Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson's Disease Patient-Specific Dopamine Neurons.

Accumulation of misfolded α-synuclein (α-syn) is a hallmark of Parkinson's disease (PD) thought to play important roles in the pathophysiology of the disease. Dendritic systems, able to modulate the folding of proteins, have emerged as promising new therapeutic strategies for PD treatment. Dendrimers have been shown to be effective at inhibiting α-syn aggregation in cell-free systems and in cell lines. Here, we set out to investigate the effects of dendrimers on endogenous α-syn accumulation in disease-relevant cell types from PD patients. For this purpose, we chose cationic carbosilane dendrimers of bow-tie topology based on their performance at inhibiting α-syn aggregation in vitro. Dopamine neurons were differentiated from induced pluripotent stem cell (iPSC) lines generated from PD patients carrying the LRRK2G2019S mutation, which reportedly display abnormal accumulation of α-syn, and from healthy individuals as controls. Treatment of PD dopamine neurons with non-cytotoxic concentrations of dendrimers was effective at preventing abnormal accumulation and aggregation of α-syn. Our results in a genuinely human experimental model of PD highlight the therapeutic potential of dendritic systems and open the way to developing safe and efficient therapies for delaying or even halting PD progression.

Inborn errors of metabolism: Lessons from iPSC models.

The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs.

MicroRNA alterations in iPSC-derived dopaminergic neurons from Parkinson disease patients.

MicroRNA (miRNA) misregulation in peripheral blood has been linked to Parkinson disease (PD) but its role in the disease progression remains elusive. We performed an explorative genome-wide study of miRNA expression levels in dopaminergic neurons (DAn) from PD patients generated by somatic cell reprogramming and induced pluripotent stem cells differentiation. We quantified expression levels of 377 miRNAs in DAn from 3 sporadic PD patients (sPD), 3 leucine-rich repeat kinase 2-associated PD patients (L2PD) (total 6 PD), and 4 healthy controls. We identified differential expression of 10 miRNA of which 5 were upregulated in PD (miR-9-5p, miR-135a-5p, miR-135b-5p, miR-449a, and miR-449b-5p) and 5 downregulated (miR-141-3p, miR-199a-5p, miR-299-5p, miR-518e-3p, and miR-519a-3p). Changes were similar in sPD and L2PD. Integrative analysis revealed significant correlations between miRNA/mRNA expression. Moreover, upregulation of miR-9-5p and miR-135b-5p was associated with downregulation of transcription factors related to the DNA hypermethylation of enhancer elements in PD DAn (FOXA1 and NR3C1). In summary, miRNA changes are associated with monogenic L2PD and sPD and co-occur with epigenetic changes in DAn from PD patients.

Inhibition of Notch rescues the angiogenic potential impaired by cardiovascular risk factors in epicardial adipose stem cells.

The epicardial adipose tissue (EAT) is a reservoir of adipose-derived stem cells (ASCs), with as yet unknown effects on myocardial and coronary arteries homeostasis. The purpose of this study was to investigate the angiogenic function of epicardial ASCs and their regulation by the common cardiovascular risk factors (CVRFs) affecting heart disease. Epicardial fat was obtained from a rodent model with clustering of CVRFs [Zucker diabetic fatty (ZDF)-Lepr(fa)] rats and from their lean control (ZDF-Crl) littermates without CVRFs, ASCs were isolated, and their function was assessed by proliferation and differentiation assays, flow cytometry, gene expression, and in vivo Matrigel angiogenesis analysis. Epicardial ASCs from both groups showed adipogenic and osteogenic differentiation capacity; however, epicardial ASCs from CVRF animals had a lesser ability to form tubular structures in vitro after endothelial differentiation, as well as a reduced angiogenic potential in vivo compared to control animals. Epicardial ASCs from CVRF rats showed up-regulation of the downstream Notch signaling genes Hes7, Hey1, and Heyl compared with control animals. The inhibition of Notch signaling by conditioning epicardial ASCs from CVRF animals with a γ-secretase inhibitor induced a reduction in Hes/Hey gene expression and rescued their angiogenic function in vivo We report for the first time the impact of CVRF burden on the ASCs of EAT and that the defective function is in part caused by increased Notch signaling. Conditioning ASCs by blocking Notch signaling rescues their angiogenic potential.-Bejar, M. T., Ferrer-Lorente, R., Peña, E., Badimon, L. Inhibition of Notch rescues the angiogenic potential impaired by cardiovascular risk factors in epicardial adipose stem cells.

