Cd + LPS. Changes in plasma nitrate concentrations and hepatic and renal Zn and Cu contents were measured together with urinary excretion rates for the metals and nitrate on 3 consecutive days: 24 h before treatment and 24 and 48 h after treatments. Cd exposure alone for 48 h caused a nearly 2-fold increase in plasma nitrate levels with no changes in urinary nitrate excretion whereas LPS treatment caused plasma nitrate levels to increase by 10-fold and urinary nitrate excretion to increase by 4-fold. Administration of LPS 24 h after Cd exposure caused a 10-fold increase in plasma nitrate concentrations and a 100-fold increase in urinary nitrate excretion compared to the rates prior to LPS administration. These results indicate a synergistic interaction between Cd and LPS toxicity. Cd exposure also caused a marked increase in hepatic Zn levels, but LPS did not cause any changes in hepatic Zn or Cu content. In sharp contrast, both Zn and Cu contents were decreased in the kidneys by 16 and 36% in animals exposed to Cd or LPS. A correlation analysis of measured variables reveals that renal Cu contents were inversely associated with plasma nitrate concentrations while urinary Cu excretion on day 3 showed a strong positive correlation with both urinary nitrate and Cd excretions on the same day. A linear regression analysis shows 20% of the variation in urinary Cu excretion was associated with urinary Cd excretion on the same day. It is concluded that reductions in renal Cu contents caused by Cd or LPS administration may be a result of Cd and NO displacement of Cu previously bound to metallothionein.

Safe levels of cadmium intake to prevent renal toxicity in human subjects.

The present review attempts to provide an update of the scientific knowledge on the renal toxicity which occurs in human subjects as a result of chronic ingestion of low-level dietary Cd. It highlights important features of Cd toxicology and sources of uncertainty in the assessment of health risk due to dietary Cd. It also discusses potential mechanisms for increased susceptibility to Cd toxicity in individuals with diabetes. Exposure assessment on the basis of Cd levels in foodstuffs reveals that vegetables and cereals are the main sources of dietary Cd, although Cd is also found in meat, albeit to a lesser extent. Cd accumulates particularly in the kidney and liver, and hence offal contains relatively high amounts. Fish contains only small quantities of Cd, while crustaceans and molluscs may accumulate larger amounts from the aquatic environment. Data on Cd accumulation in human kidney and liver obtained from autopsy studies are presented, along with results of epidemiological studies showing the relationship between renal tubular dysfunction and kidney Cd burden. These findings suggest that a kidney Cd level of 50 microg/g wet weight is a maximum tolerable level in order to avoid abnormal kidney function. This renal Cd burden corresponds to a urinary Cd excretion of 2 microg/d. Accordingly, safe daily levels of Cd intake should be kept below 30 microg per person. Individual variations in Cd absorption and sensitivity to toxicity predicts that a dietary Cd intake of 30 microg/d may result in a slight renal dysfunction in about 1% of the adult population. The previous guideline for a maximum recommended Cd intake of 1 microg/kg body weight per d is thus shown to be too high to ensure that renal dysfunction does not occur as a result of dietary Cd intake.

Relationships between the synthesis of N-nitrosodimethylamine and immune responses to chronic infection with the carcinogenic parasite, Opisthorchis viverrini, in men.

This study investigated the relationship between immune responses to infection with the liver fluke, Opisthorchis viverrini, and the synthesis of the carcinogen, N-nitrosodimethylamine (NDMA) in humans. It also examined associations between synthesis of nitric oxide (NO) and nitrosation of amines, in vivo. Antibody and T cell responses to fluke antigens and post-alcohol urinary NDMA excretion were assessed among three groups of 40-50 men with no, moderate and heavy liver fluke infection. Markers of NO synthesis (nitrate, nitrite) and nitrosation (nitrosamino acids) were also measured in biological fluids. Assessments were carried out under controlled conditions which minimised intake of exogenous nitrate and nitrite and were carried out at two time points, namely before and 4 months after elimination of the infection with praziquantel treatment. No statistically significant variation was observed in the amount of NDMA excreted between the 3 groups. However, during active infection, a strong negative association was observed between in vitro lymphoproliferative responses to some liver fluke antigens and NDMA excretion. After treatment this association was reduced. Multivariate statistical models revealed a highly significant relationship between NDMA levels and urinary nitrate, stimulation indices for two T cell responses to two parasite antigens (MW 37 kDa and 110 kDa) and gall bladder dimensions. NDMA levels after treatment were best described by the ratio between parasite-specific IgG2 and IgE, background levels of T cell proliferation, a urinary marker of nitrosation (N-nitrosothioproline) and usual level of alcohol consumption. These results suggest that individual background immunologic activity, parasite-specific responses and/or parasite products and NO synthesis are important determinants of endogenous generation of nitrosamines in O. viverrini-infected humans.

