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This review focuses on the question of metabolic syndrome (MS) being a complex, but essentially monophyletic, galaxy of associated diseases/disorders, or just a syndrome of related but rather independent pathologies. The human nature of MS (its exceptionality in Nature and its close interdependence with human action and evolution) is presented and discussed. The text also describes the close interdependence of its components, with special emphasis on the description of their interrelations (including their syndromic development and recruitment), as well as their consequences upon energy handling and partition. The main theories on MS's origin and development are presented in relation to hepatic steatosis, type 2 diabetes, and obesity, but encompass most of the MS components described so far. The differential effects of sex and its biological consequences are considered under the light of human social needs and evolution, which are also directly related to MS epidemiology, severity, and relations with senescence. The triggering and maintenance factors of MS are discussed, with especial emphasis on inflammation, a complex process affecting different levels of organization and which is a critical element for MS development. Inflammation is also related to the operation of connective tissue (including the adipose organ) and the widely studied and acknowledged influence of diet. The role of diet composition, including the transcendence of the anaplerotic maintenance of the Krebs cycle from dietary amino acid supply (and its timing), is developed in the context of testosterone and β-estradiol control of the insulin-glycaemia hepatic core system of carbohydrate-triacylglycerol energy handling. The high probability of MS acting as a unique complex biological control system (essentially monophyletic) is presented, together with additional perspectives/considerations on the treatment of this 'very' human disease. [ABSTRACT FROM AUTHOR]

The ingestion of excess lipids often produces the accumulation of liver fat. The modulation of diet energy partition affects this process and other metabolic responses, and oestrogens and androgens are implied in this process. Ten-week-old male and female rats were fed with either standard rat chow (SD), SD enriched with coconut oil (high-fat diet, HF), SD enriched with protein (high-protein diet, HP) or a 'cafeteria' diet (CAF) for 1 month. HF and CAF diets provided the same lipid-derived percentage of energy (40 %), HP diet protein energy derived was twice (40 %) that of the SD. Animals were killed under anaesthesia and samples of blood and liver were obtained. Hepatic lipid content showed sex-related differences: TAG accumulation tended to increase in HF and CAF fed males. Cholesterol content was higher only in the CAF males. Plasma oestradiol in HF and HP males was higher than in CAF. Circulating cholesterol was inversely correlated with plasma oestradiol. These changes agreed with the differences in the expression of some enzymes related to lipid and energy metabolism, such as fatty acid synthetase or phosphoglycolate phosphatase. Oestrogen protective effects extend to males with 'normal' diets, that is, not unbalanced by either lipid or protein, but this protection was not enough against the CAF diet. Oestradiol seems to actively modulate the liver core of 2C-3C partition of energy substrates, regulating cholesterol deposition and lactate production. [ABSTRACT FROM AUTHOR]

Androgens are an important and diverse group of steroid hormone molecular species. They play varied functional roles, such as the control of metabolic energy fate and partition, the maintenance of skeletal and body protein and integrity and the development of brain capabilities and behavioral setup (including those factors defining maleness). In addition, androgens are the precursors of estrogens, with which they share an extensive control of the reproductive mechanisms (in both sexes). In this review, the types of androgens, their functions and signaling are tabulated and described, including some less-known functions. The close interrelationship between corticosteroids and androgens is also analyzed, centered in the adrenal cortex, together with the main feedback control systems of the hypothalamic–hypophysis–gonads axis, and its modulation by the metabolic environment, sex, age and health. Testosterone (T) is singled out because of its high synthesis rate and turnover, but also because age-related hypogonadism is a key signal for the biologically planned early obsolescence of men, and the delayed onset of a faster rate of functional losses in women after menopause. The close collaboration of T with estradiol (E2) active in the maintenance of body metabolic systems is also presented Their parallel insufficiency has been directly related to the ravages of senescence and the metabolic syndrome constellation of disorders. The clinical use of T to correct hypoandrogenism helps maintain the functionality of core metabolism, limiting excess fat deposition, sarcopenia and cognoscitive frailty (part of these effects are due to the E2 generated from T). The effectiveness of using lipophilic T esters for T replacement treatments is analyzed in depth, and the main problems derived from their application are discussed. [ABSTRACT FROM AUTHOR]

