We investigated the effects of lactation on diurnal changes in serum leptin and hypothalamic expression of the leptin receptor isoforms, Ob-Ra, -Rb, -Rc, -Re and -Rf in rats. In non-lactating rats, serum leptin concentration was increased at night while hypothalamic mRNA levels of Ob-Rb, -Rc and -Re decreased; by contrast, expression of Ob-Ra and Ob-Rf was unchanged at night. There were significant negative correlations between serum leptin and mRNA expression of Ob-Rb (P<0.001) and Ob-Re (P<0.05), which were independent of time of day. In lactating rats, the nocturnal rise in serum leptin was attenuated. Daytime hypothalamic Ob-Rb mRNA levels were significantly lower than in non-lactating controls, and the normal nocturnal decreases in expression of Ob-Rb, -Rc and -Re were lost. The relationship between serum leptin and Ob-Re expression was not changed by lactation. Lactation had no effect on the expression of Ob-Ra mRNA in the hypothalamus. Decreased daytime Ob-Rb expression could lead to reduced hypothalamic sensitivity to leptin, and thus contribute to increased daytime appetite in lactating rats. Moreover, maintaining high levels of Ob-Re expression could, by increasing hypothalamic leptin-binding protein concentration and reducing local leptin bioavailability, further accentuate hyperphagia. Thus, selective changes in expression of specific isoforms of the leptin receptor may contribute to the hyperphagia of lactation in rats.
RG Denis, C Bing, EK Naderali, RG Vernon and G Williams
M Tena-Sempere, L Pinilla, LC Gonzalez, FF Casanueva, C Dieguez and E Aguilar
Leptin, the adipocyte-produced hormone that plays a key role in body weight homeostasis, has recently been found to be involved in the regulation of the hypothalamic-pituitary-adrenal axis. Moreover, reciprocal interactions between leptin and glucocorticoids have been described. In the present communication, two different strategies were undertaken to explore the mode of action of leptin in the direct control of rat adrenal function. First, a synthetic peptide approach demonstrated that the inhibitory effect of leptin on basal and ACTH-stimulated corticosterone secretion in vitro is, at least partially, mapped to a domain of the native protein between amino acids 116 and 130, i.e. an area of the molecule also relevant in terms of regulation of food intake and endocrine control. Secondly, semi-quantitative RT-PCR analysis indicated a complex pattern of adrenal leptin receptor (Ob-R) mRNA expression, with predominant expression of the Ob-Ra and Ob-Rb isoforms, as well as moderate levels of the Ob-Rc and Ob-Rf variants, whereas negligible signals for the Ob-Re isoform were detected. Interestingly, such an expression pattern appeared hormonally regulated as exposure to human recombinant leptin (10(-7 )M) or ACTH (10(-7 )M) significantly decreased Ob-R isoform mRNA expression. Indeed, dose-dependent ligand-induced Ob-Ra and Ob-Rb mRNA down-regulation was further confirmed by adrenal stimulation with increasing concentrations (10(-9)-10(-5 )M) of the active leptin fragment, leptin 116-130 amide. Overall, our results provide evidence for a novel regulatory step at the level of Ob-R mRNA expression in the interplay between ACTH and leptin for the tuning of rat adrenal corticosterone secretion. Furthermore, our data showing down-regulation of Ob-R mRNA expression by its cognate ligand may well be relevant to leptin physiology and its alteration in various disease states.
J Bohlender, M Rauh, J Zenk and M Groschl
Leptin plays a central role in the regulation of food intake and energy expenditure in rodents. However, it has become clear that this hormone has more than only a satiety-inducing function, and that there are other sources of leptin, such as the central nervous system, placenta and the gastrointestinal tract in addition to adipose tIssue. Knowing about the important role of the salivary glands in food intake and digestion, it was the objective of the present study to investigate how leptin and its receptor are expressed and distributed in the major salivary glands of humans. We found leptin distributed throughout the major salivary glands with obvious intracellular concentrations in granula. In contrast, immunostaining for the leptin receptor was found exclusively in the membranes of the glandular cells. A high density of the leptin receptor was localised in the epithelia of the duct lumen. PCR analysis proved the autonomous expression of leptin by the salivary glands independently from adipocytes. Accordingly the long receptor isoform was expressed by any examined tIssue. In the light of recent findings of leptin influencing the growth of rodent salivary glands, the presence and distribution of leptin and its receptor suggests an autocrine role of salivary leptin within the glands.
