Moderate calorie restriction during gestation programs offspring for lower BAT thermogenic capacity driven by thyroid and sympathetic signaling.

BACKGROUND: Maternal calorie restriction during pregnancy programs offspring for later overweight and metabolic disturbances. Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and has recently emerged as a very likely target for human obesity therapy. OBJECTIVE: Here we aimed to assess whether the detrimental effects of undernutrition during gestation could be related to impaired thermogenic capacity in BAT and to investigate the potential mechanisms involved. METHODS: Offspring of control and 20% calorie-restricted rats (days 1-12 of pregnancy) (CR) were studied at the age of 25 days. Protein levels of uncoupling protein 1 (UCP1) and tyrosine hydroxylase (TyrOH); mRNA levels of lipoprotein lipase (LPL), carnitine palmitoyltransferase 1 (CPT1) and deiodinase iodothyronine type II (DIO2) in BAT; and blood parameters including thyroid hormones, were determined. The response to 24-h cold exposure was also studied by measuring body temperature changes over time, and final BAT UCP1 levels. RESULTS: Compared with controls, CR animals displayed in BAT lower UCP1 and TyrOH protein levels and lower LPL and CPT1 mRNA levels; they also showed lower triiodothyronine (T3) plasma levels. CR males, but not females, revealed lower DIO2 mRNA levels than controls. When exposed to cold, CR rats experienced a transient decline in body temperature, but the values were reestablished after 24 h, despite having lower UCP1 levels than controls. CONCLUSIONS: These results suggest that BAT thermogenic capacity is diminished in CR animals, involving impaired BAT sympathetic innervation and thyroid hormone signaling. These alterations make animals more sensitive to cold and may contribute to long-term outcomes of gestational calorie restriction in promoting obesity and related metabolic alterations.

L-alanine transport in small intestine brush-border membrane vesicles of obese rats.

Membrane vesicles from the small intestine brush border were obtained and used to determine the possible effects of genetic or nutritional obesity on L-alanine uptake. Membrane vesicles from Zucker fa/fa obese rats and cafeteria diet-fed Zucker Fa/? rats showed the same characteristics as those of standard diet-fed lean animals. All preparations showed sodium-dependent transport as the main pathway for L-alanine uptake within the substrate concentration range tested. The apparent substrate affinity constant (Km) values and the pattern of inhibition of Na(+)-dependent L-alanine uptake by other amino acids (L-leucine and L-glutamine), suggests that system B involved in the transport of dipolar amino acids (formerly named Neutral Brush Border System) participates in the Na(+)-dependent transport of L-alanine. The affinity constant (Km) for L-alanine was essentially the same for all the groups studied (in the range of 10 mM). However, there was a higher (P < 0.05) maximal capacity (Vmax) in preparations from diet-induced obese animals (cafeteria diet) than that of genetically obese rats. These results indicate that either nutritional or genetic obesity may modify the capacity but not the affinity of transport systems for L-alanine uptake in the brush border of rat small intestine.

Plasma leptin turnover rates in lean and obese Zucker rats.

Conscious female adult lean and obese Zucker rats were injected through the jugular vein with radioactive iodine-labeled murine leptin; in the ensuing 8 min, four blood samples were sequentially extracted from the carotid artery. The samples were used in a modified RIA for leptin, in which paired tubes received the same amount of either labeled or unlabeled leptin, thus allowing us to estimate both leptin levels and specific radioactivity. The data were used to determine the decay curve parameters from which the half-life of leptin (5.46 +/- 0.23 min for lean rats and 6.99 +/- 0.75 min for obese rats) as well as the size of its circulating pool (32 pmol/kg for lean rats and 267 pmol/kg for obese rats) and the overall degradation rate (96 fkat/kg for lean rats and 645 fkat/kg for obese rats) were estimated. These values are consistent with the hormonal role of leptin and the need for speedy changes in its levels in response to metabolic challenge.

Rat insulin turnover in vivo.

Zucker lean and obese rats were injected under pentobarbital anesthesia with 125I-labeled insulin; at timed intervals from 30 to 120 sec, blood samples were extracted and used for the estimation of insulin levels by RIA. A group of rats from each series was maintained under a constant infusion of noradrenaline. For each insulin determination, a duplicate blood sample containing the same amount of insulin as that used in the RIA, but without the radioactive label, was used as a blank for insulin measurement. The radioactivity in these tubes was then used for the measurement of insulin label per ml blood. From plasma label decay curves and insulin concentrations, the insulin pool size, half-life, and rate of degradation were calculated. Obese rats had higher insulin levels (2.43 nM) and showed less effect of noradrenaline than their lean counterparts, in which insulin distribution volume shrank with noradrenaline treatment. The half-life of plasma insulin was similar in all groups (range, 226-314 sec). Pool size and overall degradation rates were higher in obese (198 femtokatals) than in lean rats (28 femtokatals). It is postulated that obese rats synthesize and cleave much more insulin than lean controls despite their higher circulating levels of insulin.