White adipose tissue (WAT) nucleated stromal cells (NSC) play important roles in regulation, defense, regeneration and metabolic control. In WAT sites, the proportions and functions of NSC change under diverse physiological or pathologic conditions. We had previously observed the massive anaerobic wasting of glucose to lactate and glycerol in rat epididymal adipocytes. To test site variability, and whether the adipocyte extensive anaerobic metabolism of glucose was found in NSC, we analyzed, in parallel, subcutaneous, mesenteric and epididymal WAT of male adult Wistar rats. Adipocytes and NSC fractions, were isolated, counted and incubated (as well as red blood cells: RBC) with glucose, and their ability to use glucose and produce lactate, glycerol, and free fatty acids was measured. Results were computed taking into account the number of cells present in WAT samples. Cell numbers were found in proportions close to 1:13:100 (respectively, for adipocytes, NSC and RBC) but their volumes followed a reversed pattern: 7,500:10:1. When counting only non-fat cell volumes, the ratios changed dramatically to 100:10:1. RBC contribution to lactate production was practically insignificant. In most samples, NSC produced more lactate than adipocytes did, but only adipocytes secreted glycerol (and fatty acids in smaller amounts). Glucose consumption was also highest in NSC, especially in mesenteric WAT. The heterogeneous NSC showed a practically anaerobic metabolism (like that already observed in adipocytes). Thus, NSC quantitative production of lactate markedly contributed (i.e. more than adipocytes) to WAT global use (wasting) of glucose. We also confirmed that glucose-derived glycerol is exclusively produced by adipocytes. [ABSTRACT FROM AUTHOR]

Background. Adult rat epididymal adipocytes are able to convert large amounts of glucose to lactate and glycerol. However, fatty acid efflux is much lower than that expected from glycerol levels if they were the product of lipolysis. Use of glucose for lipogenesis is limited, in contrast with the active glycolysis-derived lactate (and other 3-carbon substrates). In this study, we analyzed whether white adipose tissue (WAT) site and sex affect these processes. Methods. Mature adipocytes from perigonadal, mesenteric and subcutaneous WAT of female and male rats were isolated, and incubated with 7 or 14mMglucose during 1 or 2 days. Glucose consumption, metabolite efflux and gene expression of glycolytic and lipogenesis-related genes were measured. Results. The effects of medium initial glucose concentration were minimal on most parameters studied. Sex-induced differences that were more extensive; however, the most marked, distinct, effects between WAT sites, were dependent on the time of incubation. In general, the production of lactate was maintained during the incubation, but glycerol release rates increased with time, shifting from a largely glycolytic origin to its triacylglycerol (TAG) lipolytic release. Glycerol incorporation was concurrent with increased TAG turnover: lipolytic glycerol was selectively secreted, while most fatty acids were recycled again into TAG. Fatty acid efflux increased with incubation, but was, nevertheless, minimal compared with that of glycerol. Production of lactate and glycerol from glucose were maximal in mesenteric WAT. Discussion. Female rats showed a higher adipocyte metabolic activity than males. In mesenteric WAT, gene expression (and substrate efflux) data suggested that adipocyte oxidation of pyruvate to acetyl-CoA was higher in females than in males, with enhanced return of oxaloacetate to the cytoplasm for its final conversion to lactate. WAT site differences showed marked tissue specialization-related differences. Use of glucose for lipogenesis was seriously hampered over time, when TAG turnover-related lipolysis was activated. We postulate that these mechanisms may help decrease glycaemia and fat storage, producing, instead, a higher availability of less-regulated 3-carbon substrates, used for energy elsewhere. [ABSTRACT FROM AUTHOR]

White adipose tissue can metabolize large amounts of glucose to glycerol and lactate. We quantitatively traced glucose label to lactate, glycerol and fats in primary cultures of mature rat epididymal adipocytes. Cells were incubated with 7/14 mM 14C-glucose for 24/48 h. Medium metabolites and the label in them and in cells' components were measured. Gene expression analysis was done using parallel incubations. Glucose concentration did not affect lactate efflux and most parameters. Glycerol efflux increased after 24 h, coinciding with arrested lipogenesis. Steady production of lactate was maintained in parallel to glycerogenesis. Changes in adipocyte metabolism were paralleled by gene expression. Glucose use for lipogenesis was minimal, and stopped (24 h-onwards) when glycerol efflux increased because of triacylglycerol turnover. Lactate steady efflux showed that anaerobic glycolysis was the main adipocyte source of energy. We can assume that adipose tissue may play a quantitatively significant effect on glycaemia, returning 3C fragments thus minimizing lipogenesis. [ABSTRACT FROM AUTHOR]