J Wilsey and PJ Scarpace
The objectives of this study were to determine if reduced long-form leptin receptor (ObRb) expression in diet-induced obese (DIO) animals is associated with deficits in maximal leptin signaling and, secondly, to establish the effects of short-term caloric restriction (CR) on ObRb expression and function. Groups of DIO and life-long chow-fed (CHOW) F344xBN male rats, aged 6 months, were given an i.c.v. injection containing 2 micro g leptin or artificial cerebrospinal fluid (ACSF) vehicle. Leptin induced a >6-fold increase in STAT3 phosphorylation in CHOW rats, but less than 2-fold increase in DIO. Reduced maximal leptin-stimulated STAT3 phosphorylation in DIO rats was coupled with a decline in both ObRb expression and protein. At this point, subgroups of DIO and CHOW animals underwent CR for 30 days and were then tested for acute leptin responsiveness. CR resulted in a 45 and 85% increase respectively in leptin-stimulated STAT3 phosphorylation in CHOW and DIO animals. Similarly, CR increased ObRb expression and protein in both CHOW and DIO animals. To explore the role of leptin in regulating ObRb expression, we reversibly overexpressed leptin in the hypothalamus and found that ObRb mRNA inversely follows central leptin expression. By enhancing both ObRb expression and signaling capacity, CR may enhance leptin responsiveness in leptin-resistant DIO animals.
M Zerani, C Boiti, C Dall’Aglio, L Pascucci, M Maranesi, G Brecchia, C Mariottini, G Guelfi, D Zampini and A Gobbetti
. Barr VA , Lane K & Taylor SI 1999 Subcellular localization and internalisation of the four human leptin receptor isoforms. Journal of Biological Chemistry 30 21416 –21424. Boiti C , Zerani M, Zampini D & Gobbetti A
Shan-Jin Wang, Xin-Feng Li, Lei-Sheng Jiang and Li-Yang Dai
, Bertoni et al . 2009 ). The direct actions of leptin are through the leptin receptor (OBR (LEPR)), which is present in the growth plate ( Nakajima et al . 2003 , Kishida et al . 2005 ). Activation of OBR by leptin leads to the activation of many
JT Smith and BJ Waddell
Leptin, the peptide hormone product of the ob gene, regulates food intake and energy expenditure at the hypothalamic level via the long-form of the leptin receptor (Ob-Rb). Leptin also plays a key role in determining the onset of puberty, but there is controversy as to whether leptin provides a trigger for puberty or is a permissive signal. Thus, although leptin administration can advance puberty onset in rodents, circulating leptin appears stable across puberty. While these data suggest a permissive role for leptin in rat puberty, it is possible that a change in hypothalamic response to leptin (e.g. via increased Ob-Rb expression) could enhance leptin action and thus trigger puberty without a rise in circulating leptin. In the present study we assessed developmental changes in hypothalamic Ob-Rb mRNA and protein expression in female and male rats from late fetal to postpubertal life. Quantitative RT-PCR showed that Ob-Rb mRNA increased (P<0.05) by around fivefold from fetal to postpubertal life in both females and males. These increases in Ob-Rb mRNA expression were gradual, but did not increase significantly between postnatal day 30 (pre-puberty) and day 51 (post-puberty). By day 51, hypothalamic Ob-Rb mRNA expression was higher (P<0.05) in females relative to males. Hypothalamic Ob-Rb protein showed a comparable developmental pattern (approximate threefold increase from fetal to postpubertal life), although a significant increase (15%; P<0.05) was observed between days 30 and 51 in females. Plasma leptin levels exhibited a dynamic pattern in both male and female rats during the prepubertal period, characterised by a precipitous fall after birth, relative stability to day 5, then a rapid increase to a transient peak on day 12. Plasma leptin then remained unchanged from day 15 in female rats but increased in males after puberty, thus confirming the well-recognised sex difference in adult rat leptin levels. In conclusion, this study shows that developmental increases occur not only in plasma leptin but also in hypothalamic Ob-Rb expression, suggesting that both are likely to influence the timing of puberty onset. Moreover, our data show that sex differences in both hypothalamic Ob-Rb and plasma leptin emerge only after puberty.
