J. A. Fernández-López

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.

Pharmacological Approaches for the Treatment of Obesity

AbstractThe high incidence of obesity, its multifactorial nature, the complexity and lack of knowledge of the bodyweight control system, and the scarcity of adequate therapeutics have fuelled anti-obesity drug development during a considerable number of years. Irrespective of the efforts invested by researchers and companies, few products have reached a minimum level of effectiveness, and even fewer are available in medical practice. As a consequence of anti-obesity research, our knowledge of the bodyweight control system increased but, despite this, the pharmacological approaches to the treatment of obesity have not resulted yet in effective drugs.This review provides a panoramic of the multiple different approaches developed to obtain workable drugs. These approaches, however, rely in only four main lines of action: i) control of energy intake, mainly through modification of appetite; ii) control of energy expenditure, essentially through the increase of thermogenesis; iii) control of the availability of substrates to cells and tissues through hormonal and other metabolic factors controlling the fate of the available energy substrates; and iv) control of fat reserves through modulation of lipogenesis and lipolysis in white adipose tissue. A large proportion of current research is centred on neuropeptidic control of appetite, followed by the development of drugs controlling thermogenic mechanisms and analysis of the factors controlling adipocyte growth and fat storage. The adipocyte is also a fundamental source of metabolic signals, signals that can be intercepted, modulated and used to force the brain to adjust the mass of fat with the physiological means available.The large variety of different approaches used in the search for effective antiobesity drugs show both the deep involvement of researchers on this field and the large amount of resources devoted to this problem by pharmaceutical companies. Future trends in anti-obesity drug research follow closely the approaches outlined; however, the increasing mass of information on the molecular basis of bodyweight control and obesity will in the end prevail in our search for effective and harmless anti-obesity drugs.

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.

Oleoyl-estrone treatment affects the ponderostat setting differently in lean and obese Zucker rats

OBJECTIVE: To determine whether the slimming effects of treatment with oleoyl-estrone (OE) in liposomes of normal and obese rats are permanent, or disappear as soon as the treatment with the drug ceased. This study was devised to gain further knowledge on the postulated role of OE as a ponderostat signal, evaluating whether (in addition) it can lower the ponderostat setting of the rat.DESIGN: The rats were infused for 14 d (using osmotic minipumps) with oleoyl-estrone in liposomes at a dose of 3.5 μmol/kg· ·d, and were studied up to one month after the treatment ceased.SUBJECTS: Young adult lean controls (CL) or treated (TL) and obese controls (CO) or treated (TO) Zucker rats.MEASUREMENTS: Energy balance, blood glucose, liver glycogen, plasma insulin, leptin corticosterone, ACTH and estrone (free and total) concentrations, and expression of the OB gene in white adipose tissue (WAT).RESULTS: The loss of body weight caused by OE was recovered quickly in the TO, which gained weight at the same rate as the CO. TL rats, however remained at the low weight attained for one month after the treatment ceased. However, no differences were observed in calculated energy expenditure (EE) between the TL and TC rats once treatment had stopped. In TL and TO rats, liver glycogen concentrations decreased to normal shortly after treatment ceased, and leptin expression and concentrations remained normal and unchanged after the end of OE treatment. In TO rats, plasma glucose, insulin and leptin were lower than in the CO. Total estrone concentrations decreased rapidly in TL rats and more slowly in the TO, and free estrone followed a similar pattern.CONCLUSION: Continuous infusion of liposomes loaded with OE resulted in a decreased energy intake (EI), maintenance of EE and the utilization of body fat reserves in lean and obese rats alike. This process ended in obese rats as soon as the infusion ceased, so that even when the levels of free and total estrone in plasma remained high, there was a marked (and relatively fast) shift toward the basal situation, which translated into an increase in EI, maintenance of estimated EE and a marked buildup of energy stores. In lean rats, the effects of OE on leptin concentrations and OB gene expression persisted after infusion ended.

Effect of oral oleoyl-estrone on adipose tissue composition in male rats.