Objective: Sex hormone-binding globulin (SHBG) binds and transports testosterone and estradiol in plasma. The possibility that SHBG is a mixture of transporting proteins has been postulated. We analyzed in parallel the effects of obesity status on the levels and binding capacity of circulating SHBG and their relationship with testosterone and estradiol. Design: Anthropometric measures and plasma were obtained from apparently healthy young (i.e. 35 ± 7 years) premenopausal women (n = 32) and men (n = 30), with normal weight and obesity (BMI >30 kg/m2). Methods: SHBG protein (Western blot), as well as the plasma levels of testosterone, estradiol, cortisol and insulin (ELISA) were measured. Specific binding of estradiol and testosterone to plasma SHBG was analyzed using tritiumlabeled hormones. Results: Significant differences in SHBG were observed within the obesity status and gender, with discordant patterns of change in testosterone and estradiol. In men, testosterone occupied most of the binding sites. Estrogen binding was much lower in all subjects. Lower SHBG of morbidly obese (BMI >40 kg/m2) subjects affected testosterone but not estradiol. The ratio of binding sites to SHBG protein levels was constant for testosterone, but not for estradiol. The influence of gender was maximal in morbid obesity, with men showing the highest binding/SHBG ratios. Conclusions: The results reported here are compatible with SHBG being a mixture of at least two functionally different hormone-binding globulins, being affected by obesity and gender and showing different structure, affinities for testosterone and estradiol and also different immunoreac [ABSTRACT FROM AUTHOR]

Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic stimulation. [ABSTRACT FROM AUTHOR]

Female and male adult Wistar rats were fed standard chow or a simplified cafeteria diet for one month. Then, the rats were killed and the white adipose tissue (WAT) in four sites: perigonadal, retroperitoneal, mesenteric and subcutaneous (inguinal) were sampled and frozen. The complete WAT weight in each site was measured. Gene expression analysis of key lipid and glucose metabolism enzymes were analyzed, as well as tissue and plasma lactate and the activity of lactate dehydrogenase. Lactate gradients between WAT and plasma were estimated. The influence of sex and diet (and indirectly WAT mass) on lactate levels and their relationships with lactate dehydrogenase activity and gene expressions were also measured. A main conclusion is the high production of lactate by WAT, practically irrespective of site, diet or sex. Lactate production is a direct correlate of lactate dehydrogenase activity in the tissue. Furthermore, lactate dehydrogenase activity is again directly correlated with the expression of the genes Ldha and Ldhb for this enzyme. In sum, the ability to produce lactate by WAT is not directly dependent of WAT metabolic state. We postulate that, in WAT, a main function of the lactate dehydrogenase path may be that of converting excess available glucose to 3C fragments, as a way to limit tissue self-utilization as substrate, to help control glycaemia and/or providing short chain substrates for use as energy source elsewhere. More information must be gathered before a conclusive role of WAT in the control of glycaemia, and the full existence of a renewed glucose-lactate-fatty acid cycle is definitely established. [ABSTRACT FROM AUTHOR]

Adipose tissue (AT) is distributed as large differentiated masses, and smaller depots covering vessels, and organs, as well as interspersed within them. The differences between types and size of cells makes AT one of the most disperse and complex organs. Lipid storage is partly shared by other tissues such as muscle and liver. We intended to obtain an approximate estimation of the size of lipid reserves stored outside the main fat depots. Both male and female rats were made overweight by 4-weeks feeding of a cafeteria diet. Total lipid content was analyzed in brain, liver, gastrocnemius muscle, four white AT sites: subcutaneous, perigonadal, retroperitoneal and mesenteric, two brown AT sites (interscapular and perirenal) and in a pool of the rest of organs and tissues (after discarding gut contents). Organ lipid content was estimated and tabulated for each individual rat. Food intake was measured daily. There was a surprisingly high proportion of lipid not accounted for by the main macroscopic AT sites, even when brain, liver and BAT main sites were discounted. Muscle contained about 8% of body lipids, liver 1–1.4%, four white AT sites lipid 28–63% of body lipid, and the rest of the body (including muscle) 38–44%. There was a good correlation between AT lipid and body lipid, but lipid in “other organs” was highly correlated too with body lipid. Brain lipid was not. Irrespective of dietary intake, accumulation of body fat was uniform both for the main lipid storage and handling organs: large masses of AT (but also liver, muscle), as well as in the ”rest” of tissues. These storage sites, in specialized (adipose) or not-specialized (liver, muscle) tissues reacted in parallel against a hyperlipidic diet challenge. We postulate that body lipid stores are handled and regulated coordinately, with a more centralized and overall mechanisms than usually assumed. [ABSTRACT FROM AUTHOR]

White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation. [ABSTRACT FROM AUTHOR]

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids. We analyzed whether reduced urea excretion was a consequence of higherNOx; (nitrite, nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion. There were no differences in plasma nitrate or nitrite. NOx and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithine when compared with controls, whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability. [ABSTRACT FROM AUTHOR]