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming.

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.

Selective inhibition of sphingosine kinase-1 protects adipose tissue against LPS-induced inflammatory response in Zucker diabetic fatty rats.

Obesity is associated with a state of chronic inflammation. The chemokine (C-C motif) ligand 5 (CCL5) has been proposed to modulate the inflammatory response in adipose tissue (AT). However, the mechanisms underlying CCL5 upregulation in AT remain undefined. The objective of the present study was to evaluate whether the enzyme sphingosine kinase-1 (SK1) would modulate the expression of CCL5 and other inflammatory biomarkers in primary adipocytes and its potential role in lipopolysaccharide (LPS)-induced AT inflammation in a rat model of diabetes. To address this, LPS-stimulated primary adipocytes and 3T3-L1 cells were treated with a SK inhibitor, and the expression of Ccl5 and other CC chemokines were studied. Moreover, the effect of SK1 knockdown on cytokine production was analyzed in 3T3-L1 cells by transfection of SK1-specific small-interfering RNA (siRNA). The anti-inflammatory effects of SK inhibitor in AT were also investigated in vivo using the Zucker lean normoglycemic control (ZLC) rats. LPS treatment stimulated Ccl5, IL-6, pentraxin 3 (Ptx3), and Tnfα mRNA expression in primary adipocytes and 3T3-L1 cells, whereas pharmacologically and siRNA-mediated SK1 inhibition strongly reduced mRNA levels of proinflammatory cytokines in these cells. Similarly, administration of SK inhibitor to ZLC rats prevented the LPS-induced inflammatory response in AT. Our data demonstrate a role for SK1 in endotoxin-induced cytokine expression in adipocytes and suggest that inhibition of SK1 may be a potential therapeutic tool in the prevention and treatment of chronic and common metabolic disorders, including obesity, insulin-resistance, and type 2 diabetes.

Notch signaling pathway activation in normal and hyperglycemic rats differs in the stem cells of visceral and subcutaneous adipose tissue.

The precise mechanisms underlying the differential function and cardiometabolic risk of white adipose tissue (WAT) remain unclear. Visceral adipose tissue (VWAT) and subcutaneous adipose tissue (SCWAT) have different metabolic functions that seem to be ascribed to their different intrinsic expansion capacities. Here we have hypothesized that the WAT characteristics are determined by the resident adipose-derived stem cells (ASCs) found in the different WAT depots. Therefore, our objective has been to investigate adipogenesis in anatomically distinct fat depots. ASCs from five different WAT depots were characterized in both healthy lean and diabetic obese rats, showing significant differences in expression of some of genes governing the stemness and the earlier adipogenic differentiation steps. Notch-target genes [Hes (hairy and enhancer of split) and Hey (hairy/enhancer of split related with YRPW motif) families] were upregulated in ASCs derived from visceral depots. Upon adipogenic differentiation, adipocyte cell markers were downregulated in ASCs from VWAT in comparison to ASCs from SCWAT, revealing a lower adipogenic capacity in ASCs of visceral origin than in those of SCWAT in accordance with the differential activation of Notch signaling. Notch upregulation by its activator phenethyl isothiocyanate attenuated the adipogenic differentiation of ASCs from SCWAT whereas Notch inhibition by N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) increased the adipogenic differentiation of ASCs from visceral origin. In conclusion, the differential activation of Notch in ASCs is the origin of the different intrinsic WAT expansion capacities that contribute to the regional variations in WAT homeostasis and to its associated cardiometabolic risk.

Systems biology approach to identify alterations in the stem cell reservoir of subcutaneous adipose tissue in a rat model of diabetes: effects on differentiation potential and function.

