Interactions between CD36 and global intestinal alkaline phosphatase in mouse small intestine and effects of high-fat diet.

The mechanisms of the saturable component of long-chain fatty acid (LCFA) transport across the small intestinal epithelium and its regulation by a high-fat diet (HFD) are uncertain. It is hypothesized here that the putative fatty acid translocase/CD36 and intestinal alkaline phosphatases (IAPs) function together to optimize LCFA transport. Phosphorylated CD36 (pCD36) was expressed in mouse enterocytes and dephosphorylated by calf IAP (CIAP). Uptake of fluorescently tagged LCFA into isolated enteroctyes was increased when cells were treated with CIAP; this was blocked with a specific CD36 inhibitor. pCD36 colocalized in enterocytes with the global IAP (gIAP) isozyme and, specifically, coimmunoprecipitated with gIAP, but not the duodenal-specific isozyme (dIAP). Purified recombinant gIAP dephosphorylated immunoprecipitated pCD36, and antiserum to gIAP decreased initial LCFA uptake in enterocytes. Body weight, adiposity, and plasma leptin and triglycerides were significantly increased in HFD mice compared with controls fed a normal-fat diet. HFD significantly increased immunoreactive CD36 and gIAP, but not dIAP, in jejunum, but not duodenum. Uptake of LCFA was increased in a CD36-dependent manner in enterocytes from HFD mice. It is concluded that CD36 exists in its phosphorylated and dephosphorylated states in mouse enterocytes, that pCD36 is a substrate of gIAP, and that dephosphorylation by IAPs results in increased LCFA transport capability. HFD upregulates CD36 and gIAP in parallel and enhances CD36-dependent fatty acid uptake. The interactions between these proteins may be important for efficient fat transport in mouse intestine, but whether the changes in gIAP and CD36 in enterocytes contribute to HFD-induced obesity remains to be determined.

Variation in physiological stress between bridge- and cave-roosting Brazilian free-tailed bats.

Since the late 1980s, Brazilian free-tailed bats (Tadarida brasiliensis) have increasingly used bridges as roosts in the southern United States. We examined differences in blood cortisol levels, body condition, and parasite load, as measures of physiological stress in bats roosting in bridges and bats roosting in caves. We collected data during three periods, coinciding with female phases of reproduction. For all measures, bats were captured during the nightly emergence from the roost and immediately sampled. Cortisol levels were significantly higher during pregnancy and lactation and in individuals with lower body-condition scores (length of forearm to mass ratio) and significantly higher in bats roosting in caves than in those roosting in bridges. Thus, we concluded that individuals of this species that roost in bridges are not chronically stressed and seem to be unaffected by human activities present at bridges. This is a rare documented instance where a human-dominated environment does not appear to be adversely affecting the physiological health of a free-ranging animal.

Microarray and real-time PCR analysis of adrenal gland gene expression in the 7-day-old rat: effects of hypoxia from birth.

We hypothesize that changes in adrenal gene expression mediate the increased plasma corticosterone and steroidogenesis in rat pups exposed to hypoxia from birth. In the current study, rat pups (with their dams) were exposed to hypoxia from birth and compared with pups from normoxic dams fed ad libitum or pair fed to match the decreased maternal food intake that occurs during hypoxia. Microarray analysis was performed, followed by verification with real-time PCR. Furthermore, the expression of selected genes involved in adrenal function was analyzed by real-time PCR, regardless of microarray results. Hypoxia increased plasma ACTH and corticosterone, while food restriction had no effect. Microarray revealed that many of the genes affected by hypoxia encode proteins that require molecular oxygen (monooxygenases, oxidoreductases, and electron transport), whereas only a few genes known to be involved in adrenal steroidogenesis were affected. Interestingly, the expression of genes involved in mitochondrial function and intermediary metabolism was increased by hypoxia. Real-time PCR detected a small but significant increase in the expression of Cyp21a1 mRNA in the hypoxic adrenal. When decreased maternal food intake was controlled for, the effects of hypoxia were more pronounced, in that real-time PCR detected significant increases in the expression of Star (244%), Cyp21a1 (208%), and Ldlr (233%). The present study revealed that increased plasma corticosterone in rat pups was due to hypoxia per se, and not as a result of decreased food intake by the hypoxic dam. Furthermore, hypoxia induced changes in gene expression that account for more productive and efficient steroidogenesis.

Gene array analysis of the effects of chronic adrenocorticotropic hormone in vivo on immature rat adrenal glands.