The use of Transwells™ for routine cultures of 3T3L1 cells results in a much improved rate of differentiation of fibroblasts to adipocytes (100 % in 9 of 10 tests) compared with bottom-well layer cultures. Mean size of cells was not different, but the cell number and overall cell mass was 3× larger in transwell in spite of a smaller surface area. The difference between both models was the accessibility in transwells of both sides of the cells to the medium (and oxygen). Cells were counted, and their size estimated using a handheld cell counter, Scepter™, designed for blood cells, but adjusted to the larger size of adipocytes. Finally, the effect of nitric oxide was tested using spermineNONOate, a nitric oxide (NO·) donor. The product was released to cultures at a constant 1 μl/h rate for up to 3 days using osmotic Alzet™ minipumps held in wells with water and discharging their contents to the cultured cell-laden wells through a short capillary tube. A rate of 0.3 pmol/min/ml of medium did not affect the cells' size, but 0.4 pmol/min/ml significantly increased cell mass. The methodological improvements presented here allow for more uniform cultured cell yields and a more flexible environment for control of cell size and administration of signaling agents. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Background/Aims: There is ample consensus that there is a neurophysiological basis for eating disorders (ED). Traits of personality translate into behavioral traits, purging being a well-defined transversal example. The direct implication of steroid hormones on ED has seldom been studied, despite their effects on behavior. Methods: After psychological interview analysis, 57 ED female patients (31 purgative and 26 nonpurgative) and 17 female controls were studied. Metabolic parameters and analysis of androgen, estrogen and glucocorticoid hormones were determined in parallel to the psychopathological profile (EDI-2 and SCL-90-R) and anthropometric measurements. Results: Psychometric tests showed clear differences between ED and controls, but there were few hormonal-metabolic significant differences. In purgative ED there were repeated (significant) positive correlations with corticosteroid-binding globulin (CBG) and negative correlations with sex hormone-binding globulin (SHBG) versus eating and general psychopathology. In nonpurging ED there were positive correlations for deoxycortisol, free fatty acids and albumin and negative for aspartate aminotransferase and psychopathological traits. Conclusion: The data suggest that CBG/corticosteroids and sexual hormones/SHBG are involved in purging behavior and its psychopathology and severity scores. Correlations of selected psychometric data and the CBG/SHBG levels in purging may eventually result in clinical markers. This approach may provide additional clues for understanding the pathogenesis of ED. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In the metabolic syndrome, glucocorticoid activity is increased, but circulating levels show little change. Most of blood glucocorticoids are bound to corticosteroid-binding globulin (CBG), which liver expression and circulating levels are higher in females than in males. Since blood hormones are also bound to blood cells, and the size of this compartment is considerable for androgens and estrogens, we analyzed whether sex or eating a cafeteria diet altered the compartmentation of corticosterone in rat blood. The main corticosterone compartment in rat blood is that specifically bound to plasma proteins, with smaller compartments bound to blood cells or free. Cafeteria diet increased the expression of liver CBG gene, binding plasma capacity and the proportion of blood cell-bound corticosterone. There were marked sex differences in blood corticosterone compartmentation in rats, which were unrelated to testosterone. The use of a monoclonal antibody ELISA and a polyclonal Western blot for plasma CBG compared with both specific plasma binding of corticosterone and CBG gene expression suggested the existence of different forms of CBG, with varying affinities for corticosterone in males and females, since ELISA data showed higher plasma CBG for males, but binding and Western blot analyses (plus liver gene expression) and higher physiological effectiveness for females. Good cross- reactivity to the antigen for polyclonal CBG antibody suggests that in all cases we were measuring CBG.The different immunoreactivity and binding affinity may help explain the marked sex-related differences in plasma hormone binding as sex-linked different proportions of CBG forms. [ABSTRACT FROM AUTHOR]

Oleoyl-estrone (OE) is a powerful slimming agent that is also present in plasma and adipose tissue, where it is synthesized. It acts through the formation of a derivative W. OE effects (and W levels) are proportional to the dose. OE reduces food intake but maintains energy expenditure (thermogenesis). The energy gap is fulfilled with adipose tissue fat, sparing body protein and maintaining glycemia (and glycogen) with lower insulin and leptin levels. OE (in fact W) acts through specific receptors, different from those of estrogen. OE increases cholesterol catabolism, reducing hypercholesterolemia in obese rats. The main metabolic effect on adipose tissue is lowering of lipid synthesis, maintaining unchanged the intracellular lipolytic processes; the imbalance favors the progressive loss of fat, which is largely used by the muscle. OE administration induces additive effects with other antiobesity agents, such as β3-adrenergic agonists, forcing a massive loss of lipid. Corticosteroids markedly limit OE action by altering the liver control of lipogenesis. OE also inhibits the action of 17β-hydroxysteroid dehydrogenase, decreasing the synthesis of β-estradiol and testosterone. Discontinuous treatment allows for maximal efficacy both in rats and humans. OE has the advantage that the loss of fat is maintained and does not require additional dietary limitations. [ABSTRACT FROM AUTHOR]