AIMS/HYPOTHESIS: Autologous progenitor cells represent a promising option for regenerative cell-based therapies. Nevertheless, it has been shown that ageing and cardiovascular risk factors such as diabetes affect circulating endothelial and bone marrow-derived progenitor cells, limiting their therapeutic potential. However, their impact on other stem cell populations remains unclear. We therefore investigated the effects of diabetes on adipose-derived stem cells (ASCs) and whether these effects might limit the therapeutic potential of autologous ASCs. METHODS: A systems biology approach was used to analyse the expression of genes related to stem cell identification in subcutaneous adipose tissue (SAT), the stromal vascular fraction and isolated ASCs from Zucker diabetic fatty rats and their non-diabetic controls. An additional model of type 2 diabetes without obesity was also investigated. Bioinformatic approaches were used to investigate the biological significance of these changes. In addition, functional studies on cell viability and differentiation potential were performed. RESULTS: Widespread downregulation of mesenchymal stem cell markers was observed in SAT of diabetic rats. Gene expression and in silico analysis revealed a significant effect on molecules involved in the maintenance of pluripotency and self-renewal, and on the alteration of main signalling pathways important for stem cell maintenance. The viability and differentiation potential of ASCs from diabetic rats was impaired in in vitro models and in in vivo angiogenesis. CONCLUSIONS/INTERPRETATION: The impact of type 2 diabetes on ASCs might compromise the efficiency of spontaneous self-repair and direct autologous stem cell therapy.

Platelets derived from the bone marrow of diabetic animals show dysregulated endoplasmic reticulum stress proteins that contribute to increased thrombosis.

OBJECTIVE: Patients with diabetes mellitus have an increased risk of suffering atherothrombotic syndromes and are prone to clustering cardiovascular risk factors. However, despite their dysregulated glucose metabolism, intensive glycemic control has proven insufficient to reduce thrombotic complications. Therefore, we aimed to elucidate the determinants of thrombosis in a model of type 2 diabetes mellitus with cardiovascular risk factors clustering. METHODS AND RESULTS: Intravital microscopy was used to analyze thrombosis in vivo in Zucker diabetic fatty rats (ZD) and lean normoglycemic controls. Bone marrow (BM) transplants were performed to test the contribution of each compartment (blood or vessel wall) to thrombogenicity. ZD showed significantly increased thrombosis compared with lean normoglycemic controls. BM transplants demonstrated the key contribution of the hematopoietic compartment to increased thrombogenicity. Indeed, lean normoglycemic controls transplanted with ZD-BM showed increased thrombosis with normal glucose levels, whereas ZD transplanted with lean normoglycemic controls-BM showed reduced thrombosis despite presenting hyperglycemia. Significant alterations in megakaryopoiesis and platelet-endoplasmic reticulum stress proteins, protein disulfide isomerase and 78-kDa glucose-regulated protein, were detected in ZD, and increased tissue factor procoagulant activity was detected in plasma and whole blood of ZD. CONCLUSIONS: Our results indicate that diabetes mellitus with cardiovascular risk factor clustering favors BM production of hyperreactive platelets with altered protein disulfide isomerase and 78-kDa glucose-regulated protein expression that can contribute to increase thrombotic risk independently of blood glucose levels.

The subcutaneous adipose tissue reservoir of functionally active stem cells is reduced in obese patients.

It has been demonstrated that the adipose tissue, a highly functional metabolic tissue, is a reservoir of mesenchymal stem cells. The potential use of adipose-derived stem cells (ADSCs) from white adipose tissue (WAT) for organ repair and regeneration has been considered because of their obvious benefits in terms of accessibility and quantity of available sample. However, the functional capability of ADSCs from subjects with different adiposity has not been investigated. It has been our hypothesis that ADSCs from adipose tissue of patients with metabolic syndrome and high adiposity may be functionally impaired. We report that subcutaneous WAT stromal vascular fraction (SVF) from nonobese individuals had a significantly higher number of CD90+ cells than SVF from obese patients. The isolated ADSCs from WAT of obese patients had reduced differentiation potential and were less proangiogenic. Therefore, ADSCs in adipose tissue of obese patients have lower capacity for spontaneous or therapeutic repair than ADSCs from nonobese metabolically normal individuals.

