Plasma amino acids of lean and obese Zucker rats subjected to a cafeteria diet after weaning.

Plasma amino acids of Zucker obese (fa/fa) and lean (Fa/?) rats fed either a reference nonpurified pellet or a cafeteria diet have been studied from 30 to 60 days after birth. Obese rats showed higher plasma branched chain amino acid levels but similar total amino acids, urea and glucose concentrations. The ingestion of a cafeteria diet induced higher levels in many amino acids, as well as in the composite figure in lean rats, but failed to alter total 2-amino nitrogen concentrations in obese rats, despite high levels in several non-essential amino acids and lower values in essential amino acids; urea levels were much lower in rats fed the cafeteria diet. The results are consistent with an impairment of amino acid nitrogen elimination via urea cycle in cafeteria diet-fed rats. This is independent of the hyperinsulinemia-driven plasma accumulation of several essential amino acids induced by genetic obesity. The effects were, then additive.     

No significant net uptake of branched chain amino acids by the liver of fed-mid pregnant rats

The hepatic balance for valine, leucine and isoleucine has been measured in anaesthetized virgin controls and 9 and 12-day pregnant rats. The liver of non gravid animals show fractional extraction rates of over 11.6% for valine, 17.6% for leucine and 16.8% for isoleucine. Fed mid-pregnant rats do not show either a significant net uptake nor a release of these amino acids. It is proposed that higher intestinal amino acid requirements for protein synthesis during mid pregnancy than before impregnation may be, in part, the cause of a decreased hepatic uptake and, thus, a different role for the liver in the amino acid inter-organ relationships during this period is suggested.     

Individual amino acid balances in young lean and obese Zucker rats fed a cafeteria diet

The amino acid composition of the diet ingested by reference and cafeteria diet-fed lean and obese Zucker rats has been analyzed from day 30 to 60 after birth. Their body protein amino acid composition was measured, as well as the urinary and faecal losses incurred during the period studied. The protein actually selected by the rats fed the cafeteria diet had essentially the same amino acid composition as the reference diet. The mean protein amino acid composition of the rat showed only small changes with breed, age or diet.Cafeteria-fed rats had a higher dietary protein digestion/absorption efficiency than reference diet-fed rats. Obese rats wasted a high proportion of dietary amino acids when given the reference diet, but not on the cafeteria diet. In all cases, the amino acids lost as such in the urine were a minimal portion of available amino acids.In addition to breed, the rates of protein accretion are deeply influenced by diet, but even more by the age — or size — of the animals: cafeteria-fed rats grew faster, to higher body protein settings, but later protein accrual decreased considerably; this is probably due to a limitation in the blueprint for growth which restricts net protein deposition when a certain body size is attained. Obese rats, however, kept accuring protein with high rates throughout.Diet composition — and not protein availability or quality-induced deep changes in amino acid metabolism. Since the differences in the absolute levels of dietary protein or carbohydrate energy ingested by rats fed the reference or cafeteria diets were small, it can be assumed that high (lipid) energy elicits the changes observed in amino acid metabolism by the cafeteria diet. The effects induced in the fate of the nitrogen ingested were more related to the fractional protein energy proportion than to its absolute values. Cafeteria-fed rats tended to absorb more amino acids and preserve them more efficiently; these effects were shown even under conditions of genetic obesity.There were deep differences in handling of dietary amino acids by dietary or genetically obese rats. The former manage to extract and accrue larger proportions of their dietary amino acids than the latter. The effects of both models of amino acid management were largely additive, suggesting that the mechanisms underlying the development of obesity did not run in parallel to those affecting the control of amino acid utilization. Obesity may be developed in both cases despite a completely different strategy of amino acid assimilation, accrual and utilization. (Mol Cell Biochem121: 45–58, 1993)     

Amino-acid enzyme activities in liver and kidney of developing rats

The amino-acid enzymes (aspartate-, alanine- and tyrosine transaminases, serine dehydratase, glutamate dehydrogenase, glutamine synthetase, adenylate deaminase and arginase) activities in the liver and kidney of developing rats (days 19 and 21 after conception and 1, 5, 10, 20 and 30 after birth) compared with adults were determined in crude homogenates. Most enzymes attained the adult levels early after birth or at weaning, showing a marked trend towards amino-acid nitrogen conservation during late foetal and specially during the neonatal period, increasing their activity during lactation. It is postulated that these changes are closely related to availability of low grade protein in diet as well as to maturation of amino-acid homeostasis maintenance for growth.     