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 dextrancharcoal 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. [ABSTRACT FROM AUTHOR]

We intended to determine how the liver copes with the massive handling of lipids induced by OE (oleoyl-oestrone), as well as to characterize and differentiate the actual OE effects from those that may be only the consequence of decreased food intake. Thus we used male rats treated with oral OE (10 nmol/g per day) compared with a vehicle only PF (pair-fed) group and controls fed ad libitum (vehicle only). Plasma parameters, and total liver lipids, glycogen, DNA and total mRNA were measured. RNA was extracted and used for real-time PCR analysis of the gene expression of enzymes and regulatory factors of liver energy metabolism. Most hepatic proteins showed similar gene expressions in OE and controls, but the differences widened between OE and PF rats, showing that OE effects could not be merely attributed to a lower energy intake. The liver of OE-treated rats largely maintained its ability to mobilize glucose for the synthesis of fats; this was achieved in part by a peculiar combination of regulative modifications that facilitate both fatty acid disposal and restrained glucose utilization under conditions of limited food supply but ample availability of internal energy stores. In conclusion, the results presented suggest that the effect of OE on liver metabolism may be (at least in part) mediated through an insulin-sensitivity-dependent modulation of the expression of SREBP-1c (sterolregulatory- element-binding protein-1c), resulting in the unique combined effect of mildly increased (or maintained) glucose disposal but also limited enhancement of lipogenesis [ABSTRACT FROM AUTHOR]

Background: The combination of oleoyl-estrone (OE) and a selective b3-adrenergic agonist (B3A; CL316,243) treatment in rats results in a profound and rapid wasting of body reserves (lipid). Methods: In the present study we investigated the effect of OE (oral gavage) and/or B3A (subcutaneous constant infusion) administration for 10 days to overweight male rats, compared with controls, on three distinct white adipose tissue (WAT) sites: subcutaneous inguinal, retroperitoneal and epididymal. Tissue weight, DNA (and, from these values cellularity), cAMP content and the expression of several key energy handling metabolism and control genes were analyzed and computed in relation to the whole site mass. Results: Both OE and B3A significantly decreased WAT mass, with no loss of DNA (cell numbers). OE decreased and B3A increased cAMP. Gene expression patterns were markedly different for OE and B3A. OE tended to decrease expression of most genes studied, with no changes (versus controls) of lipolytic but decrease of lipogenic enzyme genes. The effects of B3A were widely different, with a generalized increase in the expression of most genes, including the adrenergic receptors, and, especially the uncoupling protein UCP1. Discussion: OE and B3A, elicit widely different responses in WAT gene expression, end producing similar effects, such as shrinking of WAT, loss of fat, maintenance of cell numbers. OE acted essentially on the balance of lipolysislipogenesis and the blocking of the uptake of substrates; its decrease of synthesis favouring lipolysis. B3A induced a shotgun increase in the expression of most regulatory systems in the adipocyte, an effect that in the end favoured again the loss of lipid; this barely selective increase probably produces inefficiency, which coupled with the increase in UCP1 expression may help WAT to waste energy through thermogenesis. Conclusions: There were considerable differences in the responses of the three WAT sites. OE in general lowered gene expression and stealthily induced a substrate imbalance. B3A increasing the expression of most genes enhanced energy waste through inefficiency rather than through specific pathway activation. There was not a synergistic effect between OE and B3A in WAT, but their combined action increased WAT energy waste. [ABSTRACT FROM AUTHOR]

In the present study we intended to determine how BAT (brown adipose tissue) maintained thermogenesis under treatment with OE (oleoyl-oestrone), a powerful slimming hormone that sheds off body lipid but maintains the metabolic rate. Overweight male rats were subjected to daily gavages of 10 nmol/g of OE or vehicle (control) for 10 days. A PF (pair-fed) vehicle-receiving group was used to discount the effects attributable to energy availability limitation. Interscapular BAT mass, lipid, DNA, mRNA and the RT-PCR (real-time PCR) expression of lipid and energy metabolism genes for enzymes and regulatory proteins were measured. BAT mass and lipid were decreased in OE and PF, with the latter showing a marked reduction in tissue mRNA. Maintenance of perilipin gene expression in PF and OE rats despite the loss of lipid suggests the preservation of the vacuolar interactive surface, a critical factor for thermogenic responsiveness. OE and, to a lesser extent, PF maintained the expression of genes controlling lipolysis and fatty acid oxidation, but markedly decreased the expression of those genes involved in lipogenic and acyl-glycerol synthesis. OE did not affect UCP1 (uncoupling protein 1) (decreased in PF), β3 adrenergic receptors or hormone-sensitive lipase gene mRNAs, which may translate in maintaining a full thermogenic system potential. OE rats were able to maintain a less energetically stressed BAT (probably through glucose utilization) than PF rats. These changes were not paralleled in PF rats, in which lower thermogenesis and glucose preservation resulted in a heavier toll on internal fat stores. Thus the mechanism of action of OE is more complex and tissue-specific than previously assumed. [ABSTRACT FROM AUTHOR]