Development of a mature adrenocortical phenotype is a critical event in the transition of mammals from fetal to postnatal life. We previously reported that the functional maturation of the adrenal glands of newborn rats is accelerated by adrenocorticotropic hormone (ACTH). We report here that chronic exposure of neonatal/juvenile rat pups to ACTH in vivo results in significant changes in expression of over 200 genes in the adrenal glands. ACTH significantly upregulated genes associated with cell signaling, gene transcription, cell migration and tissue remodeling. In addition, ACTH significantly downregulated several genes associated with de novo cholesterol biosynthesis and cholesterol trafficking. Finally, ACTH upregulated genes associated with intracellular metabolism and inactivation of glucocorticoids. The results demonstrate that the developmental effects of ACTH alter expression of a broad range of genes involved not solely in steroid synthesis, but in cellular functions related to growth and differentiation of the glands. In addition, the negative effects of ACTH on genes required for cholesterol synthesis and production of active glucocorticoids, suggests a mechanism whereby excessive production of glucocorticoids, which may have deleterious actions on developing structures like the central nervous system, is prevented.

Impaired leptin expression and abnormal response to fasting in corticotropin-releasing hormone-deficient mice.

Leptin has been postulated to comprise part of an adipostat, whereby during states of excessive energy storage, elevated levels of the hormone prevent further weight gain by inhibiting appetite. A physiological role for leptin in this regard remains unclear because the presence of excessive food, and therefore the need to restrain overeating under natural conditions, is doubtful. We have previously shown that CRH-deficient (Crh(-/-)) mice have glucocorticoid insufficiency and lack the fasting-induced increase in glucocorticoid, a hormone important in stimulating leptin synthesis and secretion. We hypothesized that these mice might have low circulating leptin. Indeed, Crh(-/-) mice exhibited no diurnal variation of leptin, whereas normal littermates showed a clear rhythm, and their leptin levels were lower than their counterparts. A continuous peripheral CRH infusion to Crh(-/-) mice not only restored corticosterone levels, but it also increased leptin expression to normal. Surprisingly, 36 h of fasting elevated leptin levels in Crh(-/-) mice, rather than falling as in normal mice. This abnormal leptin change during fasting in Crh(-/-) mice was corrected by corticosterone replacement. Furthermore, Crh(-/-) mice lost less body weight during 24 h of fasting and ate less food during refeeding than normal littermates. Taken together, we conclude that glucocorticoid insufficiency in Crh(-/-) mice results in impaired leptin production as well as an abnormal increase in leptin during fasting, and propose that the fast-induced physiological reduction in leptin may play an important role to stimulate food intake during the recovery from fasting.

Basal and adrenocorticotropin-stimulated corticosterone in the neonatal rat exposed to hypoxia from birth: modulation by chemical sympathectomy.

We previously demonstrated that 7-d-old rat pups exposed to hypoxia from birth exhibit ACTH-independent increases in corticosterone associated with an increase in steroidogenic acute regulatory (StAR) and peripheral-type benzodiazepine receptor (PBR) proteins. The purpose of the present study was to determine whether this increase in corticosterone could be attenuated by chemical sympathectomy induced with guanethidine treatment. Rat pups were exposed to normoxia or hypoxia from birth and treated with vehicle or guanethidine and studied at 7 d of age. Hypoxia per se resulted in an increase in plasma corticosterone without a change in plasma ACTH. Guanethidine treatment attenuated the increase in basal corticosterone in hypoxic pups but did not attenuate ACTH-stimulated corticosterone production. This effect was specific as basal and ACTH-stimulated aldosterone was not affected. Guanethidine also attenuated the increase in StAR protein induced by hypoxia. Neither the effect of hypoxia nor that of guanethidine could be explained by changes in the levels of adrenal tyrosine hydroxylase, StAR, or P450scc mRNA, adrenal tyrosine hydroxylase immunohistochemistry, or adrenal catecholamine content. We conclude that chemical sympathectomy normalizes basal corticosterone levels but has no effect on ACTH-stimulated corticosterone levels in 7-d-old rats exposed to hypoxia from birth. The mechanism of the effect of guanethidine to normalize hypoxia-stimulated basal corticosterone remains to be identified, although StAR protein may be an important mediator. This ACTH-independent increase in corticosterone may be a mechanism by which the neonate can increase circulating glucocorticoids necessary for survival while bypassing the hyporesponsiveness of the neonatal hypothalamic-pituitary-adrenal axis.