Insulin degradation by adipose tissue is increased in human obesity.

White adipose tissue samples from obese and lean patients were used for the estimation of insulin protease and insulin:glutathione transhydrogenase using 125I-labeled insulin. There was no activity detected in the absence of reduced glutathione, which indicates that insulin is cleaved in human adipose tissue through reduction of the disulfide bridge between the chains. Obese patients showed higher transhydrogenase activity (per U tissue protein wt, per U tissue wt, and in the total adipose tissue mass) than the lean group. There is a significant correlation between the activity per U tissue wt, and protein and total activity in the whole adipose tissue with respect to body mass index, with a higher activity in obese patients. The potential of insulin cleavage by adipose tissue in obese patients was a mean 5.6-fold higher than that in controls. The coexistence of high insulinemia and high cleavage capability implies that insulin secretion and turnover are increased in the obese. Thus, white adipose tissue may be crucial in the control of energy availability through modulation of insulin cleavage.

Carrier-mediated uptake of L-(+)-lactate in plasma membrane vesicles from rat liver.

Plasma membrane vesicles from rat liver transported L-lactate into the inner vesicular space. Kinetic analysis of L-lactate uptake gave a Km value of approx. 2.9 mM. Selective inhibition was found in a similar pattern to that described for the hepatic lactate carrier. L-Lactate transport was enhanced when a pH gradient was created across the plasma membrane. Vesicles obtained from fasted rats showed a higher uptake of L-lactate than those from fed rats, when incubated with physiological concentrations of L-lactate.

Is leptin an insulin counter-regulatory hormone?

Leptin, the product of the ob gene, controls appetite through the hypothalamus and may affect many other tissues because of the widespread distribution of its receptors. Leptin is synthesized by white adipose tissue (WAT) under conditions of high energy availability and insulin stimulus. Glucocorticoids enhance this synthesis and catecholamines hamper leptin production. Leptin diminishes insulin secretion by the pancreatic beta cells and induces insulin resistance. In fact leptin hampers insulin action on WAT itself in a negative feedback loop. The evidence acquired in studies on diabetics, starvation, refeeding and insulin and glucose clamps supports this interpretation, which may also explain part of the difficulties encountered by the current postulate that links leptin to WAT mass size signalling to the brain. Leptin may be, essentially, a counter-regulatory hormone limiting the insulin drive to store energy in the form of fat, its effects reaching from a decrease in food intake to lower insulin secretion and increased resistance to insulin and lower glucose uptake and fat synthesis by WAT.

Effect of oleoyl-estrone administration on corticosterone binding to tissues of lean and obese Zucker rats.

A group of female Zucker lean and obese rats was treated with 3.5 micromol/day kg of oleoyl-estrone in liposomes (OE) injected i.v. continuously for 14 days with inserted osmotic minipumps. Samples of liver were extracted on days 0, 3, 6, 10 and 14 and the expression of corticosterone-binding globulin (CBG) was determined by Northern blot. On the same dates, the total binding capacity of plasma, liver, periovaric white adipose tissue (WAT) and subcutaneous WAT was also determined using tritium-labelled corticosterone. Treatment with OE resulted in diminished CBG gene expression in the liver, this being more marked in the obese rats. Basal (time 0) corticosterone binding was higher in the plasma, liver and WAT of lean rats. Treatment with OE resulted in a gradual and general loss of binding capacity in the plasma and all tissues studied, for lean and obese rats alike. Since CBG decreases may result in enhanced glucocorticoid availability (and effects), the global decrease in corticosterone binding observed can be interpreted as a counteractive response to the energy imbalance elicited by OE.

Alanine uptake by liver of mid-lactating rats.

L-Alanine transport in liver plasma membrane vesicle preparations from fed virgin and 15-day-lactating rats was studied. Lactation was found to induce a decrease of the maximal rate (Vmax) of a high-capacity-low-affinity component of the Na(+)-dependent L-alanine uptake. However, a high-affinity-low-capacity agency was significantly induced in lactating-rat livers. L-Alanine uptake was differentially inhibited by other amino acids in those preparations from lactating rats, and showed different sensitivity to Li+ as a cosubstrate instead of Na+ and to inhibition by sulfhydryl modifying reagents (N-ethylmaleimide [NEM] and p-chloromercuribenzosulfonate [PCMBS]). All of these observations taken together suggest that system A is upregulated in lactating-rat livers, thus resulting in a different contribution of both agencies A and ASC to the total Na(+)-dependent alanine transport into liver plasma membrane vesicles. This was demonstrated using the analogue alpha-methyl-aminoisobutyric acid (MeAIB), a specific system A substrate. L-Alanine uptake rates, as calculated from plasma membrane enzyme marker recoveries, were also enhanced in the physiologic range of alanine concentrations in blood. Our results prove that the physiologic adaptation to lactation involves modulation of system A activity in the liver.