Oleoyl-estrone (OE) decreases energy intake while maintaining glucose homeostasis, and energy expenditure at the expense of body fat. White adipose tissue (WAT) depots behave differently under starvation, postprandial state and pharmacologically induced lipolysis. To understand the mechanism of massive lipid loss from WAT elicited by OE treatment. We used overweight male rats. Rats receiving OE (10 nmol/g) gavages were compared with controls and a pair-fed group. Whole fat pads from the mesenteric, retroperitoneal, epididymal and inguinal subcutaneous sites were excised and analyzed for lipid, DNA, mRNA and the expression of lipogenic, fatty acid transporters and lipase genes. In OE and pair-fed rats, WAT weights decreased, with the limited loss of cells. Patterns of gene expression in most WAT sites were similar for OE and PF, suggesting a shared mechanism of fat mobilization, but in mesenteric WAT, PF increased lipogenic and fatty acid transporter gene expressions. However, OE inhibited lipogenic expressions more deeply than PF. White adipose tissue sites showed different expression patterns, hinting at relatively specialized functions in fat storage; thus, single site analyses cannot be extrapolated to whole WAT. Differences between mesenteric and the other sites suggest that ‘visceral fat’ should be reserved for this site only, and not applied to other abdominal fat depots (epididymal, retroperitoneal). [ABSTRACT FROM AUTHOR]

Protein contributes significantly to the human daily energy budget. The high diversity in composition, digestibility and dietary proportion complicates the estimation of its actual energy contribution. In practical terms we continue using the energy equivalents estimated by Atwater. This results in a persistent source of imprecision in the calculation of dietary energy that at least can be partially corrected. We used experimentally obtained data to compute an algorithm that will allow to estimate the gross energy content of a protein which composition is known. The relationship between gross energy (i.e. bomb calorimeter-derived) of protein is not a direct correlate of its metabolic efficacy as energy supplier. Thus we estimated the metabolic energy yield (i.e. ATP equivalents) of amino acid residues, using the data to compute the estimated protein metabolic energy yield. Both approaches were to be used to propose a corrected protein energy equivalence factor that will increase the precision in the calculation of dietary protein energy, especially when information on protein composition is available. The gross energy content of amino acids was measured with a bomb-calorimeter, and compared with that of glucose. Amino acid estimated metabolizable energy yield, in moles of ATP per mol of amino acid residue, was also calculated. The net heat yield of all amino acids were used to compute the theoretical heat production of albumin, collagen, gelatin and whole rat protein, which gross energy was also measured experimentally. The mean estimated energy yield (both gross and metabolizable) for each amino acid residue were used to compute the theoretical energy of a number of dietary protein sources which composition was available in the literature. Calculated energy yield of a few selected proteins coincided with the data directly measured in the bomb calorimeter. The computed gross energy yield and metabolizable energy yield for a number of dietary protein sources was estimated. There were minor differences between both parameters: the proportion of aromatic and branched chain amino acids was the main factor affecting the gross energy yield of a given protein; conversely, the higher proportion of nitrogen, especially, but not exclusively, related to arginine and glycine content correlated with lower metabolizable energy. These parameters, corrected by the gross and metabolizable energy of glucose were used to compute a mean energy equivalence for dietary protein: 19 kJ/g protein (i.e. 4.55 kcal/g protein). This value, higher than the current Atwater factor, does not include protein digestibility (as Atwater value did), but included the cost of nitrogen excretion. The methodology presented allows the approximate calculation of both the purported heat production of a protein (pure or mixture) for which we know its amino acid composition (and even get a good estimate if we only know its proportion of nitrogen), and its metabolic energy equivalence. We also propose the use of a new energy correlate of dietary protein; this can be further tuned if the proportion of nitrogen in the protein is known, and even further if its amino acid composition is available. As a consequence of its application to dietary proteins, their energy yield may be higher than usually considered, which may influence the calculations of energy balance. [ABSTRACT FROM AUTHOR]