Comparative effects of oleoyl-estrone and a specific beta3-adrenergic agonist (CL316, 243) on the expression of genes involved in energy metabolism of rat white adipose tissue.

BACKGROUND: The combination of oleoyl-estrone (OE) and a selective beta3-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 lipolysis-lipogenesis 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.

Short-term oral oleoyl-estrone decreases the expression of ghrelin in the rat stomach.

Oleoyl-estrone (OE) mobilizes body fat and decreases food intake. The precise mechanism of its modulation of appetite is unknown. Since the effects of OE on food intake appear early, here we studied the effect of OE on the expression of gut peptides that affect short-term ingestive behavior: ghrelin, leptin, CCK, PYY, and GLP-1. Two hours after a single OE dose, adult male rats were killed and their stomach fundus and intestine sections were dissected and processed for real-time PCR amplification. Semi-quantitative estimation of gene mRNA tissue levels showed that OE markedly decreased ghrelin expression in the stomach; leptin mRNA was unchanged; CCK mRNA decreased in the proximal intestine while PYY and GLP-1 expression in the intestine was not altered. Our results indicate that the short-term decrease in food intake induced by OE may be essentially the consequence of a marked decrease in the expression of ghrelin in the stomach.

Effects of combined oleoyl-estrone and rimonabant on overweight rats.

Oleoyl-estrone (OE) decreases appetite, maintains energy expediture, induces lipolysis (sparing protein), and decreases cholesterolemia and insulin resistance. Rimonabant (SR141716) is a cannabinoid-receptor inhibitor that decreases appetite and mobilizes fat. We studied whether their combination improves their slimming effects. Male overweight rats received daily gavages of 5.3 mg/kg OE, 10 mg/kg rimonabant, or both drugs during 10 days. Body weight and composition, energy balance, adipose tissue weight, and serum hormones and metabolites were measured. OE halved food intake and maintained energy expenditure at the expense of body fat. Rimonabant effects on appetite and energy balance were less marked, resulting in lower lipid mobilization. OE and rimonabant followed the OE pattern, with no additive or synergic effects. Glycemia was maintained, but OE decreased insulin, GLP-1, and cholesterol, whilst rimonabant increased cholecystokinin and cholesterol, and decreased NEFA. Both drugs decreased leptin and triacylglycerols; ghrelin was unchanged. The results hint at different mechanisms of action of both drugs: we can assume that OE effects do not involve the cannabinoid pathway. OE does not seem to act, either, after 10 days, through the secretion of ghrelin or the intestinal appetite-controlling peptides tested.

Estimation of the metabolizable energy equivalence of dietary proteins.

BACKGROUND: 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. AIM OF THE STUDY: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Combined effects of oleoyl-estrone and a beta3-adrenergic agonist (CL316,243) on lipid stores of diet-induced overweight male Wistar rats.

Oleoyl-estrone (OE) decreases appetite, induces adipose tissue wasting and resets the ponderostat setting, sparing glucose and protein. The beta3-adrenergic agonists increase energy expenditure and lipolysis. We studied the combination of both treatments to enhance fat mobilization. Overweight male rats received oral OE for 10 days; they were compared with controls and rats receiving a beta3-adrenergic agonist, CL316,243 (B3A); another group received both OE and B3A. Serum 3-hydroxybutyrate, NEFA, triacylglycerols and glucose showed only slight changes in all groups vs. controls; OE-treated rats showed lower cholesterol. OE decreased food intake and B3A increased energy expenditure. OE rats lost about 15%, B3A 24%, and those receiving both compounds lost 39% of their initial total body energy. In all cases, most of this energy imbalance was accounted for by the loss of body lipid. The combined treatment of OE and B3A reduced food intake, nevertheless maintaining a high energy expenditure. The combination of a beta3-adrenergic agonist with OE may help compensate the short-lived effects of the agonist and enhance the lipid mobilization action of OE. The eventual combination of both compounds should be explored as a way to obtain faster and more effective ways to treat obesity.

