Endothelial cells (ECs) are key metabolic gatekeepers, yet their role in metabolic health remains unclear. Given their central involvement in energy metabolism, mitochondria are ideally positioned to enable ECs to adapt to ever-changing metabolic requirements. Here, we explore the hypothesis that mitochondrial dynamics proteins in ECs influence whole-body metabolic status. Genetic deficiency of Mfn2 in ECs (Mfn2iΔEC), but not of Mfn1iΔEC, induces a mitohormetic response in the adipose vasculature, enhancing antioxidant defenses, mitochondrial fitness, and lipid oxidation, ultimately improving metabolic outcomes. Cultured ECs secrete the mitokine growth differentiation factor 15 (GDF15) via a forkhead box O1 (FOXO1)-dependent axis, a response also observed under stress conditions in vivo. Notably, Mfn2iΔEC mice exhibited elevated endothelial and circulating GDF15 levels, and neutralization of GDF15 partly attenuated their metabolic benefits. Consistent with mitohormetic activation, Mfn2iΔEC mice showed protection against diet-induced obesity and delayed age-related decline. Hence, vascular mitohormetic adaptations emerge as a novel mechanism promoting systemic metabolic health.

Despite considerable research on the differential metabolic effects of diets, information regarding the dietary energy partition process remains scarce and controversial, complicating the analysis of its main metabolic regulatory factors, including diet composition. We utilized a highly controlled dietary setup to analyze relative changes in nutrient content, measuring 17β-estradiol (E2) and testosterone (T) levels. This study integrates recalculated data from previous homologous publications with new, unpublished material, completing a multi-year investigation. We used a common chow-based model: ST or standard diet, HF high-fat and HP or high-protein. A CF, cafeteria-type diet was added for comparison. Rats (female and male) were fed the diets for 30 d. Nutrient intake and body composition were measured. Plasma glucose, lactate, T and E2 were also measured. The energy intake of non-CF groups, was largely based in carbohydrates (CH), yielding hexoses (6C units), which were fully oxidized, or accrued as 2C fragments (via their aggregation as fatty acids). 3C glycolytic units were eventually stored, in part, as glycerol in TAG (triacyl-glycerols). In 30 d, accrual of 2C exceeded about ten-fold that of 3C. Amino acid (AA) catabolism primarily yielded 2C, 3C, and 4C–5C fragments; the latter, potentially acting as Krebs cycle anaplerotic intermediates (KCAI), facilitated 2C oxidation and reduced TAG accumulation. Lactate levels correlated with lactate dehydrogenase activities in muscle and liver. TAG accrual was maximal in CF, quite differently from HF despite having a similar dietary lipid intake proportion. Maximal rates of lipid storage were observed in the ST (and CF) groups. Females showed a higher ability to oxidize CH and to store less TAG. T was correlated with protein deposition, whilst E2 decreased this by favoring AA oxidation and raising the availability of KCAI. Results in rats suggest that an effective partition scheme requires nearly half of the energy from carbohydrates (polysaccharides). Furthermore, initial protein oxidation, yielding KCAI, facilitates 2C oxidation from carbohydrates, thereby reducing their conversion to TAG storage.

SCOPE:The aim of this study is to investigate the effect of time-of-day on serum hormones and gene expression in adrenal glands, studying the impact of sex, obesogenic diet, and timing of proanthocyanidins administration, with a focus on glucocorticoids synthesis by this gland. METHODS AND RESULTS:Female and male rats, assigned to a standard chow or a cafeteria diet-fed group, receive a daily oral dose of a grape seed proanthocyanidin extract (GSPE), or a vehicle (when light is turned on, or when light is turned off). Corticosterone, estradiol, and testosterone serum levels, and the expression analysis of clock genes and genes related to corticosterone synthesis pathway, are assessed. Serum hormone levels exhibited a marked time-of-day effect also see in the expression of scavenger receptor class B member 1 (Scarb1) and cyp11b genes. The correlation between these two genes and period circadian regulator 2 (Per2) is also extended to other clock genes, although to a lesser extent: cryptochrome (Cry) and nuclear receptor subfamily 1 group D member 1 (Rev-erba). CONCLUSION:The strong correlations found suggest an important role of local Per2 (but also of Cry and Rev-erbA) in regulating the expression of the enzymes involved in the corticosterone synthesis pathway. The expression of clock genes in adrenals is influenced by sex and diet but not by GSPE.

