Dysregulation of macrophage PEPD in obesity determines adipose tissue fibro-inflammation and insulin resistance.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes.

Extracellular vesicles released by steatotic hepatocytes alter adipocyte metabolism.

The composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes by characterizing EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal markers in comparison with their lean counterpart. Moreover, proteomic analysis revealed that those EVs reflect the metabolic state of the parent cell in that the majority of proteins upregulated relate to fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway. In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity in recipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations in different adipocyte metabolic pathways in cells treated with hepatic EVs. In summary, our work showed that steatosis has a significant impact in the amount and composition of EVs secreted by hepatocytes. Moreover, our data point to the involvement of hepatic-EVs in the development of pathologies associated with MetS.

Defective glucose and lipid metabolism in rheumatoid arthritis is determined by chronic inflammation in metabolic tissues.

BACKGROUND: Rheumatoid arthritis (RA) patients are at increased risk of insulin resistance (IR); however, the specific mechanisms mediating this association are currently unknown. OBJECTIVE: To investigate whether the inflammatory activity associated with RA accounts for the observed defective glucose metabolism and lipid metabolism in these patients. METHODS: We followed two main strategies: (i) extensive metabolic profiling of a RA cohort of 100 patients and 50 healthy control subjects and (ii) mechanistic studies carried out in both a collagen-induced arthritis mouse model and 3T3-L1 adipocytes treated with conditioned serum from RA patients. RESULTS: Following the exclusion of obese and diabetic subjects, data from RA patients demonstrated a strong link between the degree of systemic inflammation and the development of IR. These results were strengthened by the observation that induction of arthritis in mice resulted in a global inflammatory state characterized by defective carbohydrate and lipid metabolism in different tissues. Adipose tissue was most susceptible to the RA-induced metabolic alterations. These metabolic effects were confirmed in adipocytes treated with serum from RA patients. CONCLUSIONS: Our results show that the metabolic disturbances associated with RA depend on the degree of inflammation and identify inflammation of adipose tissue as the initial target leading to IR and the associated molecular disorders of carbohydrate and lipid homeostasis. Thus, we anticipate that therapeutic strategies based on tighter control of inflammation and flares could provide promising approaches to normalize and/or prevent metabolic alterations associated with RA.

Sphingolipids and glycerophospholipids - The "ying and yang" of lipotoxicity in metabolic diseases.

Sphingolipids in general and ceramides in particular, contribute to pathophysiological mechanisms by modifying signalling and metabolic pathways. Here, we present the available evidence for a bidirectional homeostatic crosstalk between sphingolipids and glycerophospholipids, whose dysregulation contributes to lipotoxicity induced metabolic stress. The initial evidence for this crosstalk originates from simulated models designed to investigate the biophysical properties of sphingolipids in plasma membrane representations. In this review, we reinterpret some of the original findings and conceptualise them as a sort of "ying/yang" interaction model of opposed/complementary forces, which is consistent with the current knowledge of lipid homeostasis and pathophysiology. We also propose that the dysregulation of the balance between sphingolipids and glycerophospholipids results in a lipotoxic insult relevant in the pathophysiology of common metabolic diseases, typically characterised by their increased ceramide/sphingosine pools.

Dihydroceramide desaturase 1, the gatekeeper of ceramide induced lipotoxicity.

The pathogenic relevance of sphingolipid metabolism is increasingly being recognised. Here we elaborate on a new player within the sphingolipid field: the degs1 enzyme, a recently discovered enzyme that catalyses the final step in the de novo biosynthesis of ceramides controlling the step from dihydroceramides to ceramides. Here, we describe its function and dysregulation by factors such as oxidative stress, hypoxia and inflammation and provide evidence indicating that dihydroceramides constitute a biologically active molecule from the sphingolipid family with certain differential characteristics with respect to its delta-4 unsaturated counterparts, the ceramides. Finally we present pathophysiological scenarios characterised by specific increases in dihydroceramide that challenge the concept that "all ceramides species are the same". This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

Expression of mitochondrial biogenesis-signaling factors in brown adipocytes is influenced specifically by 17beta-estradiol, testosterone, and progesterone.