Insulin attenuates atrophy of unweighted soleus muscle by amplified inhibition of protein degradation.

Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.

Induction of cytochrome P450 2A6 expression in humans by the carcinogenic parasite infection, opisthorchiasis viverrini.

The purpose of this study was to examine in vivo the activity of cytochrome P450 (CYP) 2A6, an enzyme capable of activating carcinogens, including N-nitrosodimethylamine, in humans with the carcinogenic liver fluke infection, opisthorchiasis viverrini, before and after treatment with the antiparasitic agent, praziquantel. Coumarin hydroxylase activity of CYP 2A6 was assessed by administering a probe drug, coumarin, and measuring its metabolite, 7-hydroxycoumarin, in urines collected between 0-2 h and 2-4 h of 106 people with varying intensities of Opisthorchis viverrini infection. Five individuals who did not excrete any detectable 7-hydroxy coumarin (and have a genetic defect probably leading to an absence of catalytic activity of the CYP 2A6 protein) were excluded from analysis. Infected people excreted an average of 22.7 mumol of 7-hydroxycoumarin in the first 2 h after taking the drug, whereas the mean of the uninfected group was 19.4 mumol; this difference did not reach statistical significance (P = 0.10). However, a highly significant increase in CYP 2A6-related activity was observed in infected individuals who also had radiological evidence of biliary fibrosis (28.1 mumol) compared to those without (19.4 mumol; P = 0.01). Reassessments of coumarin hydroxylase activity of CYP 2A6 made 2 months after praziquantel treatment showed highly significant reductions in the amount of 7-hydroxycoumarin excreted among the infected groups but no difference in the uninfected group. These results suggest that expression of CYP 2A6 is induced among chronically infected people who also have fibrosis of the intrahepatic bile duct. As already demonstrated in an animal model and now observed in humans for the first time, this increase in CYP 2A6-related enzyme activity may represent an important mechanistic link between inflammatory products of chronic liver fluke infection (e.g., DNA alkylation damage from endogenously formed N-nitrosamines) and the high risk of cholangiocarcinoma faced by infected individuals.

Thiocyanate-independent nitrosation in humans with carcinogenic parasite infection.

Infection with the liver fluke, Opisthorchis viverrini, is a causative agent of cholangiocarcinoma. One possible contributing factor in this carcinogenesis is the chronic, local generation of nitric oxide by inflammatory cells expressing inducible nitric oxide synthase and the production of N-nitroso compounds via the reaction between amines and nitrosating agents derived from nitric oxide. Our previous studies provided evidence that nitric oxide synthesis is elevated during human liver fluke infection. Here we present data on the same sample of men which definitively demonstrates increased nitrosation of proline and thioproline (thiazolidine-4-carboxylic acid) among infected men compared to uninfected control subjects on a low nitrate diet. This difference was specifically abolished by co-administration of ascorbic acid with proline and by elimination of parasites by praziquantel treatment. Multivariate statistical models demonstrate the importance of salivary thiocyanate levels to variation in the nitrosation of proline among uninfected individuals, but not among those with current fluke infection. This suggests that considerable generation of nitrosating agents (N203/N204) in infected people may be occurring via oxidation of arginine by nitric oxide synthase in inflamed tissue which is thiocyanate insensitive. Analyses revealed positive associations between N-nitrosoproline excretion and nitrate/nitrite levels in urine, plasma and saliva and with usual alcohol intake; with variation in these trends between groups. In conclusion, we have confirmed the relationship between O.viverrini infection and enhanced endogenous nitrosation, showing evidence of its extragastric site. New information is also provided on the determinants of N-nitrosamino acid excretion in men on a controlled low nitrate diet without smoking, conditions which reduce exogenous sources of nitrosating agents.

Parasite-associated morbidity: liver fluke infection and bile duct cancer in northeast Thailand.

