Clinical Implications and Molecular Features of Extracellular Matrix Networks in Soft Tissue Sarcomas.

PURPOSE: The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterized. We aimed to investigate the tumor ECM and adhesion signaling networks present in STS and their clinical implications. EXPERIMENTAL DESIGN: Proteomic and clinical data from 321 patients across 11 histological subtypes were analyzed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS), and undifferentiated pleomorphic sarcomas (UPS). RESULTS: This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct coregulated ECM networks which are associated with tumor malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the lymphocyte cytosolic protein 1 cytoskeletal protein as a prognostic factor in LMS. Characterization of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signaling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodeling proteins as candidate antistromal therapeutic targets. Finally, we define a proteoglycan signature that is an independent prognostic factor for overall survival in DDLPS and UPS. CONCLUSIONS: STS comprise heterogeneous ECM signaling networks and matrix-specific features that have utility for risk stratification and therapy selection, which could in future guide precision medicine in these rare cancers.

Chemoautotrophy, symbiosis and sedimented diatoms support high biomass of benthic molluscs in the Namibian shelf.

The molluscs Lucinoma capensis, Lembulus bicuspidatus and Nassarius vinctus are highly abundant in Namibian oxygen minimum zone sediments. To understand which nutritional strategies allow them to reach such impressive abundances in this extreme habitat we investigated their trophic diversity, including a chemosymbiosis in L. capensis, focussing on nitrogen biochemical pathways of the symbionts. We combined results of bulk nitrogen and carbon (δ13C and δ15N) and of compound-specific isotope analyses of amino acid nitrogen (AAs-δ15NPhe and δ15NGlu), with 16S rRNA gene sequencing of L. capensis tissues and also with exploratory results of ammonium, nitrate and nitrite turnover. The trophic position (TP) of the bivalve L. capensis is placed between autotrophy and mixotrophy, consistent with its proposed symbiosis with sulfur-oxidizing Candidatus Thiodiazotropha sp. symbionts. The symbionts are here revealed to perform nitrate reduction and ammonium uptake, with clear indications of ammonium host-symbionts recycling, but surprisingly unable to fix nitrogen. The TP of the bivalve L. bicuspidatus is placed in between mixotrophy and herbivory. The TP of the gastropod N. vinctus reflected omnivory. Multiple lines of evidences in combination with current ecosystem knowledge point to sedimented diatoms as important components of L. bicuspidatus and N. vinctus' diet, likely supplemented at times with chemoautotrophic bacteria. This study highlights the importance of benthic-pelagic coupling that fosters the dietary base for macrozoobenthos in the OMZ. It further unveils that, in contrast to all shallow water lucinid symbionts, deeper water lucinid symbionts rely on ammonium assimilation rather than dinitrogen fixation to obtain nitrogen for growth.

The perplexing role of immuno-oncology drugs in osteosarcoma.

Osteosarcoma is a rare, primary tumour of bone. Curative treatment consists of multi-agent chemotherapy and complete surgical resection. Despite the use of multi-agent chemotherapy, the risk of recurrence is high. Survival outcomes for patients with osteosarcoma have not changed since the 1980's. Based on biologic rationale, there has been interest in adding immunotherapies to upfront curative intent chemotherapy, including mifamurtide (a macrophage activator) and interferon. However, results to date have been disappointing. In the metastatic setting, checkpoint inhibitors alone have not proven effective. Ongoing translational work is needed to further understand which patients may benefit from immune-oncology approaches with standard cytotoxic chemotherapy.

[A case of sole supportive care of severe neuroparalytic foodborne botulism due to homemade pesto]. / Fall einer rein supportiven Behandlung eines schweren neuroparalytischen Nahrungsmittelbotulismus durch hausgemachtes Pesto.

In this article, a case of severe foodborne botulism in a 78-year-old man due to homemade pesto is presented. His initial symptoms were gastrointestinal problems. Later he suffered of cranial nerve palsies, muscle weakness of the upper extremities and respiratory failure, so he had to be admitted to the intensive care unit for mechanical ventilation. The botulism was confirmed by serology and culture. Because of spontaneously improving neurological deficits, we decided not to treat with the botulism antitoxin and the patient had complete clinical remission.