Background Although prenatal and postnatal programming of metabolic diseases in adulthood is well established, the mechanisms underpinning metabolic programming are not. Dlk1, a key regulator of fetal development, inhibits adipocyte differentiation and restricts fetal growth. Methods Assess DLk1 expression in a Wistar rat model of catch-up growth following intrauterine restriction. Dams fed ad libitum delivered control pups (C) and dams on a 50% calorie-restricted diet delivered pups with low birth weight (R). Restricted offspring fed a standard rat chow showed catch-up growth (R/C) but those kept on a calorie-restricted diet did not (R/R). Results Decreased Dlk1 expression was observed in adipose tissue and skeletal muscle of R/C pups along with excessive visceral fat accumulation, decreased circulating adiponectin, increased triglycerides and HOMA-IR (from p < 0.05 to p < 0.0001). Moreover, in R/C pups the reduced Dlk1 expression in adipose tissue and skeletal muscle correlated with visceral fat ( r = −0.820, p < 00001) and HOMA-IR ( r = −0.745, p = 0.002). Conclusions Decreased Dlk1 expression relates to visceral fat expansion and insulin resistance in a rat model of catch-up growth following prenatal growth restriction. Modulation of Dlk1 expression could be among the targets for the early prevention of fetal programming of adult metabolic disorders.

Proanthocyanidins (PAs) are one of the most commonly ingested polyphenols in the human diet, with a wide range of beneficial health effects. Remarkably, PAs have been reported to influence core and peripheral clock genes expression, and their effects may change in a time-of-day dependent manner. Therefore, the aim of this study was to investigate whether the capacity of PAs to modulate the metabolome is conditioned by the time-of-day in which these compounds are consumed in a diet- and sex-dependent manner. To do this, a grape seed proanthocyanidin extract (GSPE) was administered to female and male Fischer 344 rats at ZT0 (in the morning) and ZT12 (at night) and the GSPE administration time effect was evaluated on clock genes expression, melatonin hormone and serum metabolite levels in a healthy and obesogenic context. The results showed an administration time effect of GSPE on the metabolome in a sex and diet-dependent manner. Specifically, there was an effect on amino acid, lipid and cholate metabolite levels that correlated with the central clock genes expression. Therefore, this study shows a strong influence of sex and diet on the PAs effects on the metabolome, modulated in turn by the time-of-day.

The relative proportions between nutrients affects the fate of them for metabolic interconversion, storage and turnover or, essentially for energy, in a process of partition modulated by hormonal and metabolic factors. A basic common chow model was used: ST or standard diet, HF high-fat and HP or high-protein had a common substrate. A CF cafeteria-type diet was added for comparison. Rats (female and male) received the diet for 30d. Nutrient intake and body composition were measured. Plasma glucose, lactate, testosterone (T) and estradiol (E2) were measured. Common groups energy intake, was largely based in carbohydrates CH, yielding 6C units, which were fully oxidized or stored as 2C fragments (fatty acids). Lactate (3C) was partially stored as glycerol of TAG. Accrual of 2C far exceeded about ten-fold that of 3C. Amino acid (AA) catabolism yielded 2C, 3C and 4C-5C fragments, the latter acting as Krebs cycle anaplerotic intermediates (KCAI), that facilitated the oxidation of 2C in detriment of TAG accumulation Lactate levels, were related to lactic dehydrogenase activities in muscle and liver. TAG accrual was maximal in CF, quite differently from HF with similar diet lipid proportion. Maximal rates of lipid storage were found in ST (and CF) groups. Females showed a higher ability to oxidize CH and to store less TAG. T increased N and protein deposition, whilst E2 decreased them favoring AA oxidation and the availability of KCAI. In the rat, an effective energy partition scheme requires that about half of the energy is provided by CH (polysaccharide), and needs enough protein to provide KCAI for a smooth 2C oxidation.

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.

Accelerated catch-up growth following intrauterine restriction increases the risk of developing visceral adiposity and metabolic abnormalities. However, the underlying molecular mechanisms of such metabolic programming are still poorly understood. A Wistar rat model of catch-up growth following intrauterine restriction was used. A gene expression array was performed in the retroperitoneal adipose tissue sampled at postnatal day (PD) 42. Five hundred and forty-six differentially expressed genes (DEGs) were identified (adjusted p value < 0.05). Gene ontology enrichment analysis identified pathways related to immune and lipid metabolic processes, brown fat cell differentiation, and regulation of PI3K. Ccl21, Npr3, Serpina3n, Pnpla3, Slc2a4, and Serpina12 were validated to be upregulated in catch-up pups (all p < 0.01) and related to several fat expansion and metabolic parameters, including body weight at PD42, postnatal body weight gain, white and brown adipose tissue mass, plasma triglycerides, and insulin resistance index (all p < 0.05). Genes related to immune and metabolic processes were upregulated in retroperitoneal adipose tissue following catch-up growth in juvenile rats and were found to be associated with fat expansion and metabolic parameters. Our results provide evidence for several dysregulated genes in white adipose tissue that could help develop novel strategies to prevent the metabolic abnormalities associated with catch-up growth.