Involvement of CD36 and intestinal alkaline phosphatases in fatty acid transport in enterocytes, and the response to a high-fat diet.

The vertebrate intestine is notable for its plasticity in response to environmental, pathologic, reproductive, and dietary challenges. The molecular mechanisms of intestinal adaptations typically involve both morphologic and functional changes. In response to chronic ingestion of a high-fat diet, for example, the mammalian small intestine quickly adapts to efficiently accommodate increased transport of long-chain fatty acids across the mucosa. Whereas this may be adaptive in the short term, in the long term it may contribute to the pathologies associated with chronic high-fat diets in humans and other mammals. This review focuses on some of the known and putative mechanisms by which fatty acids are transported across the intestinal epithelium in addition to simple diffusion, and how these mechanisms may be regulated in part by a high-fat diet. A model is proposed in which two key proteins, CD36 and the enzyme intestinal alkaline phosphatase, work in a coordinated manner to optimize fatty acid transport across enterocytes in mice.

Macro- and microgeographic variation in metabolism and hormone correlates in big brown bats (Eptesicus fuscus).

To better understand intraspecific variation in basal metabolic rate (BMR), we examined environmental, physiological, and/or cellular bases for residual variation in BMR in big brown bats, Eptesicus fuscus. We measured BMR and plasma levels of thyroid hormone (T(3)) and leptin in bats captured in maternity colonies in eastern Massachusetts (MA; northern population) and in Alabama and Georgia (ALGA; southern population) to assess macrogeographic (between- or among-population) and microgeographic (within-population) variation in those traits. After accounting for effects of body mass, stage of pregnancy, and within-population variation, bats from the northern population did not differ significantly in BMR, T(3), or leptin values from those in the southern population. However, after accounting for the effects of body mass and stage of pregnancy, a test for differences in all traits among colonies from both populations was significant. For BMR, bats differed significantly among the northern colonies. Moreover, after removing the effects of body mass and stage of pregnancy, bats from the AL colony had significantly higher BMR than did bats from all other colonies except one in MA, and they had significantly higher T(3) levels but lower leptin levels than did bats from two other colonies. The presence of among-colony and within-population variation for these traits suggests that proximate (nonevolutionary) factors (e.g., microhabitat differences such as roost type) play an important role in shaping intraspecific variation in BMR and its hormone correlates.

High-fat diet-induced changes in body mass and hypothalamic gene expression in wild-type and leptin-deficient mice.

We tested whether diet-induced obesity results from increased energy consumption, is associated with changes in expression of genes involved in leptin signal transduction, and is altered by hyperleptinemia. C57BL/6 mice were fed a low-fat diet (LFD) or high-fat diet (HFD) for up to 15 weeks. HFD mice weighed significantly more than LFD controls by 3 weeks, despite consuming less energy. HFD mice had significantly greater leptin, insulin, and glucose levels than LFD mice, suggesting leptin and insulin resistance. Adiponectin levels declined with age but were unaffected by diet. HFD was associated with altered hypothalamic expression of genes whose products regulate the activity or nuclear translocation of STAT3, an important mediator of leptin actions. Expression of two isoforms of the leptin receptor decreased at 15 weeks in hypothalami of HFD mice in a tissue-specific manner. The type of fat (saturated versus unsaturated) did not influence weight gain on an HFD, but animals on LFD gained significantly more weight and adiposity if the dietary fat consisted mostly of saturated fats; this occurred despite no difference in energy consumption or absorption. Replacement of leptin to leptin-deficient ob/ob mice decreased hypothalamic leptin receptor expression and did not prevent HFD-induced weight gain. It is concluded that (1) increased energy consumption is not required for HFD-induced obesity in C57BL/6 mice, (2) HFD results in weight gain partly by modulating hypothalamic leptin-signaling pathways, (3) saturated fats induce weight gain even when total fat content of the diet is low, and (4) the effects of HFD are manifest in the presence or absence of circulating leptin.

Changes in body mass, serum leptin, and mRNA levels of leptin receptor isoforms during the premigratory period in Myotis lucifugus.