Development of a marine fish model for studying in vivo molecular responses in ecotoxicology.

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5microm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, V(v)) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8+/-0.2 mgO(2)L(-1) for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p<0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

Leptin alters the structural and functional characteristics of adipose tissue before birth.

This study aimed to determine for the first time whether leptin can act to alter the structural and functional characteristics of adipose tissue before birth. Leptin (0.48 mg/kg/day) or saline was infused intravenously into fetal sheep for 4 days from either 136 or 137 days of gestation (term=147+/-3 days). Circulating leptin concentrations were increased approximately four- to fivefold by leptin infusion. Leptin infusion resulted in a significant increase in the proportion of smaller lipid locules present within fetal perirenal adipose tissue (PAT), and this was associated with a significant increase in the proportion of multilocular tissue and a significant decrease in the proportion and relative mass of unilocular tissue in fetal PAT. The relative abundance of leptin mRNA in fetal PAT was significantly lower in the leptin-infused group, and there was a positive correlation between the relative abundance of leptin mRNA and the proportion of unilocular adipose tissue in fetal PAT. The amount of uncoupling protein 1 tended to be higher (P=0.06) in leptin-infused compared with saline-infused fetuses. This is the first demonstration that leptin can act to regulate the lipid storage characteristics, leptin synthetic capacity, and potential thermogenic functions of fat before birth.

[Antioxidant vitamin supplementation in the prevention of cardiovascular disease]. / Antioxidanzien und Vitamine in der Prävention von kardiovaskulären Erkrankungen.

Oxidative stress, in particular oxidative modification of LDL-cholesterol, appears to be of great importance in the pathogenesis of atherosclerosis. Various observational epidemiological studies have suggested that antioxidant vitamin intake is associated with reduced cardiovascular morbidity and mortality. Also, experimental studies in animals have demonstrated that antioxidant vitamins slow the progression of atherosclerosis. However, prospective controlled clinical trials have failed to demonstrate a benefit of antioxidant vitamin supplementation in primary or secondary prevention of cardiovascular disease. Thus, the use of antioxidants and vitamin supplements as a preventive or therapeutic intervention can not be recommended.

Mechanisms involved in ATP-evoked Ca2+ oscillations in isolated human granulosa-luteal cells.

Using single-cell microfluorimetry, we have shown that ATP evoked repetitive Ca2+ oscillations in intact Fura-2 loaded human granulosa-luteal cells (hG/LCs) in the absence of extracellular Ca2+. Sustained increases in [Ca2+]i required extracellular Ca2+ and ATP depleted stores were refreshed by brief (2 min) incubation with external Ca2+. Basal [Ca2+]i was unaffected by caffeine (1 mM), but 20 mM caffeine inhibited ATP-evoked Ca2+ release in the absence of external Ca2+. Thimerosal (10 microM) evoked repetitive Ca2+ spikes, under Ca(2+)-free conditions, which fused to form an elevated plateau when external Ca2+ was replaced. Thimerosal-induced changes in [Ca2+]i were reversibly inhibited by the thiol reducing agent dithiothreitol (1 mM). The periodicity and amplitude of the [Ca2+]i oscillations produced by thimerosal and ATP differ. ATP- or thimerosal-evoked changes in [Ca2+]i were unaffected by dantrolene sodium (10 microM). The Ca(2+)-ATPase inhibitor thapsigargin (1 microM) increased [Ca2+]i and attenuated subsequent ATP-evoked changes in [Ca2+]i. We conclude that ATP stimulates an oscillatory release of Ca2+ from InsP3-sensitive stores in hG/LCs.

Suppression of luteinizing hormone secretion by atrial and brain natriuretic peptides in ovariectomized rats.

Brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) are present in brain regions regulating LH secretion. Similarities in the molecular structure of these peptides suggest similar physiological function within the brain. This study examined the effects of centrally administered BNP and ANP on LH secretion in mature ovariectomized (OVX) rats. Intracerebroventricular (icv) administration of 2 nmol ANP or BNP decreased mean plasma LH concentration and LH pulse amplitude (P less than 0.05 for ANP; P less than 0.01 for BNP; n = 8/group) and frequency (P less than 0.01 for ANP and BNP). LH secretion was not affected by ANP or BNP at a lower concentration (0.2 nmol, icv; P greater than 0.05; n = 8/group). It was concluded that ANP and BNP may be involved in central regulation of LH secretion. Mechanisms possibly involved in suppression of LH secretion by ANP and BNP were also examined. OVX rats were treated with an opioid antagonist, naloxone (0.5 mg, iv), 45, 75, and 105 min after ANP or BNP (2 nmol, icv). Naloxone eliminated suppression of mean plasma LH concentration and LH pulse amplitude by 2 nmol ANP and BNP (P less than 0.05; n = 7/group). OVX rats were treated with a dopamine antagonist, pimozide (0.6 mg/kg, sc), 90 min before treatment with ANP or BNP (2 nmol, icv). Pimozide pretreatment blocked suppression of LH secretion by ANP or BNP (P less than 0.05; n = 9/group). Naloxone alone did not affect LH secretion (P greater than 0.05; n = 5). It was concluded that components of ANP and BNP suppression of LH secretion may depend upon opioid and dopamine activity.

Stimulation of progesterone secretion by recombinant follistatin-288 in human granulosa cells.

The effects of recombinant human follistatin (follistatin-288) on basal and hCG-stimulated progesterone secretion were examined in cultured human granulosa cells. Follistatin increased progesterone secretion in a dose-dependent manner. However, follistatin did not augment hCG- or cAMP-stimulated progesterone secretion. Time-course analysis revealed that follistatin increased progesterone secretion after 24 h of incubation. Follistatin also enhanced basal, but not hCG-stimulated, 20 alpha-hydroxyprogesterone accumulation, indicating that the increase in progesterone accumulation was not due to a blockade of the 20 alpha-hydroxylase metabolic pathway. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, follistatin significantly increased intracellular cAMP accumulation to levels comparable to those induced by 1 IU/ml hCG. These results provide the first evidence of a stimulatory action of follistatin on progestin secretion in the human ovary, which is accompanied by an increased accumulation of intracellular cAMP levels. Follistatin may well be another potential regulator of steroid hormone production in human granulosa cells during the periovulatory period.

Effect of prostaglandin F2 alpha on cytosolic free calcium ion concentrations in rat luteal cells.

Changes in cytosolic free calcium concentrations [( Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) were measured in single rat luteal cells, using the calcium-sensitive fluorescent dye fura-2. A total of 112 cells were studied in 20 experiments. The average resting [Ca2+]i was 113 +/- 6.4 nM. In 59 cells (53%), there was a 4.6 +/- 0.2-fold increase in [Ca2+]i within 29.3 +/- 1.0 sec of PGF2 alpha administration, and the cells recovered by 78.0 +/- 4.5 sec. The magnitude of the increase in [Ca2+]i in response to PGF2 alpha was not changed by an increase in the concentration of PGF2 alpha. Perifusion with low calcium buffer reduced, then eliminated, the [Ca2+]i response to PGF2 alpha. Perifusion of cells with PGF2 alpha resulted in a single transient [Ca2+]i response, similar to that after short term exposure to PGF2 alpha. Many (67%) of the cells that responded to PGF2 alpha also responded to GnRH. No additive increase in [Ca2+]i was seen when PGF2 alpha and GnRH were administered together. The source of the calcium appears to be intracellular stores that are shared by GnRH and PGF2 alpha.

P2-purinoreceptor evoked changes in intracellular calcium oscillations in single isolated human granulosa-lutein cells.