Control of mitochondrial biogenesis in brown adipose tissue (BAT), as part of the thermogenesis program, is a complex process that requires the integration of multiple transcription factors to orchestrate mitochondrial and nuclear gene expression. Despite the knowledge of the role of sex hormones on BAT physiology, little is known about the effect of these hormones on the mitochondrial biogenic program. The aim of this study was to determine the effect of testosterone, 17beta-estradiol, and progesterone on the expression of nuclear factors involved in the control of mitochondrial biogenesis and thermogenic function such as ppargamma, pgc1alpha, nrf1, gabpa, and tfam, and also an inhibitor of PI3K-Akt pathway, recently found to be involved in the control of mitochondrial recruitment (pten). For this purpose, an in vitro assay using cell-cultured brown adipocytes was used to address the role of steroid hormones, progesterone, testosterone, and 17beta-estradiol on the mRNA expression of these factors by real-time PCR. Thus 17beta-estradiol seemed to exert a dual effect, activating the PI3K-Akt pathway by inhibiting pten mRNA expression and also inhibiting nrf1 and tfam mRNA expression. Progesterone seemed to positively stimulate mitochondriogenesis and BAT differentiation by increasing the mRNA expression of the gabpa-tfam axis and ppargamma, respectively, but also exerted a negative output by increasing pten mRNA levels. Finally, testosterone inhibited the transcription of pgc1alpha, the master factor involved in UCP1 expression and mitochondrial biogenesis. In conclusion, our results support the idea that sex hormones have direct effects on different mediators of the mitochondriogenesis program.

Sex steroid receptor expression in different adipose depots is modified during midpregnancy.

Sex hormone signalling is key in the understanding of adipose tissue metabolism during pregnancy. Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of lipolysis and lipogenesis. We analyze steroid receptor mRNA levels in different rat adipose depots and mammary fat pad, as well as the sex hormone profile during midpregnancy, coinciding with the placentation process. Thus, progesterone and estradiol plasma levels were increased as well as testosterone levels. This hormonal profile was accompanied by low glucose to insulin ratio. PR-B, ERalpha and AR receptor densities during midpregnancy were dependent on adipose depot location. In mammary fat pad, the mRNA levels of sex hormone receptors were correlated with the growth of the depot. These results demonstrate that sex steroid hormone receptor mRNA expression during midpregnancy is tissue-specific. Our results agree with the idea that the increased estrogenic and androgenic signalling could be addressed to reducing the lipogenic state in early pregnancy exerted mainly by progesterone and to prepare adipose tissue for the beginning of the catabolic phase in late pregnancy in a depot-specific manner.

Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu.

Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17beta-estradiol (17beta-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17beta-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17beta-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER)alpha and ERbeta densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ERalpha predominated over ERbeta expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17beta-E2 increased ERalpha (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.

Gender- and site-related effects on lipolytic capacity of rat white adipose tissue.

Gender- and site-related differences in the lipolytic capacity, at the different steps of the adrenergic pathway, in gonadal and inguinal white adipose tissue (WAT), were assessed by studying alpha2A-adrenergic receptor (AR), beta3-AR and hormone-sensitive lipase (HSL) protein levels, and by determining the lipolytic response to different agents. Gonadal WAT showed a lower alpha2A/beta3-AR ratio, a greater lipolytic capacity in response to AR agonists, and higher HSL activity and protein levels than inguinal WAT. In female rats, we found greater alpha2A-AR protein levels and alpha2A/beta3-AR ratio compared to their male counterparts, but, on the other hand, a higher lipolytic response to beta-AR agonists and a greater lipolytic capacity at the postreceptor level, including a more activated HSL protein. Thus, the lipolytic capacity was clearly higher in gonadal than in inguinal WAT, at the different steps of the adrenergic pathway studied. Moreover, in both tissues, females showed a greater inhibition of lipolysis via alpha2-AR, which was counteracted by the higher lipolytic capacity at the postreceptor level.

Using the Web to aid biochemists and microbiologists: an example of computer-based learning using PCR as an example.