Migration and hibernation in mammals may be preceded by a period of leptin resistance, which may in part account for the increasing adiposity and body mass that occurs during these periods. We hypothesized that hypothalamic expression of leptin receptor mRNA would decrease during the premigration (PM) period in the little brown myotis, Myotis lucifugus. Body mass of M. lucifugus increased during the PM period, but serum leptin levels did not change during that time. Hypothalamic mRNA levels for both the short (ObRa) and fully active long (ObRb) forms of the leptin receptor increased during PM, but the relative increase in ObRa was larger and occurred sooner than ObRb. mRNA levels of an inhibitor of leptin signaling (protein inhibitor of activated STAT3: PIAS3) increased in hypothalami during the PM period in bats. Adiponectin is an adipokine that has been linked to obesity in rodents; normally, serum levels of adiponectin decrease in obesity. In M. lucifugus, adiponectin mRNA levels decreased in adipose tissue during the period of mass gain, but circulating adiponectin levels did not change. We conclude that the relative changes in leptin receptor isoform expression during the PM fattening period may favor binding of leptin to the less active short isoform. Coupled with increased expression of PIAS3 and the dissociation of serum leptin levels from body mass and adiposity, these changes could account in part for the adaptive fattening during the PM period. In addition, the adipokine profiles of M. lucifugus during the PM period and that of obesity in non-hibernating mammals are strikingly dissimilar.

Inhibition of trophoblast invasiveness in vitro by immunoneutralization of leptin in the bat, Myotis lucifugus (Chiroptera).

In addition to effects on metabolism and appetite, leptin is a reproductive hormone produced and secreted by the placenta of many, but not all mammalian species. In mice, in which the placenta does not secrete leptin, exogenously added leptin stimulates invasiveness of early (but not late)-gestation trophoblast cells. We report a similar phenomenon occurs in Myotis lucifugus (little brown myotis), a species in which the placenta synthesizes and secretes leptin. Immunoneutralization of endogenously secreted leptin from cultured M. lucifugus trophoblast cells inhibited the ability of these cells to invade a matrigel matrix. The effect was not due to an inhibitory effect of the antibody on cell proliferation, nor was it a non-specific effect of antibody administration. Cell invasion was significantly reduced in untreated cells obtained from late-gestation placentas, and the antibody had no effect at that time. This occurred despite continued expression throughout gestation of the long (OBRb) and short (OBRa) isoforms of leptin receptor mRNA. This study suggests that an important function of leptin during pregnancy is an effect on trophoblast cell invasiveness, at a time when the placenta is becoming established. That this occurs in two phylogenetically unrelated and distant species, regardless of whether the placenta is a source of secreted leptin, suggests that this is a highly conserved reproductive action of leptin.

The hormonal and behavioral response to group formation, seasonal changes, and restraint stress in the highly social Malayan Flying Fox (Pteropus vampyrus) and the less social Little Golden-mantled Flying Fox (Pteropus pumilus) (Chiroptera: Pteropodidae).

This study examined behavioral and physiological responses (changes in inter-animal spacing, total glucocorticoids, testosterone, and body mass) to the formation of breeding and same-sex groups in two bat species, the socially gregarious Malayan Flying Fox (Pteropus vampyrus) and the less social Little Golden-mantled Flying Fox (Pteropus pumilus). We hypothesized that social instability, especially in the breeding groups and especially in P. vampyrus, would result in elevated glucocorticoids and that social facilitation of breeding and/or male-male competition would result in persistently higher levels of testosterone in breeding males. Seasonal rhythms in all measures were also predicted, and the glucocorticoid stress response was expected to vary by sex, season, and group type. Nearly all animals responded to group formation with elevated glucocorticoids, but, for breeding animals (especially aggressive male P. vampyrus), these responses persisted over time. In both species, breeding group formation resulted in elevated testosterone in males. Glucocorticoids, testosterone, testes volume, and body mass generally peaked in the breeding season in males (late summer and early autumn), but the seasonal glucocorticoid peak in females occurred in late winter and early spring. All animals responded to restraint stress with elevations in glucocorticoids that largely did not differ by sex, time of year, reproductive condition, group type, or, in lactating females, the presence of her pup. Changes in both behavior and physiology were more evident in P. vampyrus than in P. pumilus, and we believe that their underlying social differences influenced their responses to group formation and to the changing seasonal environment.

Characterization of pituitary-adrenocortical activity in the Malayan flying fox (Pteropus vampyrus).