OBJECTIVE: To determine whether the oral administration of oleoyl-estrone has similar mass-decreasing effects on the main different sites of white adipose tissue (WAT).DESIGN: Adult male Zucker lean rats were given a daily oral gavage of oleoyl-estrone (OE, 10 µmol/kg) in 0.2 ml of sunflower oil for 10 days, and were compared with controls receiving only the oil. The mass of the main WAT sites: subcutaneous, epididymal, mesenteric, retroperitoneal, gluteal, perirenal and interscapular, as well as perirenal and interscapular brown adipose tissue (BAT), were dissected and studied.MEASUREMENTS: The tissue weight, DNA, protein, lipid and total cholesterol content, together with the levels of leptin and acyl-estrone in the larger WAT and BAT masses, were measured.RESULTS: The weights of WAT depots were correlated with body weight but those of BAT were not. Cell size was maximal for epididymal and mesenteric and minimal for subcutaneous and retroperitoneal WAT and BAT. Differences were detected in DNA, and in protein and lipid content between distinct WAT sites. OE treatment tended to decrease cell number and cell size in WAT; only small differences in composition were found between WAT locations inside the visceral cavity and those outside. Decreases in lipid content were maximal in mesenteric fat. Leptin and acyl-estrone content were fairly uniform at the different WAT sites, except for high concentrations in gluteal WAT. OE induced a greater decrease in leptin and acyl-estrone than in DNA and lipids; changes in these hormones were fairly parallel in all sites.CONCLUSIONS: In general, the differences in composition between visceral and peripheral subcutaneous WAT and their responses to OE were less marked than the individual differences observed between specific sites, regardless of location. WAT sites are fairly diverse in composition, but their response to OE treatment was uniform. OE decreased the weight of WAT through reduction of both cell numbers and size; but did not change the mass or composition of BAT significantly. The effects of OE are more marked in the hormonal signals (leptin and acyl-estrone) from the tissue than in its composition and mass.

Oral oleoyl-estrone induces the rapid loss of body fat in Zucker lean rats fed a hyperlipidic diet.

OBJECTIVE: To test whether oleoyl-estrone affects body weight when given orally, which may help curtail the secondary growth-boosting effects of derived estrone.DESIGN: The rats were fed for 15 days with a powdered hyperlipidic diet (16.97 MJ/kg metabolizable energy) in which 46.6% was lipid-derived and 16.1% protein-derived energy (HL group), containing 1.23±0.39 μmol/kg of fatty-acyl esters of estrone. This diet was supplemented with additional oleoyl-estrone to produce diets with 2.5 μmol/kg (diet OE2.5), 4.4 μmol/kg (diet OE4.4), and 33.3 μmol/kg content in fatty-acyl estrone (diet OE33).SUBJECTS: Twelve-week old female Zucker lean (Fa/?) rats initially weighing 200–235 g.MEASUREMENTS: Food intake and body weight changes; urine and droppings production and nitrogen content. Body composition (water, lipid, protein) and total energy. Energy and nitrogen balances. Plasma chemistry including free amino acids.RESULTS: Oral administration of oleoyl-estrone in a hyperlipidic diet resulted in significant losses of fat, energy and, ultimately, weight, which were dependent on the dose of oleoyl-estrone ingested. Treatment induced the maintenance of energy expenditure combined with lower food intake, creating an energy gap that was filled with internal fat stores whilst preserving body protein. The decrease in food intake was not a consequence of food aversion but of diminished appetite. Energy expenditure was practically constant for all groups except for the OE33, which showed values about 25% lower than the controls. In most of the groups studied, there was a net protein deposition in spite of severe lipid and energy drainage. Amino acid levels agreed with this N-sparing shift. In spite of lowered energy intake, the N balance was positive or near zero in all groups, with a sizeable N-gap in controls and in lower-dose groups that disappeared in the OE33 group.CONCLUSION: Treatment of rats with a hyperlipidic diet containing added oleoyl-estrone resulted in the dose-related loss of fat reserves with scant modification of other metabolic parameters and preservation of body protein. The results agree with the postulated role of oleoyl-estrone as a ponderostat signal and open the way for its development as anti-obesity drug.