The aim of this study was two-fold: to make available to the medical community a fast, efficient tool for troubleshooting PCR problems, and to demonstrate that hypermedia allow one to approach this kind of problem in a new, more useful way. The Web provides access to an enormous amount of information in the medical area, and in particular enables troubleshooting of new techniques in the medical laboratory. Currently it is possible to find textual information but it is not always easy to find hypermedia information. In many cases authors change only the means of dissemination of a book or paper to the Web, but do not adapt the information to the features of this new medium. The prototype of our hypermedia application was developed using Director and Flash programs (Macromedia), the application was developed from a decision tree built as a result of information compiled from expert PCR users and the existing literature. It was implemented on a website using Dreamweaver (Macromedia). The usefulness and possibilities of this application have been tested with physicians, medical laboratory technicians, and health science students, and have been proven to be more effective than traditional methods, both in education processes and in helping researchers and medical laboratory technicians in their daily work. The advance and popularization of the Internet in the medical profession requires information on the Web to adapt to the advantages offered by hypermedia. This new step must be taken, and the Web must offer medical users not only a different medium of information distribution but also a new way of handling and obtaining information.

Sexual dimorphism in the adrenergic control of rat brown adipose tissue response to overfeeding.

Gender-related differences in the brown adipose tissue (BAT) response to overfeeding rats on a cafeteria diet were studied by assessing the balance between the expression of beta-adrenoceptors (beta1-, beta2-, beta3-AR) and alpha2A-AR and their relation to the expression of uncoupling proteins (UCP1, UCP2, UCP3). Cafeteria diet feeding for 15 days, which involved a similar degree of hyperphagia in both sexes, led to a greater body weight excess in females than in males and a lower activation of thermogenesis. Gender-related differences were found for different adrenoceptor expression and protein levels, which might explain, in part, sex differences in the thermogenic parameters. The lower expression of alpha2A-AR in females than in males could be responsible for the higher expression of UCP1 and thermogenic capacity under non-hyperphagic conditions. However, in a situation of high adrenergic stimulation--as occurs with overfeeding--as there is a preferential recruitment of the beta3-AR by noradrenaline compared with other adrenergic receptors, the higher levels of beta3-AR in males rats than in females could be responsible for the greater thermogenic capacity and the lesser weight gain in males. Thus, the alpha2/beta3 balance in BAT could be a key in the thermogenic control.

Diferencias sexuales en la distribución y función de los tejidos adiposos / Gender differences in adipose tissues distribution and function

La heterogeneidad de los depósitos de tejido adiposo viene determinada por la combinación de factores genéticos y ambientales, en particular, por el perfil hormonal.En los últimos años numerosos estudios han sugerido un importante papel de las hormonas sexuales en el control del metabolismo lipídico y en la determinación de los perfiles de distribución grasa en hombres y mujeres.En los depósitos adiposos del hombre, la testosterona, estimula la lipólisis (incrementa la sensibilidad beta-adrenérgica) y limita la incorporación de ácidos grasos en los depósitos (mediante la modulación de la actividad de la LPL), a través del receptor de andrógenos, siendo este efecto más pronunciado en las regiones viscerales donde predominan los receptores de andrógenos respecto a otras regiones; sin embargo, la presencia de una mayor actividad LPL en la región visceral en comparación con los depósitos adiposos subcutáneos contrarresta este efecto, determinando que en el varón estos depósitos almacenen más ácidos grasos que el resto. Datos en consonancia con el hecho de que con la edad, caracterizada por una disminución de los niveles de testosterona, se incrementa el tamaño de los depósitos viscerales.En mujeres, los estrógenos también ejercen una función lipolítica incrementando la sensibilidad a la noradrenalina en los depósitos grasos. Paralelamente los estrógenos inducen la síntesis de la LPL al menos en la región glúteo-femoral, que además contiene una mayor densidad de receptores adrenérgicos alfa2, lo que implicaría una acumulación preferencial de ácidos grasos en esa zona. Los efectos de los estrógenos vienen mediados por los receptores de estrógenos, aunque no se han encontrados diferencias en la densidad de estos receptores en los depósitos estudiados (AU)