Metabolic surgery modulates the enterohormone profile, which leads, among other effects, to changes in food intake. Bitter taste receptors (TAS2Rs) have been identified in the gastrointestinal tract and specific stimulation of these has been linked to the control of ghrelin secretion. We hypothesize that optimal stimulation of TAS2Rs could help to modulate enteroendocrine secretions and thus regulate food intake. To determine this, we have assayed the response to specific agonists for hTAS2R5, hTAS2R14 and hTAS2R39 on enteroendocrine secretions from intestinal segments and food intake in rats. We found that hTAS2R5 agonists stimulate glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK), and reduce food intake. hTAS2R14 agonists induce GLP1, while hTASR39 agonists tend to increase peptide YY (PYY) but fail to reduce food intake. The effect of simultaneously activating several receptors is heterogeneous depending on the relative affinity of the agonists for each receptor. Although detailed mechanisms are not clear, bitter compounds can stimulate differentially enteroendocrine secretions that modulate food intake in rats.

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.

SCOPE:Eating large amounts of fat is usually associated with fat accumulation. However, different types of diets (not only lipids) elicit different metabolic responses.METHODS AND RESULTS:Male and female rats (10 week-old) are distributed in four groups and fed for 1 month a standard diet (SD), or this diet enriched with either lipid (high-fat diet, HF) or protein (high-protein diet, HP), or a cafeteria diet (CAF). Both HF and CAF diets share the percentage of energy from lipids (40%) but these are different. Protein-derived energy in the HP diet is also 40%. Feeding SD, HF, and HP diets does not result in differences in energy intake, energy expenditure, total body weight, or lipid content. However, the CAF-fed groups show increases in these parameters, which are more marked in the male rats. The CAF diet increases the mass of adipose tissue while the HF diet does not.CONCLUSION:Different diets produce substantial changes in the fate of ingested nutrient energy. Dietary lipids are not essential for sustaining an increase in body lipid (or adipose tissue) content. Body protein accrual is unrelated to dietary lipids and overall energy intake. Both protein and lipid accrual are more efficient in male rats.

The ingestion of excess fat often produces an increased body weight, because of higher adiposity and accumulation of fat in the liver. However, modulation of diet energy partition may affect differently the body metabolic responses and the extent of lipid deposition. Ten-week-old male and female rats were fed with either standard rat chow (SD), standard diet enriched with coconut oil (high-fat diet, HF), standard diet enriched with protein (high-protein diet, HP) or a self-selected “cafeteria” diet (CAF) for one month. Both HF and CAF diets provided the same lipid-derived percentage of energy (40%) HP diet protein-energy derived was twice (40%) than those of the SD diet. After the treatment, CAF groups showed significant weight increases. Hepatic lipid content also showed sex-related differences; triacylglycerol accumulation was significant in HF and CAF fed males. Cholesterol content was higher only in the CAF male group. Plasma estradiol in HF and HP males was higher than in CAF. Circulating cholesterol was inversely correlated with estradiol levels, which were proportional to lactate levels. These changes agreed with the differences found in the expression of key hepatic enzymes of lipid and energy metabolism. The protective effect of estrogens preventing excess liver lipid deposition, is also effective in males with ‘normal’ diets unbalanced by lipid or protein, but is not sufficient to protect males from the massive changes produced by a markedly obesogenic cafeteria-type diet. Estradiol protective effects are exerted at the root of energy metabolism, on the partition of substrates distributed from or entering the liver.

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.

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.

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.

Background: Glucose is the main inter-organ energy supplying metabolite in humans and other vertebrates. In clinical analyses, its measurement is probably the most performed and used for diagnostic, monitoring and control of the physiological status. However, glucose chemical structure, specially its anomeric forms (alpha/beta), may deeply interfere in their own analyses, often resulting in misleading results. Methods: These effects on glucose estimation were studied by using a common glucose oxidase/peroxidase based method, in the presence or absence of added mutarotase, which speeds up the alpha/beta conversion rate. Glucose concentrations were measured in pure standards and plasma samples from control and cafeteria diet-fed rats. Results: The addition of mutarotase resulted in higher glucose readings, independently of glucose concentration and its initial anomeric proportions in the sample. In the absence of mutarotase, cafeteria-fed rats had higher glucose levels than controls, but the differences disappeared in its presence, because under experimental conditions, a proportion of the alpha-anomer was not isomerized and thus was not measured. Conclusions: Diet altered the proportion of anomers, suggesting that glucose usage by physiological processes affects the anomers’ ratio and may have an important metabolic meaning, which deserves a detailed study in addition to the need to correct the methods in use to obtain real “total glucose” readings. J Endocrinol Metab. 2019;9(3):63-70 doi: https://doi.org/10.14740/jem555

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 14 mM glucose 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.

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 7/14mM C-14-glucose for 24/48h. 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 24h, 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 (24h-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.