Oleoyl-estrone does not alter hypothalamic neuropeptide Y in Zucker lean and obese rats.

Female Zucker lean and obese rats were treated for 14 days with 3.5 micromol/kg oleoyl-estrone (OE) in liposomes (Merlin-2). After 0, 3, 6, 10, and 14 days of treatment, the rats were killed and hypothalamic nuclei (lateral preoptic, median preoptic, paraventricular, ventromedial and arcuate) were used for neuropeptide Y (NPY) radioimmunoassay. In 14 days, OE decreased food intake by 26% in lean and 38% in obese rats and energy expenditure by 6% in lean and 47% in obese rats; the body weight gap between controls and treated rats becoming -17.8% of initial b.wt. in the lean and -13.6% in the obese rats. Obese rats showed higher NPY levels in all the nuclei than the lean rats. Despite a negative energy balance and decreased food intake, there were practically no changes in NPY with OE treatment. The results indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis in lean and genetically obese rats.

The hepatic amino acid system A transport activity, is up-regulated in obese Zucker rats.

The utilization of L-alanine by liver is dependent on amino acid uptake from blood. This uptake, mainly mediated by the A transport system, may be regulated by different nutritional and physiologic conditions. The regulation of this transport system by diets with different protein content was tested in lean and obese Zucker rats. High-protein (HP) and low-protein (LP) diets led to changes in the rats' growth patterns, especially in lean animals. However, homeostasis was relatively well maintained, as seen in plasma values, in spite of the increased urea production in the HP groups and increased triacylglycerides in the LP groups. The obese animals took up L-alanine at a higher rate than the lean animals. Obesity led to the emergence of a high-affinity component (K(M) approximately 0.1-0.2 mM) in the transport system, which was not dependent on the protein content of the diet. This component has a 10-fold increase in affinity for L-alanine, but with an approximately 3- to 5-fold reduction in maximal velocity of transport.

Zucker obese rats are insensitive to the CRH-increasing effect of oleoyl-estrone.

Adult female Zucker lean and obese rats were treated for 14 days with 3.5 nm/kg oleoyl-estrone (OE) in liposomes (Merlin-2) through continuous i.v. injection with osmotic minipumps. Rat wt. and food intake were measured daily. On days 0, 3, 6, 10, and 14, groups of rats were killed and their hypothalamic nuclei [lateral preoptic (LPO), median preoptic (MPO), paraventricular (PVN), ventromedial (VMH), and arcuate (ARC)] were dissected, homogenized, and used for the measurement of corticosterone-releasing hormone (CRH) by radioimmunoassay. The OE treatment decreased food intake by 67.4% in lean and 62.6% in obese rats (means for 14 days). Body wt. decreased steadily in lean and obese rats, the gap between controls and treated rats becoming 11.5% of initial body wt. in the lean and 12.4% in the obese. The levels of CRH in the ARC nucleus were at least 10-fold higher than in the other nuclei. No changes in CRH were observed in any of the nuclei of obese rats, with levels up to day 6 similar to those of lean rats. In the lean rats, the LPO and ARC nuclei showed peaks on day 10, while the MPO showed no changes and the PVN and VMH nuclei showed a progressive increase, to a maximum at the end of the study (day 14). This contrasted with the peak of plasma adrenocorticotropic hormone (ACTH) and corticosterone (day 6 in lean and day 14 in obese rats). There was a definite lack of correlation between the plasma levels of these two hormones and the levels of CRH in the hypothalamic nuclei, and between the latter and the decreases in appetite in the rats. The loss of appetite induced by OE is not necessarily mediated by CRH, because the obese rats show an intense decrease in voluntary food intake but their hypothalamic nuclei CRH levels do not change at all. Hypothalamic nuclei CRH does not, necessarily, mediate the rise in glucocorticoids induced by OE treatment, because this is observed in lean and obese rats, lean rats increases being mismatched with those of hypothalamic CRH. The OE induced changes in hypothalamic CRH require a fully functional leptinergic pathway, because it is not observed in Zucker fa/fa rats lacking a working leptin receptor. This--indirectly--shows that leptin is needed for its synthesis or modulation.