Intestinal handling of an oral oleoyl-estrone gavage by the rat

Adult Zucker lean (Fa/?) female rats received a single 250 nmol oral gavage of 3H-labelled oleoylestrone in 0.2 ml of sunflower oil. After one hour, samples of arterial, portal and suprahepatic blood, and lymph were obtained and fractioned to determine the amount of radioactivity present in the form of free estrone, acyl-estrone and hydrophilic estrone esters in the blood of each vessel. Lipoprotein fractions (chylomicra + VLDL, LDL, HDL and lipoprotein-depleted plasma) were also analysed as well as the distribution of absorbed 3H-estrone in the intestine, specific organs and carcass. About one third of the oleoyl-estrone dose recovered was found in the tissues, mainly in the blood, the rest remaining relatively untouched in the intestinal content. High hypothalamic estrone uptake (compared with the rest of the brain) was observed. Data from non-radioactive estrone measurements showed a similar pattern of absorption and tissue distribution to that obtained by 3H-estrone tracking alone. In both cases, most of the estrone present in the intestinal lumen was absorbed as intact oleoyl-estrone, but a significant part was absorbed as free estrone. There is a net transfer of 3H-estrone into portal blood HDL, and part of the 3H-estrone is also loaded into lymph-carried chylomicra. A large share of free estrone is filtered by the liver, but most of the acyl-estrone absorbed passes unaltered. The oral administration of oleoyl-estrone results in significant absorption of the unaltered molecule, which is transferred to lymph-carried chylomicra and also directly to plasma HDLs. It may be inferred that the HDL fraction contains the physiological carrier of oleoyl-estrone in its role of ponderostat signal.     

Ammonium uptake and urea production in hepatocytes from lean and obese Zucker rats

The metabolic differences in vitro between genetic and dietary obese rats in the uptake of ammonium and amino acids by the liver and their use for ureogenesis have been assayed using hepatocytes isolated from Lean, Obese Zucker (Genetic obese) rats and Dietary obese rats. The hepatocytes of genetic obese animals took up more ammonium and produced higher amounts of urea from ammonium and alanine than those of lean and dietary obese groups (2 and 5 times more respectively). In the lean and dietary obese groups urea synthesis accounted for almost all the nitrogen taken up as ammonium. Thus, dietary and genetic obesity show a widely different handling of nitrogen, and the genetic obese rats need to break down protein to maintain their hepatocyte function.     

Short-term treatment with estrone oleate in liposomes (Merlin-2) does not affect the expression of the ob gene in Zucker obese rats

Young female Zucker fa/fa rats of 370-430 g were implanted with osmotic minipumps releasing 3.5 mol/dayúkg of estrone oleate in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a sustained loss of appetite, and a decrease in body weight of 3.5%, which contrasts with the 8.2% increase in controls during the period studied. Plasma insulin, glucose and urea decreased, and liver glycogen increased with Merlin-2 treatment. Plasma ACTH and corticosterone increased to a maximum at the end of the experiment. The expression of the ob gene in adipose tissue was unchanged, and plasma leptin levels were also unchanged by treatment. Estrone levels increased more than 1500-fold, and estrone oleate rose 100-fold during treatment. The fact that estrone oleate had no effect on the leptin levels or expression in obese rats, in contrast with the marked inhibition observed in the lean suggests that the functionality of the leptin receptor is essential for estrone oleate inhibition of the ob gene. This also suggests that leptin may control ob gene expression in white adipose tissue and that estrone oleate may activate this process. The slimming effect of estrone oleate is, thus, not directly dependent on leptin, since both normoleptinemic and hyperleptinemic animals lose fat following treatment nor are the effects on appetite and energy expenditure mediated by leptin. However, leptin levels and the expression of the ob gene are directly linked with estrone oleate function. A possible involvement of leptin in estrone oleate action is postulated. The results support the participation of estrone oleate in the control of body weight and hint at the complexity of its regulation by leptin and glucocorticoids.     