In this study, we have demonstrated that P2-purinoreceptor agonists evoke oscillatory intracellular calcium ([Ca2+]i) responses in human granulosa-lutein cells (GLCs). Intracellular calcium was measured using microspectrofluorimetric techniques. ATP at concentrations of 1-100 microM increased [Ca2+]i, whereas neither adenosine nor AMP evoked changes in [Ca2+]i. The nonhydrolysable ATP analogue, ATP gamma S, also elevated [Ca2+]i with an efficacy similar to that of ATP, indicating that the changes in Ca2+ were not due to ATP hydrolysis, but that human GLCs possess functional P2-purinoreceptors. Uridine triphosphate (UTP) was equipotent to ATP at stimulating [Ca2+]i, and both ATP and UTP were consistently more effective at eliciting a response than ADP, suggesting that human GLCs possess the P2U class of purinergic receptors (ATP = UTP > > ADP > > AMP = adenosine). We have demonstrated that the purinergic agonist-induced changes in [Ca2+]i involve both Ca2+ influx and Ca2+ mobilization from cytosolic stores. Prolonged ATP treatment in Ca(2+)-free buffer (1 mM EGTA) still evokes transient oscillatory changes in [Ca2+]i in a pertussis toxin-insensitive manner. In Ca(2+)-containing conditions, the sustained phase of the response was generally unaffected by verapamil (10 microM), suggesting that influx is not occurring through voltage-dependent Ca(2+)-channels. These findings are consistent with the hypothesis that ATP and other P2-purinergic receptor agonists elicit changes in [Ca2+]i in human ovarian cells and that these events are initiated by the release of Ca2+ from cytosolic stores, and sustained by extracellular calcium ([Ca2+]e) influx. This is the first time that oscillatory patterns of [Ca2+]i have been reported in human GLCs.

Cytosolic free calcium increased by prostaglandin F2 alpha (PGF2 alpha), gonadotropin-releasing hormone, and angiotensin II in rat granulosa cells and PGF2 alpha in human granulosa cells.

Cytosolic [Ca2+]i was measured using a microspectrofluorimetric technique. Prostaglandin F2 alpha (PGF2 alpha, 10(-6) M) transiently increased the concentration of free cytosolic Ca2+ ([Ca2+]i) in individual rat and human granulosa cells. In a study examining a total of 170 individual rat and human granulosa cells, approximately 100% of rat granulosa cells and 80% of human granulosa cells tested responded to PGF2 alpha (10(-6) M). In a dose-response trial, the magnitude of the [Ca2+]i response did not vary, although a decreasing number of cells responded to decreasing PGF2 alpha concentrations (10(-5) to 10(-9) M). PGE2 (10(-4) to 10(-6) M) did not affect [Ca2+]i in rat or human granulosa cells. GnRH (10(-6) M) increased [Ca2+]i in rat but not human granulosa cells. Over 90% of rat granulosa cells tested responded. Angiotensin II (ANG II, 10(-5) M) increased [Ca2+]i in approximately 25% of rat, but not human granulosa cells. Individual rat granulosa cells which responded to GnRH responded to PGF2 alpha and vice versa. Individual rat granulosa cells which responded to ANG II responded to PGF2 alpha and GnRH. Conversely, less than 30% of individual rat granulosa cells which responded to PGF2 alpha and GnRH responded to ANG II. Desensitization (pretreatment) of rat granulosa cells by continuous hormone perifusion suggested that effects of PGF2 alpha, GnRH, and ANG II on [Ca2+]i were receptor specific. However, the effects of combined hormone treatments on [Ca2+]i were not additive. The transient increase in [Ca2+]i in response to PGF2 alpha or GnRH, alone, may be maximal. Results of this study suggested that effects of PGF2 alpha, GnRH, and ANG II receptor-ligand interactions may be at least partially mediated by transient increases in [Ca2+]i in rat granulosa cells. Similarly, effects of PGF2 alpha, but not GnRH or ANG II, receptor-ligand interactions may be mediated by transient increases in [Ca2+]i in human granulosa cells.

Acute stimulation of human chorionic gonadotropin secretion by recombinant human activin-A in first trimester human trophoblast.

Inhibin and activin are synthesized by the placenta, raising questions as to their functions in this tissue. The possibility of local regulation of other placental hormones critical during pregnancy was investigated by examining the effects of recombinant human inhibin-A and activin-A on hCG secretion. Potential interactions with GnRH-stimulated hCG secretion were also explored. First trimester human placental tissue was physically dissociated to isolate trophoblast cells. Cells were cultured on microcarrier beads for 7-10 days and loaded into 1.5-ml chambers in a perifusion system. After a 1-h control period, hormone treatments were administered in the perifusion medium for 5 min, and perifusate was collected for an additional 55 min. Perifusate fractions were analyzed for hCG concentration by RIA. Both activin and GnRH stimulated a rapid and transient hCG secretory response in the perifusion system. Unlike the effect of GnRH, the activin-stimulated hCG response was not blocked by concomitant treatment with a GnRH antagonist. The hCG RIA results were confirmed by preliminary experiments using a novel hCG bioassay. This suggested that activin did not facilitate hCG secretion by stimulation of endogenous GnRH release. Inhibin alone did not affect hCG secretion or GnRH-stimulated hCG secretion. However, treatment with inhibin and activin in combination resulted in a transient increase in hCG, followed by a decrease in hCG secretion to below pretreatment levels. The results suggested that in addition to GnRH, activin may play a role in the regulation of hCG secretion in first trimester placenta.