Distribution of oleyl-anilide hydrolysing activity in rat and human tissues.

A method has been developed and tested for the measurement of anilide hydrolysing activity in rat tissues. A concentrated solution of labelled oleyl anilide in isopropanol is added to the tissue homogenates and after incubation, the chloroform/methanol extract of the samples is chromatographed on Silicagel TLC plates and the oleic acid radioactivity is measured. The activity is time-, homogenate- and temperature-dependent, the optimal pH for measurement is 8 and there is no significant spontaneous anilide degradation. In the rat, the activity is widely distributed, with highest protein specific activity in the adipose tissues. The tissue activities of a same animal are fairly well inter-correlated, with rats showing very low activity in all tissues compared with others presenting high overall activity. The levels of activity found can easily explain the fast elimination of anilides administered to rats and their scant toxic effects. Human adipose tissue samples showed a wide range of anilide hydrolase activities per gram of protein, in general lower than in rats and with some values very low. It is postulated that this lack of anilide-hydrolising capability in some humans may be related to the incidence of the toxic oil syndrome.

Effects of chronic ethanol treatment on amino acid uptake and enzyme activities in the lactating rat mammary gland.

The effects of chronic ethanol consumption on mammary gland amino acid uptake at the 15th day of lactation in the rat have been studied. Ethanol treatment decreased the arterial levels of Ala, Asp, Gly, Pro, Lys and Met, and increased those of Gln and alpha-amino-butyrate. Chronic ethanol treatment produced a decrease in the arteriovenous differences of Asp, Thr, Arg, Met and Phe, and increased those of Ala, Gln, Gly, Pro and Tyr. The combination of the calculated values of relative extraction and the arteriovenous differences indicate that these alterations in amino acid uptake are related to changes in the transport process for Ala, Asp, Thr, Pro, Arg, Asn, Gly, Tyr, and Phe, and that the alterations in the arteriovenous differences of Gln, Lys and Met are due to the affected arterial levels of these amino acids. Measurements of enzymatic activities in the mammary gland show that these alterations in the amino acid transport process cannot be ascribed to changes in the gamma-glutamyl cycle.

Estrogen effects on blood amino acid compartmentation.

The present paper focuses on the study of blood amino acid compartmentation in healthy men (lean and obese) and women, with special emphasis on the estimation of the recently described blood-cell adsorbed amino acid pool. The wide range of changes found in this pool on comparing different physiological situations may be attributable to its proposed characteristic high dynamism on the one hand, but also to the influence of other factors such as hormones. Along these lines, the sex- and obesity-linked variations found here in human blood led to the speculation as to whether these differences could be related to the influence of estrogens. This hypothesis was further tested by chronically treating a group of male rats with estrone and checking their subsequent blood amino acid compartment changes (which yielded a greater difference in the adsorbed pool). From the overall results obtained it may be concluded that the higher production of estrogens in women and obese men affects amino acid availability to the tissues by modulating the blood-cell adsorbed amino acid pool through a mechanism that is, at present, unknown.

Formaldehyde derived from dietary aspartame binds to tissue components in vivo.

Adult male rats were given an oral dose of 10 mg/kg aspartame 14C-labelled in the methanol carbon. At timed intervals of up to 6 hours, the radioactivity in plasma and several organs was investigated. Most of the radioactivity found (>98% in plasma, >75% in liver) was bound to protein. Label present in liver, plasma and kidney was in the range of 1-2% of total radioactivity administered per g or mL, changing little with time. Other organs (brown and white adipose tissues, muscle, brain, cornea and retina) contained levels of label in the range of 1/12 to 1/10th of that of liver. In all, the rat retained, 6 hours after administration about 5% of the label, half of it in the liver. The specific radioactivity of tissue protein, RNA and DNA was quite uniform. The protein label was concentrated in amino acids, different from methionine, and largely coincident with the result of protein exposure to labelled formaldehyde. DNA radioactivity was essentially in a single different adduct base, different from the normal bases present in DNA. The nature of the tissue label accumulated was, thus, a direct consequence of formaldehyde binding to tissue structures. The administration of labelled aspartame to a group of cirrhotic rats resulted in comparable label retention by tissue components, which suggests that liver function (or its defect) has little effect on formaldehyde formation from aspartame and binding to biological components. The chronic treatment of a series of rats with 200 mg/kg of non-labelled aspartame during 10 days resulted in the accumulation of even more label when given the radioactive bolus, suggesting that the amount of formaldehyde adducts coming from aspartame in tissue proteins and nucleic acids may be cumulative. It is concluded that aspartame consumption may constitute a hazard because of its contribution to the formation of formaldehyde adducts.