Background: Current 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. Methods: With 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. Results: The 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. Conclusion: The 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. [ABSTRACT FROM AUTHOR]

Background: Dehydroepiandrosterone (DHEA) released by adrenal glands may be converted to androgens and estrogens mainly in the gonadal, adipose, mammary, hepatic and nervous tissue. DHEA is also a key neurosteroid and has antiglucocorticoid activity. DHEA has been used for the treatment of a number of diseases, including obesity; its pharmacological effects depend on large oral doses, which effect rapidly wanes in part because of its short half-life in plasma. Since steroid hormone esters circulate for longer periods, we have studied here whether the administration of DHEA oleoyl ester may extend its pharmacologic availability by keeping high circulating levels. Results: Tritium-labelled oleoyl-DHEA was given to Wistar male and female rats by gastric tube. The kinetics of appearance of the label in plasma was unrelated to sex; the pattern being largely coincident with the levels of DHEA-sulfate only in females, and after 2 h undistinguishable from the results obtained using labelled DHEA gavages; in the short term, practically no lipophilic DHEA label was found in plasma. After 24 h only a small fraction of the label remained in the rat organs, with a different sex-related distribution pattern coincident for oleoyl- and free- DHEA gavages. The rapid conversion of oleoyl-DHEA into circulating DHEA-sulfate was investigated using stomach, liver and intestine homogenates; which hydrolysed oleoyl-DHEA optimally near pH 8. Duodenum and ileum contained the highest esterase activities. Pure hog pancreas cholesterol-esterase broke down oleoyl-DHEA at rates similar to those of oleoyl-cholesterol. The intestinal and liver esterases were differently activated by taurocholate and showed different pH-activity patterns than cholesterol esterase, suggesting that oleoyl-DHEA can be hydrolysed by a number of esterases in the lumen (e.g. cholesterol-esterase), in the intestinal wall and the liver. Conclusion: The esterase activities found may condition the pharmacological availability (and depot effect) of orally administered steroid hormone fatty acid esters such as oleoyl-DHEA. The oral administration of oleoyl-DHEA in order to extend DHEA plasma availability has not been proved effective, since the ester is rapidly hydrolysed, probably in the intestine itself, and mainly converted to DHEA-sulfate at least in females. [ABSTRACT FROM AUTHOR]

Corticosterone binding (CB) capacity was determined in visceral and subcutaneous white adipose tissue (WAT), as well as in plasma of lean Zucker rats. Perfusion of rats with saline eliminated most liver and kidney corticosterone binding but did not affect CB in WAT. The cytosol extracts of isolated cells, however, did not bind corticosterone in detectable amounts. By means of a RT-PCR procedure it was found that corticosterone-binding globulin (CBG) was expressed in WAT. By immunohistochemical detection in WAT sections, CBG was seen in a thin layer surrounding the cells near the plasma membrane. These data suggest that the CBG layer surrounding the cells may act as a protective barrier limiting the access of glucocorticoids to adipocytes. [ABSTRACT FROM AUTHOR]

Adult female rats received a constant i.v. infusion of oleoyl estrone (3.5 μmol/kg day) in a lipidic suspension for 14 days. On days 0 (no treatment), 3, 6, 10 and 14, as well as on day 14 for controls (receiving only the lipid); the rats were killed and the expression of the β1 , β2 and β3 adrenoceptor genes, in brown adipose tissue and in subcutaneous and periovaric white adipose tissue, were measured by RNA protection assay, and compared with that of cyclophyllin. The β3 adrenoceptor was the most expressed in all adipose tissues, whereas β2 was the less expressed in brown adipose tissue. Oleoyl estrone significantly, but moderately, increased the expression of β adrenoceptors in the three adipose tissues: β1 increased in subcutaneous, β2 and β3 in periovaric and β3 in brown adipose tissue. Oleoyl estrone also decreased β3 expression in subcutaneous white adipose tissue. On day 10, adipocytes isolated from periovaric white adipose tissue of oleoyl estrone treated rats showed higher cAMP response to an isoproterenol challenge than the controls. The mechanism by which oleoyl estrone elicits the wasting of fat reserves could be mediated by adrenergic pathways, at least in part. [ABSTRACT FROM AUTHOR]

The amino acid composition of the protein from three strains of rat (Wistar, Zucker lean and Zucker obese), subjected to reference and high-fat diets has been used to determine the mean empirical formula, molecular weight and N content of whole-rat protein. The combined whole protein of the rat was uniform for the six experimental groups, containing an estimate of 17.3% N and a mean aminoacyl residue molecular weight of 103.7. This suggests that the appropriate protein factor for the calculation of rat protein from its N content should be 5.77 instead of the classical 6.25. In addition, an estimate of the size of the non-protein N mass in the whole rat gave a figure in the range of 5.5 % of all N. The combination of the two calculations gives a protein factor of 5.5 for the conversion of total N into rat protein. [ABSTRACT FROM PUBLISHER]