Pituitary-adrenocortical and gonadal endocrine activity was investigated in a captive colony of Pteropus vampyrus, a highly social Old World fruit bat. Both cortisol and corticosterone were present in plasma, at a ratio of approximately 5:1, respectively. Glucocorticoid but not testosterone levels significantly increased prior to and concomitant with the evening active period. Restraint stress for 15-60 min resulted in a significant and rapid increase in plasma levels of adrenocorticotropic hormone (ACTH) and glucocorticoids. ACTH levels quickly returned to baseline following restraint whereas glucocorticoid levels remained elevated for at least 30 min after restraint ended. Plasma ACTH levels after stress were similar to levels reported after stress in other mammals. Stress-induced glucocorticoid levels were several-fold greater than those reported for most mammals. Restraint for 15 min significantly inhibited testosterone levels. Restraint stress did not affect hormone levels on the morning following restraint. Brief capture, handling, and release of the animals did not elicit increases in these hormones. The physiological responsiveness of the pituitary and adrenal glands, along with P. vampyrus's documented seasonality and range of social behaviors, makes these bats an excellent model for exploring the general physiology of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes, as well as social influences on these axes.

The effect of leptin on mouse trophoblast cell invasion.

The hormone leptin is produced by adipose tissue and can function as a signal of nutritional status to the reproductive system. The expression of leptin receptor and, in some species, leptin, in the placenta suggests a role for leptin in placental development, but this role has not been elucidated. Leptin is required at the time of embryo implantation in the leptin-deficient ob/ ob mouse and has been shown to upregulate expression of matrix metalloproteinases (MMPs), enzymes involved in trophoblast invasion, in cultured human trophoblast cells. This led us to the hypothesis that leptin promotes the invasiveness of trophoblast cells crucial to placental development. We found that leptin stimulated mouse trophoblast cell invasion through a matrigel-coated insert on Day 10, but not Day 18 of pregnancy. Optimal stimulation occurred at a concentration of 50 ng/ml leptin, similar to the peak plasma leptin concentration during pregnancy in the mouse. Leptin treatment did not stimulate proliferation of mouse trophoblast cells in primary culture. Leptin stimulation of invasion was prevented by 25 muM GM6001, an inhibitor of MMP activity. Our results suggest that leptin may play a role in the establishment of the placenta during early pregnancy and that this function is dependent on MMP activity. This effect of leptin may represent one mechanism by which body condition affects placental development.

Baseline and stress-induced glucocorticoids during reproduction in the variable flying fox, Pteropus hypomelanus (Chiroptera: Pteropodidae).

Baseline and stress-responsive glucocorticoid (GC) levels were assessed during early pregnancy, late pregnancy, and lactation in female variable flying foxes (Pteropus hypomelanus) and in males over the same time period. Animals were maintained in a breeding colony in captivity. High levels of both cortisol and corticosterone were detected, with total plasma GC levels being among the highest documented in vertebrates (up to 3000 ng/ml in individual animals, with cortisol being the primary GC, accounting for approximately 78% of total GCs), and significantly greater in males than in females. Plasma levels of cortisol and corticosterone showed nearly identical profiles within each sex, with the exception of females in late pregnancy, in which corticosterone, but not cortisol, increased significantly. Baseline levels of plasma cortisol were highest in September (when pups were between 1 and 2 months of age) in both sexes, which may be related to the approaching onset of the mating period. There was a continuum in the magnitude of the response to stress (handling and sampling) over time in females, with the greatest stress response in early pregnancy, a dampened response during late pregnancy, and no significant stress response during lactation. Surprisingly, males failed to exhibit elevated GCs after this stress, but did have significant stress-induced hyperglycemia and suppression of plasma testosterone levels. This may be due to their high (perhaps maximal) baseline levels, which suggests that being in a breeding group was chronically stressful for males.

Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR.