Infection with the liver fluke, Opisthorchis viverrini, remains a major public health problem in Northeast Thailand, where approximately one-third of the population is infected. The northeast region is largely populated by Laos-descendent Thais who enjoy eating raw fish, which harbour the infective stage of the fluke. The parasite has maintained its presence in the population despite the widespread use of praziquantel and dissemination of health education material throughout the region by vigorous government-sponsored programs in recent years. The most severe consequence of liver fluke infection is cholangiocarcinoma, i.e. cancer of the bile duct epithelium. Although mortality due to the parasites alone appears to be uncommon, cholangiocarcinoma arising as a result of infection is one of the leading causes of death in the region. This paper reviews the pathogenesis of infection and the geographic, hospital-based and community studies which demonstrate the close relationship between infection and cancer. In addition, data from the Cancer Registry of Khon Kaen, Northeast Thailand and population-based studies using ultrasonography to visualize early tumours which illuminate the very high frequency of the cancer among heavily infected individuals and communities are discussed. Finally, the paper will close with a brief commentary on the prospects for control of the parasite and its likely impact on the frequency of cancer given the current epidemiological situation of liver fluke infection.

Liver fluke infection and cholangiocarcinoma: model of endogenous nitric oxide and extragastric nitrosation in human carcinogenesis.

Cancers arising during bacterial, viral and parasitic infection provide useful models to investigate the link between inflammation and carcinogenesis. Because the inflammatory agent is known, relationships between immune responses, the production of DNA-damaging agents, such as nitric oxide, oxygen radicles and N-nitroso compounds, and cancer risk can be explored. This paper first describes the close relationship between infection with the liver fluke, Opisthorchis viverrini, and cholangiocarcinoma in humans. Data are then presented which demonstrate an elevation in levels of salivary nitrite and urinary and plasma nitrate among men with moderate and heavy liver fluke infections compared to uninfected controls which was absent 4 months after the parasites were cleared with praziquantel. Because of the strict control over subject selection and dietary intake plus the absence of the increase following treatment, we conclude that the higher levels of nitrate and nitrite reflect endogenous generation of nitric oxide resulting from liver fluke infection. Excess nitric oxide generation in the inflamed tissue is likely to lead directly to the formation of N-nitroso compounds mediated by activated macrophages. Further work will attempt to demonstrate a link between this increase and both parasite-specific immune responses and the risk of cancer.

Time course of the response of myofibrillar and sarcoplasmic protein metabolism to unweighting of the soleus muscle.

Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of 3H-phenylalanine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show (1) acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and (2) sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

Cholangiocarcinomas in Japanese and Thai patients: difference in etiology and incidence of point mutation of the c-Ki-ras proto-oncogene.

Point-mutational activation of the c-Ki-ras proto-oncogene has been shown to be rare in human hepatocellular carcinoma, the most common primary liver cancer and one usually associated with chronic viral infection. To reveal the association of c-Ki-ras activation with cholangiocarcinogenesis under different etiological backgrounds, the incidence of point mutation at codons 12 and 13 of the c-Ki-ras proto-oncogene was examined in three groups of human liver cancers with differentiation to biliary epithelial cells: Group 1, cholangiocellular carcinoma in Japanese with normal livers; Group 2, cholangiocellular carcinoma in Thais who had lived in an area where the liver fluke Opisthorchis viverrini is endemic; and Group 3, combined hepatocellular-cholangiocellular carcinoma, a rare type showing features of both hepatocellular and biliary epithelial differentiation, in Japanese with chronic viral hepatitis with or without cirrhosis. The polymerase chain reaction and direct sequencing of its product were used to detect the mutation. Point mutation at codon 12 of the c-Ki-ras gene was detected in five (56%) of nine cases in Group 1. In contrast, the mutation was not detected in any of the cases in Groups 2 and 3. Therefore, point-mutational activation of c-Ki-ras did not seem to be involved in the development of primary liver cancers associated with apparent chronic irritation of liver cells or biliary epithelial cells caused by exogenous liver-fluke or viral infection. On the other hand, point-mutational activation of the c-Ki-ras proto-oncogene may be involved in cholangiocarcinogenesis in liver without preexisting liver-fluke or viral infection.

Insulin effects in denervated and non-weight-bearing rat soleus muscle.

Previous reports indicated that glucose uptake in denervated muscle is resistant to insulin, while in non-weight-bearing (unweighted) muscle this effect of insulin is enhanced. To extend the comparison of these differences, insulin effects on amino acid uptake and protein metabolism were studied in soleus muscles subjected to denervation or unweighting. Denervated muscle showed insulin resistance of both 2-deoxy[1,2-3H]glucose and alpha-[methyl-3H]aminoisobutyric acid uptake whereas unweighted muscle showed an increased or normal response, respectively. Atrophy was greater in denervated than in unweighted muscle, apparently due to faster protein degradation. The stimulation of protein synthesis and the inhibition of protein degradation by insulin was generally less in denervated than in unweighted muscle. Since metabolic measurements in denervated-unweighted muscles did not differ from those in denervated-weight-bearing muscles, effects of denervation must be independent of leg posture.