Differential Short-Term Distribution of Estrone and Oleoyl-Estrone Administered in Liposomes to Lean and Obese Zucker Rats

Thirteen-week-old female Zucker lean (Fa/Fa) and obese (fa/fa) rats were injected through a cannula inserted in the left jugular vein with 1 mL/kg of ³H-labeled oleoyl-estrone in liposomes (Merlin-2) (i.e., 670 fmol, 84 kBq). The rats were killed 10 minutes later and dissected. The presence of intact or hydrolyzed oleoyl-estrone was later determined in all samples. The pattern of distribution of estrone was quite different from that of oleoyl-estrone both in rats that were lean and in those that were obese. Estrone was better retained by white adipose tissue than oleoyl-estrone. Liver, spleen, and lungs accumulated more oleoyl-estrone and split part of it, from 4.7% (lung, obese) to 27% (liver, lean). The overall high retention of estrone by the rat tissues results in its very low circulating levels. The fast splitting of liposome-carried oleoyl-estrone by most tissues (up to more than 67% by intestine and skin of lean rats) may help explain the rise in blood free estrone. The differences between lean and obese Zucker rats are mainly quantitative in the case of estrone, the main differences being found in blood and adipose tissues. However, when we compare the data for oleoyl-estrone, the differences cannot be dismissed simply as due to differences in body size or the extent of fat deposits. A large portion of the label remained in the blood of the rats that were obese but not in those that were lean, the tissues of which took up more label. Brown adipose tissue shows a fair affinity for oleoyl-estrone in the rats that were lean but practically does not retain label in the rats that were obese, suggesting that oleoyl-estrone may have a direct effect on brown adipose tissue. The decreased uptake of oleoyl-estrone in rats that were obese shows that the mechanism regulating the turnover or disposal of this signal is altered in this type of genetic obesity.     

Clock polymorphisms and circadian rhythms phenotypes in a sample of the Brazilian population

A Clock polymorphism T to C situated in the 3' untranslated region (3'-UTR) has been associated with human diurnal preference. At first, Clock 3111C had been reported as a marker for evening preference. However these data are controversial, and data both corroborating and denying them have been reported. This study hypothesizes that differences in Clock genotypes could be observed if extreme morning-type subjects were compared with extreme evening-type subjects, and the T3111C and T257G polymorphisms were studied. The possible relationship between both polymorphisms and delayed sleep phase syndrome (DSPS) was also investigated. An interesting and almost complete linkage disequilibrium between the polymorphisms T257G in the 5' UTR region and the T3111C in the 3' UTR region of the Clock gene is described. Almost always, a G in position 257 corresponds to a C in position 3111, and a T in position 257 corresponds to a T in position 3111. The possibility of an interaction of these two regions in the Clock messenger RNA structure that could affect gene expression was analyzed using computer software. The analyses did not reveal an interaction between those two regions, and it is unlikely that this full allele correspondence affects Clock gene expression. These results show that there is no association between either polymorphism T3111C or T257G in the Clock gene with diurnal preference or delayed sleep phase syndrome (DSPS). These controversial data could result from the possible effects of latitude and clock genes interaction on circadian phenotypes.     

Effect of sex and prior exposure to a cafeteria diet on the distribution of sex hormones between plasma and blood cells

It is generally assumed that steroid hormones are carried in the blood free and/or bound to plasma proteins. We investigated whether blood cells were also able to bind/carry sex-related hormones: estrone, estradiol, DHEA and testosterone. Wistar male and female rats were fed a cafeteria diet for 30 days, which induced overweight. The rats were fed the standard rat diet for 15 additional days to minimize the immediate effects of excess ingested energy. Controls were always kept on standard diet. After the rats were killed, their blood was used for 1) measuring plasma hormone levels, 2) determining the binding of labeled hormones to washed red blood cells (RBC), 3) incubating whole blood with labeled hormones and determining the distribution of label between plasma and packed cells, discounting the trapped plasma volume, 4) determining free plasma hormone using labeled hormones, both through membrane ultrafiltration and dextran-charcoal removal. The results were computed individually for each rat. Cells retained up to 32% estrone, and down to 10% of testosterone, with marked differences due to sex and diet (the latter only for estrogens, not for DHEA and testosterone). Sex and diet also affected the concentrations of all hormones, with no significant diet effects for estradiol and DHEA, but with considerable interaction between both factors. Binding to RBC was non-specific for all hormones. Estrogen distribution in plasma compartments was affected by sex and diet. In conclusion: a) there is a large non-specific RBC-carried compartment for estrone, estradiol, DHEA and testosterone deeply affected by sex; b) Prior exposure to a cafeteria (hyperlipidic) diet induced hormone distribution changes, affected by sex, which hint at sex-related structural differences in RBC membranes; c) We postulate that the RBC compartment may contribute to maintain free (i.e., fully active) sex hormone levels in a way similar to plasma proteins non-specific binding.