Effect of oral oleoyl-estrone treatment on plasma lipoproteins and tissue lipase activities of Zucker lean and obese female rats

Objective: To study the effect of oral oleoyl-estrone on the plasma lipoprotein profile and tissue lipase activities in order to determine the handling of circulating lipids by adipose tissue, liver and muscle of obese female rats.Design: Lean (Fa/?) and obese (fa/fa) female Zucker rats treated for 10 days with a daily gavage of 0.2 ml sunflower oil containing 0 (controls) or 10 µmol/kg of oleoyl-estrone. After sacrifice, samples of tissues and plasma were taken.Measurements: Plasma lipoprotein classes and composition; lipoprotein lipase and hepatic lipase activities in plasma, liver, skeletal muscle and periovaric and mesenteric white adipose tissue (WAT).Results: Oleoyl-estrone decreased plasma cholesterol (mainly in HDLs: 76%) of lean rats, but dramatically decreased all lipid classes in obese rats, in which chylomicra and VLDL lost most of their triacylglycerols (95 and 81%, respectively). Hepatic lipase activity decreased markedly with oleoyl-estrone in all groups, both in plasma (79% lean, 100% obese) and liver (62% in both groups). Lipoprotein lipase activity was largely unchanged by oleoyl-estrone in lean rats, but in the obese it decreased in WAT (82% in periovaric, and 49% in mesenteric), and increased in plasma (×4) and in skeletal muscle (×5); liver levels showed no change.Conclusions: The shift observed in obese rats from a decrease in liver and WAT lipoprotein lipase and hepatic lipase activities to an increase in muscle lipoprotein lipase is coincident with the hypolipemic effect of oleoyl-estrone, especially in obese rats, and indicates that muscle is a key site for the disposal of endogenous fat mobilized due to oleoyl-estrone treatment.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

BackgroundCurrent methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions.MethodsWith a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell.ResultsThe application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation.ConclusionThe method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

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 the slimming agent oleoyl-estrone in liposomes on the body weight of Zucker obese rats

OBJECTIVE: To determine whether the mechanisms by which estrone acyl-esters carried by lipoproteins induce the loss of body fat can affect Zucker fa/fa rats, since they are hyperphagic and could not eliminate excess energy through thermogenesis, two aspects essential for the slimming effect of oleoyl-estrone in normal rats. DESIGN: The rats were infused for 28 d (osmotic minipumps) with oleoyl-estrone in liposomes (Merlin-2) at a dose of 3.5 mmol/day·kg. SUBJECTS: Lean (L) and obese (O) Zucker rats. MEASUREMENTS: Body weight changes. Oxygen consumption, body composition (water, lipid, protein), nitrogen balance, plasma chemistry. RESULTS: Treatment resulted in loss of body weight: 12.0 % (28 g) L, 9.4 % (34 g) O, mainly due to fat: 37.5 % (10.8 g) L, 11.7 % (15.5 g) O and water, preventing further increases in body weight and fat storage. Untreated rats increased their body weight: 10.5 % (24 g) L, 32.2 % (101 g) O and lipid stores: 20.3 % (5.9 g) L, 39.8 % (49.0 g) O, making the differences more marked. On day 28, glucose levels were maintained in all groups; in L, triacylglycerols increased and total cholesterol decreased; O showed no changes in plasma composition. In all rats, food intake decreased with treatment, and heat production (oxygen consumption) was unchanged (L) or slightly decreased (O). Energy expenditure per unit of fat-free mass remained unchanged. Protein balance was maintained in all groups; slimming was achieved without loss of body protein. CONCLUSION: Treatment of genetically obese rats with oleoyl-estrone in liposomes (Merlin-2) results in sustained loss of body weight – mainly lipid, sparing protein – for up to 28 d, essentially preventing further increase in body weight and accumulation of lipid and protein. This is achieved through lower food intake and relatively small changes (if any) in energy expenditure.