Effects of oral estrone on rat energy balance.

Oral doses of estrone from 10 nmol/(kg day) to 10 micromol/(kg day) were given to adult Wistar male rats for 10 days. Body composition, energy balance, total body estrone balance and plasma metabolites and hormones were measured at the end of the treatment. Body weight (as well as food intake, body energy, fat and water accrual) increased at doses in the 10--100 nmol/(kg day) range, but decreased at higher doses. Energy expenditure decreased with increasing doses of estrone. Plasma metabolite changes suggested the maintenance of energy homeostasis, and lipid parameters indicated that lipid mobilization increased with the increasing doses of estrone. Plasma estrone, acyl-estrone and estradiol levels decreased at low doses and increased at high doses of estrone. We conclude that: (a) repeated estrone gavages, even at very high doses, do not result in the accrual of estrone in the body; (b) low doses of estrone promote growth and high doses decrease body mass and fat accretion; (c) administration of estrone at low doses decreases its circulating levels and the levels of estradiol and acyl-estrone, a situation reverted at higher doses and (d) estrone administration induces a dose-dependent shift towards lower energy expenditure.

Effects of oleoyl-estrone with dexfenfluramine, sibutramine or phentermine on overweight rats.

We studied the combination of oleoyl-estrone with either dexfenfluramine, sibutramine or phentermine in overweight male rats treated for 10 days in order to determine whether they shared a mechanism of action. Oleoyl-estrone, dexfenfluramine and sibutramine decreased body weight and energy (essentially lipids); losses were higher when combined with oleoyl-estrone. Glycemia was maintained except under phentermine; oleoyl-estrone induced decreases in triacylglycerols, cholesterol, insulin and HOMA (homeostasis model assessment). Combination of oleoyl-estrone and sibutramine resulted in the loss of up to 29% body energy in 10 days. Energy expenditure was maintained. The effects of oleoyl-estrone and dexfenfluramine or sibutramine on appetite were substantially additive. All oleoyl-estrone-treated rats showed increased insulin sensitivity. In conclusion, combined treatment of overweight rats with oleoyl-estrone and sibutramine or dexfenfluramine results in a dramatic loss of weight and fat, whilst maintaining circulating energy homoeostasis.

Tamoxifen does not prevent the mobilization of body lipids elicited by oleoyl-estrone.

Oleoyl-estrone is a powerful, slimming adipose tissue-derived signal that has biological effects widely opposed to those of its estrone moiety. The present experiment was designed to determine whether oleoyl-estrone effects on body energy are mediated by the estrogen receptor, blocked with the antagonist tamoxifen. Male Wistar rats were given daily oral doses of 10 micromol/kg d of oleoyl-estrone in oil containing 0 or 0.40 mg/kg d of tamoxifen. The data were compared with controls receiving only oil or 50 nmol/kg d of free estrone. After 10 days, the rats were killed, and their body composition and plasma metabolites and hormones were analyzed. Rats receiving estrone increased their body energy and lipid content compared with controls. Both groups of oleoyl-estrone-treated rats lost body weight, energy, and lipid; the losses in the rats receiving tamoxifen alone were less marked than in those receiving oleoyl-estrone. No significant changes in plasma glucose or triacylglycerols were observed. The patterns of change of estrone sulphate, estradiol, and oleoyl-estrone were consistent with a noticeable hydrolysis of oleoyl-estrone. The lack of differences in the fat mass in oleoyl-estrone-treated rats irrespective of the presence of tamoxifen suggested that the estrogenic pathway was not responsible for the slimming effects observed. Thus, it can be concluded that oleoyl-estrone effects are not mediated through its conversion to estrone or estradiol acting through the estrogen receptor. Tamoxifen partly mimicked the slimming effects of oleoyl-estrone; this could be speculatively explained by tamoxifen acting through the oleoyl-estrone signalling pathway.