The adrenocortical response to hypoxia may be a critical component of the adaptation to this common neonatal stress. Little is known about adrenal function in vivo in hypoxic neonates. The purpose of this study was to evaluate adrenocortical responses to ACTH in suckling rat pups exposed to hypoxia from birth to 5-7 days of age compared with normoxic controls. We also evaluated potential cellular controllers of steroidogenic function in situ. In 7-day-old pups at 0800, hypoxia from birth resulted in increased basal (12.2 +/- 1.4 ng/ml; n = 12) and ACTH-stimulated (94.0 +/- 9.4 ng/ml; n = 14) corticosterone levels compared with normoxic controls (basal = 8.3 +/- 0.5 ng/ml; n = 11; stimulated = 51.3 +/- 3.8 ng/ml; n = 8). This augmentation occurred despite no significant difference in plasma ACTH levels in normoxic vs. hypoxic pups before (85 +/- 4 vs. 78 +/- 8 pg/ml) or after (481 +/- 73 vs. 498 +/- 52 pg/ml) porcine ACTH injection (20 microg/kg). This effect was similar in the afternoon at 6 days of age and even greater at 5 days of age at 0800. The aldosterone response to ACTH was not augmented by exposure to hypoxia from birth. Adrenocortical hypoxia-inducible factor (HIF)-1alpha mRNA was undetectable by RT-PCR. Steroidogenic acute regulatory (StAR) protein in adrenal subcapsules (zona fasciculata/reticularis) was augmented by exposure to hypoxia; this effect was greatest at 5 days of age. Peripheral-type benzodiazepine receptor (PBR) protein was also increased at 6 and 7 days of age in pups exposed to hypoxia from birth. We conclude that hypoxia from birth results in an augmentation of the corticosterone but not aldosterone response to ACTH. This effect appears to be mediated at least in part by an increase in controllers of mitochondrial cholesterol transport (StAR and PBR) and to occur independently of measurable changes in endogenous plasma ACTH. The augmentation of the corticosterone response to acute increases in ACTH in hypoxic pups is likely to be an important component of the overall physiological adaptation to hypoxia in the neonate.

Changes in baseline and stress-induced glucocorticoid levels during the active period in free-ranging male and female little brown myotis, Myotis lucifugus (Chiroptera: Vespertilionidae).

Baseline and stress-responsive glucocorticoid (GC) levels were characterized during the active period in free-ranging male and reproductive female little brown myotis (Myotis lucifugus). Bats were trapped and blood was sampled within 3 min of capture at two maternity sites during the summer and at one swarming site prior to hibernation in New England. Both GC hormones, cortisol and corticosterone, were detected, with cortisol accounting for an average of approximately 95% of total circulating GCs. Samples collected at the dusk emergence and after the first return from feeding showed significant seasonal differences across the active period (early pregnancy, mid-to-late pregnancy, lactation [and comparable mid-summer times for males], and pre-hibernation) within and between each sex. Elevated baseline values were found in mid-to-late pregnancy females at emergence, and in both males and females at the swarming site compared to other groups. Female GC values during mid-to-late pregnancy and during the pre-hibernation period were greater than those for males. Significantly higher GC levels following 15 min of restraint were exhibited by all animals in the summer and prior to hibernation. There was little variation between groups or sexes in the total GC levels reached following restraint. Taken together, these results suggest that: (1) GCs may be involved in the increased feeding and/or fat deposition characteristic of pregnancy and the pre-hibernation period, (2) GCs may be related to mating and to the generally increased levels of activity that occur during the pre-hibernation period, and (3) regardless of sex or reproductive condition, all animals maximally respond to restraint stress.

Comparative analysis of expression and secretion of placental leptin in mammals.

Increased plasma level of leptin appears to be a ubiquitous feature of pregnant mammals. The mechanisms by which leptin levels are increased may be species specific, however, with some species upregulating adipose leptin production and others expressing leptin in the placenta. Placental leptin expression was examined in representative species of the two most abundant mammalian orders, Rodentia and Chiroptera, and in cultured human choriocarcinoma (BeWo) cells. Leptin mRNA was expressed in BeWo cells and in placentas of Myotis lucifugus (little brown bat), Eptesicus fuscus (big brown bat), and Rattus norvegicus (laboratory rat), but not the common laboratory mouse Mus musculus. cAMP stimulated secretion of leptin from BeWo cells and also stimulated leptin mRNA expression in the cells. In addition to adipose and placental tissue, leptin transcript in M. lucifugus was detectable in heart, spleen, and liver, but not in lung, brain, and kidney. Hepatic expression was also observed in E. fuscus, but not in mice or rats, and did not appear to result from hepatic fat deposition. Leptin cDNA was cloned and sequenced from M. lucifugus placenta and shared up to 95% homology with other mammalian leptin cDNAs. It is concluded that 1) placental leptin expression and secretion are species-specific traits, 2) placental leptin production represents one of three major mechanisms for achieving high circulating maternal leptin levels during pregnancy, the others being upregulation of adipose leptin production and production of circulating leptin-binding proteins, and 3) hepatic leptin expression in pregnant insectivorous bats may be an adaptation resulting from the presence of extremely low amounts of subcutaneous fat during pregnancy and lactation in these species.