Cytosolic free Ca2+ in human syncytiotrophoblast cells increased by gonadotropin-releasing hormone.

Effects of GnRH on free cytosolic Ca2+ concentrations ([Ca2+]i) were examined in individual first trimester human cytotrophoblast and syncytiotrophoblast cells by fura-2 microspectrofluorimetry. GnRH (10(-6) M) did not affect [Ca2+]i in cytotrophoblasts on days 2-9 of culture, with 50 cells tested each day. GnRH (10(-6) M) did not affect [Ca2+]i on days 2-3, but increased [Ca2+]i in 15% of culture-derived syncytiotrophoblasts on day 4 (8 of 52 cells) and in 48% on days 5-9 of culture (158 of 332 cells). Culture-derived syncytiotrophoblasts originated from four first trimester placentae. GnRH increased [Ca2+]i in a preliminary trial using syncytiotrophoblasts derived directly from a single first trimester placenta and cultured for 3 days (13 of 33 cells). Culture-derived first trimester syncytiotrophoblast cells that responded to 10(-6) M GnRH (28 of 63 cells) on day 6 also responded to GnRH at 10(-7) M (26 of 28 of the above cells), 10(-8) M (24 of 28 cells, 10(-9) M (22 of 28 cells), and 10(-10) M (3 of 28 cells). No cells responded to GnRH below a concentration of 10(-10) M. Desensitization of syncytiotrophoblasts by continuous GnRH perifusion (10(-6) M) and blockade of GnRH by competitive antagonism with Nal-Glu-GnRH (10(-6) M) suggested that effects of GnRH were receptor specific. The results provide direct evidence supporting the contention that the intracellular signaling resultant from GnRH receptor-ligand interactions in syncytiotrophoblasts may be at least partially mediated by transient increases in [Ca2+]i.

Luteinizing hormone-releasing hormone (LHRH)- and (hydroxyproline9)LHRH-stimulated human chorionic gonadotropin secretion from perifused first trimester placental cells.

(Hydroxyproline9)LHRH [(Hyp9)LHRH] has been isolated from human, sheep, rodent, and frog hypothalamus. (Hyp9)LHRH is the major LHRH moiety in fetal rat hypothalamus. This study compared 1) synthetic LHRH-stimulated hCG secretion from term trophoblast vs. first trimester placental cells and 2) the ability and specificity with which synthetic LHRH and (Hyp9)LHRH could stimulate hCG secretion from 8- to 12-week gestation placenta. Physically dissociated cells from multiple placentae were pooled, plated on microcarrier beads, and perifused in 1.5-ml chambers (1.5 x 10(6) cells/chamber). Effluent fractions were analyzed for hCG. Each chamber was its own control. Basal hCG secretion did not depend upon exogenous LHRH stimulation. The amplitude of LHRH-stimulated hCG pulses was greater from first trimester placental than term trophoblast cells (mean +/- SEM, 6.99 +/- 1.47 vs. 0.50 +/- 0.05 mIU/ml perifusate; peak minus basal; n = 4 chambers; P less than 0.01). LHRH and (Hyp9)LHRH (10(-9) M) increased hCG secretion from first trimester placental cells (5.02 +/- 1.29 vs. 8.64 +/- 1.61 and 4.36 +/- 0.58 vs. 7.44 +/- 1.01 mIU/ml; n = 15 and 9, respectively; P less than 0.01). At the concentrations used, LHRH and (Hyp9)LHRH seemed to stimulate hCG secretion equipotently (P greater than 0.05). Simultaneous perifusion with an LHRH antagonist, (Nal-Glu)LHRH blocked the hCG secretory response to LHRH or (Hyp9)LHRH (equimolar 10(-9) M concentrations; n = 5; P less than 0.05). (Nal-Glu)LHRH alone (10(-9) M) did not affect hCG secretion (n = 5; P greater than 0.05). The results suggested that first trimester placental cells are more responsive to LHRH than are term trophoblast cells. (Hyp9)LHRH is a potential physiological secretagogue of hCG.