Aspartate transaminase, alanine transaminase, glutamate dehydrogenase, arginase, serine dehydratase, tyrosine transaminase, glutamine synthetase, glutaminase and adenylate deaminase activities were measured in crude homogenates of 12, 19 and 21-day rat placentae. There is a considerable quantitative importance in enzymes able to produce free ammonia, such as adenylate deaminase and glutamate dehydrogenase, activity that progressively decrease with the age of placenta. The glutamine synthetase and tyrosine transaminase activities increase with age, while serine dehydratase decreases considerably and aspartate and alanine transaminase do not change practically. Arginase shows a maximum at 19, with lower 12 and 21-day activities. No measurable glutaminase activity has been found. The possible implications of the enzymes studied upon the ammonia-producing activity of rat placenta are discussed together with the relative decreasing role of placenta for the overall metabolic activity of the foetus, especially during the last phases of its development. [ABSTRACT FROM AUTHOR]

Humans have developed effective survival mechanisms under conditions of nutrient (and energy) scarcity. Nevertheless, today, most humans face a quite different situation: excess of nutrients, especially those high in amino-nitrogen and energy (largely fat). The lack of mechanisms to prevent energy overload and the effective persistence of the mechanisms hoarding key nutrients such as amino acids has resulted in deep disorders of substrate handling. There is too often a massive untreatable accumulation of body fat in the presence of severe metabolic disorders of energy utilization and disposal, which become chronic and go much beyond the most obvious problems: diabetes, circulatory, renal and nervous disorders included loosely within the metabolic syndrome. We lack basic knowledge on diet nutrient dynamics at the tissue-cell metabolism level, and this adds to widely used medical procedures lacking sufficient scientific support, with limited or nil success. In the present longitudinal analysis of the fate of dietary nutrients, we have focused on glucose as an example of a largely unknown entity. Even most studies on hyper-energetic diets or their later consequences tend to ignore the critical role of carbohydrate (and nitrogen disposal) as (probably) the two main factors affecting the substrate partition and metabolism. [ABSTRACT FROM AUTHOR]

Under normoxic conditions, adipocytes in primary culture convert huge amounts of glucose to lactate and glycerol. This "wasting" of glucose may help to diminish hyperglycemia. Given the importance of insulin in the metabolism, we have studied how it affects adipocyte response to varying glucose levels, and whether the high basal conversion of glucose to 3-carbon fragments is affected by insulin. Rat fat cells were incubated for 24 h in the presence or absence of 175 nM insulin and 3.5, 7, or 14 mM glucose; half of the wells contained 14C-glucose. We analyzed glucose label fate, medium metabolites, and the expression of key genes controlling glucose and lipid metabolism. Insulin increased both glucose uptake and the flow of carbon through glycolysis and lipogenesis. Lactate excretion was related to medium glucose levels, which agrees with the purported role of disposing excess (circulating) glucose. When medium glucose was low, most basal glycerol came from lipolysis, but when glucose was high, release of glycerol via breakup of glycerol-3P was predominant. Although insulin promotes lipogenesis, it also limited the synthesis of glycerol-3P from glucose and its incorporation into acyl-glycerols. We assume that this is a mechanism of adipose tissue defense to avoid crippling fat accumulation which has not yet been described. [ABSTRACT FROM AUTHOR]

Nitrogen balance studies have shown that a portion of the N ingested but not excreted is not accounted for. We compared several diets (standard, high-fat, high-protein, and self-selected cafeteria) to determine how diet-dependent energy sources affect nitrogen handling, i.e., the liver urea cycle. Diet components and rat homogenates were used for nitrogen, lipid, and energy analyses. Plasma urea and individual amino acids, as well as liver urea cycle enzyme activities, were determined. Despite ample differences in N intake, circulating amino acids remained practically unchanged in contrast to marked changes in plasma urea. The finding of significant correlations between circulating urea and arginine-succinate synthase and lyase activities supported their regulatory role of urea synthesis, the main N excretion pathway. The cycle operation also correlated with the food protein/energy ratio, in contraposition to total nitrogen losses and estimated balance essentially independent of dietary energy load. The different regulation mechanisms observed have potentially important nutritional consequences, hinting at nitrogen disposal mechanisms able to eliminate excess nitrogen under conditions of high availability of both energy and proteins. Their operation reduces urea synthesis to allow for a safe (albeit unknown) mechanism of N/energy excess accommodation. [ABSTRACT FROM AUTHOR]