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.

Oleoyl-estrone does not have direct estrogenic effects on rats.

The estrogenic effects of oleoyl-estrone (OE) administration, either though continuous i.v. infusion with osmotic minipumps or administered by daily oral gavage, were studied. Binding of OE to human recombinant purified alpha receptors was negligible, and that of estrone (E1) was only a fraction of 17beta-estradiol (E2) binding. Intravenous--but not oral--OE administration resulted in marked increases of both E1 and E2 in rat plasma, but oral OE did not induce significant changes in either plasma hormone in Wistar or Zucker rats. The weight of uteri and ovaries increased with time of administration in Zucker rats treated with i.v. OE, but inguinal mammary gland proliferation between subcutaneous adipose tissue was even more marked. Oral administration of OE, however, did not increase either uterine weight or mammary gland proliferation, even at doses (10 micromol/kg x d) higher than those given i.v. (3.5 micromol/kg x d). The results indicate that i.v. administration of OE resulted in limited estrogenic effects mainly due to the high accumulation of E1 giving rise to significant increases in E2. On the other hand, oral administration of OE, even at higher daily doses, did not increase the circulating levels of either estrogen and, therefore, there were no significant effects on mammary gland proliferation or uterine weight. The oral administration of OE as a slimming drug, then, do not result in estrogenic side effects over a wide range of daily doses.

Structural determinants of oleoyl-estrone slimming effects.

Female adult 9-week old Wistar rats were implanted with osmotic minipumps releasing for 14 days a liposome suspension (controls) loaded with oleoyl-estrone or other compounds of the Merlin series: estrone, estradiol, oleoyl-estradiol, oleoyl-DHEA, stearoyl-estrone, palmitoyl-estrone, oleoyl-diethylstilbestrol (DES), estrone oleoyl-ether and oleoyl-3-methoxy-estrone. All compounds were given at the same dose of 3.5 micromol/day x kg for 14 days. The effects on body weight and food intake were recorded. In the case of estrone esters, the body composition and nitrogen balance were also determined. The chronic administration of oleoyl-estrone in liposomes to rats lowers food intake, maintaining energy consumption, thus inducing the active utilization of internal stores and, consequently, the loss of body weight. This loss is mainly due to a decrease in fat, with lower proportional losses of water and a limited consumption of body protein. Free estrone had no effects on body weight, but estradiol did induce a decrease in body weight, similar to that of oleoyl-estradiol. Oleoyl-DHEA had no significant effect on body weight nor in food intake. Oleoyl-DES mimicked fairly well the effects of oleoyl-estrone, both affecting food intake and body weight. There was a relative lack of effects of estrone oleoyl-ether and of oleoyl-3-methoxy-estrone. The effects of oleoyl-estrone were in part mimicked by stearoyl- and palmitoyl-estrone, but their activity on a molar basis was lower, which suggests that the fatty acid moiety significantly influences the activity of the estrone ester as a slimming agent. The differences observed in the appetite suppression and overall slimming power of the stearoyl and palmitoyl-estrone clearly indicate that the sites of action of the physiological agonist oleoyl-estrone are at least two; the shape of the molecule, thus, may elicit a different degree of response of the systems controlled by oleoyl-estrone levels. From this interaction a series of global effects are elicited, such as appetite suppression and the loss of body (fat) weight, the latter in part (but not only) due to decreased food intake. The results shown here also suggest that the overall configuration of fatty acyl-estrone is more constrictive for its function as slimming agent than for its role as appetite suppressant, which hints to different target organs or sites of action endowed with receptors showing different degrees of fulfilling the structural constrictions of the agonist molecule.

Effect of a cafeteria diet on energy intake and balance in Wistar rats.

The energy balance and nutrient selection strategies of 30-day-old Wistar rats offered a reference pellet and a seven-item cafeteria diet were studied in two consecutive 15-day periods: 30-45 and 45-60 days after birth. Cafeteria-fed rats grew faster, incorporating more fat and water, but a similar amount of protein to reference-fed animals. In the second 15 days all rats ate less and produced less heat than in the first 15 days. Reference-fed rats also deposited less energy in their bodies, in contrast to the tendency towards higher carcass energy deposition in cafeteria-fed rats. Cafeteria-fed rats selected much more fat and sugars than controls, with similar protein and less starch; in the second period studied, cafeteria-fed rats significantly increased their sugar consumption, with no change in fat or protein. It is suggested that the switch to selecting more sugars may be an essential factor in the shift towards increased fat deposition at the expense of heat production in cafeteria-fed rats.