Induction of polyphosphoinositide breakdown in rat corpus luteum by prostaglandin F2 alpha.

The present study examines the possibility that, in the rat corpus luteum, an initial action of prostaglandin F2 alpha (PGF2 alpha) is to induce a ligand-stimulated breakdown of membrane inositol phospholipids. Luteal cells in primary culture were prepared from immature rats after PMSG and human CG priming. In 32P-prelabeled cells, PGF2 alpha caused a rapid decrease in the level of radiolabel found in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, as early as 20 sec after addition of the hormones. At 1 and 2.5 min, the effect of 10(-6) M PGF2 alpha on phosphatidylinositol 4,5-bisphosphate was significantly greater than that caused by 10(-6) M LHRH in identical cell cultures. By contrast, the levels of the 32P-prelabeled phosphatidylinositol and phosphatidic acid were increased at 5 min by PGF2 alpha or LHRH. Concomitant with the alterations in cellular levels of 32P-prelabeled phospholipids, PGF2 alpha markedly enhanced the accumulation of 3H-labeled inositol phosphates, i.e. inositol 1-phosphate, inositol diphosphate, and inositol triphosphate, during a 5-min incubation. A significant increase of radiolabeled inositol diphosphate was seen as early as 1 min after the addition of either PGF2 alpha or LHRH; PGF2 alpha was more effective than LHRH in this regard. The stimulatory effect of LHRH on inositol phosphate accumulation could be blocked completely by the concomitant presence of a potent LHRH antagonist, and at the concentration used (10(-6) M) the effects of PGF2 alpha and LHRH were not additive. Interestingly, the addition of an exogenous phospholipase C also caused a similar enhancement of inositol phosphate accumulation in identical cell cultures. For the first time, these data suggest that, at the level of the corpus luteum, hydrolysis of phosphoinositides may immediately follow PGF2 alpha (and to a lesser extent LHRH) receptor binding, and this in turn may lead to the generation of 1,2-diacylglycerol and inositol phosphates, resynthesis of phosphatidic acid and phosphatidylinositol, and mobilization of Ca2+.

Determinants of fetal leptin synthesis, fat mass, and circulating leptin concentrations in well-nourished ewes in late pregnancy.

We have investigated the factors regulating leptin synthesis, fat deposition, and circulating leptin concentrations in fetuses of well nourished ewes in late pregnancy. Vascular catheters were surgically inserted in 17 pregnant ewes and their fetuses at 103-120 d gestation (term = 147 +/- 3 d). Ewes were fed a diet providing either 100% (control; n = 9) or approximately 155% (well fed; n = 8) of the maintenance energy requirements and fetal perirenal and interscapular fat depots were collected at 139-141 d gestation. There was a significant relationship between the relative mass of fetal unilocular fat and fetal glucose (relative mass of unilocular fat, 1.14; fetal glucose, +0.16; r = 0.50; P < 0.04; n = 17), but not insulin, concentrations in the control and well-fed groups. In contrast to the controls, there was a positive relationship between the relative abundance of leptin mRNA and fetal insulin, but not glucose, concentrations in fetal perirenal adipose tissue in the well-fed group. A moderate increase in maternal nutrition also resulted in a strong reciprocal relationship between uncoupling protein 1 and leptin expression in fetal perirenal adipose tissue in late gestation (well-fed group: uncoupling protein 1 mRNA:18S rRNA, -0.51; leptin mRNA:beta-actin mRNA, +1.53; r = 0.80; P < 0.02; n = 8). These studies provide evidence that fetal glucose and insulin differentially regulate fetal fat deposition and leptin mRNA expression within the fetal perirenal fat depot in the well nourished animal during late gestation.