Different mechanisms of increased proteolysis in atrophy induced by denervation or unweighting of rat soleus muscle.

Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents (eg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which antagonizes sarcoplasmic reticulum release of Ca2+, reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to denervation. Calcium ionophore (A23187) increased proteolysis more so in unweighted than control muscles whether or not Ca2+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (1) atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome, and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2(+)-dependent proteolysis and/or thiol proteases.

Effects of stretching and disuse on amino acids in muscles of rat hind limbs.

Effects of stretching on muscle amino acids were tested in unloaded soleus by casting the foot in dorsiflexion on one limb of tail-casted, hindquarter-suspended rats. For comparison with unloading, amino acids also were measured in shortened extensor digitorum longus (EDL) in the same casted limb and in denervated leg muscles. Concentrations of tyrosine and glutamate were lower, while aspartate, ammonia, and the ratio of glutamine to glutamate were greater in the stretched than in the freely moving, unloaded soleus, but stretched did not differ from weight-bearing, control muscle. Therefore, stretching the soleus muscle prevented changes in certain amino acids due to unloading. Aspartate, ammonia, glutamine, and the ratio of glutamine to glutamate were lower in the shortened EDL than in the freely moving muscle of the contralateral limb, or in the control muscle. When denervated, these leg muscles also showed lower aspartate, ammonia, and ratio of glutamine to glutamate relative to innervated muscles. Since muscle shortening or denervation produced amino acid changes that mimicked the effects of unloading on the soleus, these responses must reflect the effect of muscle disuse. These data suggested that lower ammonia might cause the lower ratio of glutamine to glutamate with disuse. Because the fresh muscle energy charge, one factor which controls AMP deaminase, generally was not affected by disuse, altered deamination of glutamate via glutamate dehydrogenase may explain the variations in muscle ammonia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of immobilization on rat hind limb muscles under non-weight-bearing conditions.

Dorsiflexion of one unloaded hind limb caused hypertrophy of the soleus relative to weight-bearing controls and faster growth of the plantaris and gastrocnemius muscles relative to the contralateral freely moving muscles. Unloading of the soleus muscle diminished primarily myofibrillar proteins whereas stretching increased all proteins. Stretching the soleus increased RNA, accelerated, especially, in vitro synthesis of sarcoplasmic proteins, and diminished in vitro proteolysis. Both in vivo and in vitro results showed slower synthesis and faster degradation in the freely moving than in the weight-bearing soleus muscle, faster synthesis and slower degradation in the stretched than in the freely moving soleus muscle, and faster degradation in the stretched than in the weight-bearing soleus muscle. Hence, stretching of the soleus muscle prevented changes in mass and protein metabolism produced by unloading. Shortening of the extensor digitorum longus muscle produced less muscle growth, slowed in vitro protein synthesis, and lowered RNA relative to the contralateral, freely moving muscle.

Problems in analysis of data from muscles of rats flown in space.

Comparison of hindlimb muscles of rats flown on Spacelab-3 or tail-traction-suspended showed that 11-17 h reloading post-flight might have altered the results. Soleus atrophied, plantaris, gastrocnemius and extensor digitorum longus grew slower, and tibialis anterior grew normally. In both flight and simulated soleus and plantaris, higher tyrosine and greater glutamine/glutamate ratio indicated negative protein balance and increased glutamine production, respectively, relative to controls. Aspartate was lower in these muscles. Reloading generally decreased tyrosine, but increased aspartate and glutamine/glutamate. These data showed that at 12 h of reloading after flight is characterized by reversal to varying extents of effects of unloading.

Rat muscle protein turnover and redox state in progressive diabetes.

Protein synthesis and degradation, and redox state were measured in soleus and extensor digitorum longus muscles of rats up to 12 days after injection of streptozotocin. Muscle growth was slower in these animals apparently due to slower protein synthesis throughout the duration of diabetes. Up to day 4 after injection of streptozotocin or withdrawal of insulin from treated, diabetic animals, the muscle ratio of lactate/pyruvate, an indicator of the cytoplasmic NAD+ redox couple, was lower and protein degradation was faster than in control muscles. Thereafter, the ratio of lactate/pyruvate was greater and protein degradation was slower than in size- or age-matched control muscles. Insulin treatment in vitro or in vivo increased lactate/pyruvate and decreased proteolysis. Therefore, in muscles of streptozotocin-diabetic rats, the initial increase and later fall in proteolysis, and the inhibition of proteolysis by insulin, may correlate with opposite changes in NADH/NAD+.