M Tena-Sempere, PR Manna, FP Zhang, L Pinilla, LC Gonzalez, C Dieguez, I Huhtaniemi and E Aguilar
Leptin, the product of the ob gene, is a pivotal signal in the regulation of neuroendocrine function and fertility. Although much of the action of leptin in the control of the reproductive axis is exerted at the hypothalamic level, some direct effects of leptin on male and female gonads have also been reported. Indeed, recent evidence demonstrated that leptin is able to inhibit testosterone secretion at the testicular level. However, the molecular mechanisms behind this effect remain unclear. The focus of this study was twofold: (1) to identify potential targets for leptin-induced inhibition of steroidogenesis, and (2) to characterize in detail the pattern of expression and cellular distribution of leptin receptor (Ob-R) mRNA in adult rat testis. In pursuit of the first goal, slices of testicular tissue from adult rats were incubated with increasing concentrations of recombinant leptin (10(-9)--10(-7 )M) in the presence of human chorionic gonadotropin (hCG; 10 IU/ml). In this setting, testosterone secretion in vitro was monitored, and expression levels of mRNAs encoding steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450 scc) and 17 beta-hydroxysteroid dehydrogenase type III (17 beta-HSD) were assessed by Northern hybridization. In pursuit of the second goal, the pattern of cellular expression of the Ob-R gene in adult rat testis was evaluated by in situ hybridization using a riboprobe complementary to all Ob-R isoforms. In addition, testicular expression levels of the different Ob-R isoforms, previously identified in the hypothalamus, were analyzed by means of semi-quantitative RT-PCR. In keeping with our previous data, recombinant leptin significantly inhibited hCG-stimulated testosterone secretion. In this context, leptin, in a dose-dependent manner, was able to co-ordinately decrease the hCG-stimulated expression levels of SF-1, StAR and P450 scc mRNAs, but it did not affect those of 17 beta-HSD type III. In situ hybridization analysis showed a scattered pattern of cellular expression of the Ob-R gene within the adult rat testis, including Leydig and Sertoli cells. In addition, assessment of the pattern of expression of Ob-R subtypes revealed that the long Ob-Rb isoform was abundantly expressed in adult rat testis. However, variable levels of expression of Ob-Ra, Ob-Re, and Ob-Rf mRNAs were also detected, whereas those of the Ob-Rc variant were nearly negligible. In conclusion, our results indicate that decreased expression of mRNAs encoding several up-stream elements in the steroidogenic pathway may contribute, at least partially, to leptin-induced inhibition of testicular steroidogenesis. In addition, our data on the pattern of testicular expression of Ob-R isoforms and cellular distribution of Ob-R mRNA may help to further elucidate the molecular mechanisms of leptin action in rat testis.
Yoshihiro Suzuki, Yohei Kurose, Hideyuki Takahashi, Sadaki Asakuma, Yoshiyuki Azuma and Shigeki Kobayashi
). Furthermore, lactating rats exhibit a decrease in leptin receptor mRNA expression in the hypothalamus ( Brogan et al . 2000 , Denis et al . 2003 b ). On the other hand, leptin receptors and mRNAs encoding leptin receptor isoforms are detected in the rat
M P Di Yorio, M G Bilbao, M C Pustovrh, J P Prestifilippo and A G Faletti
secreted from adipose cells and transported via blood to act in specific central and peripheral systems. Leptin is recognized by the leptin receptor (Ob-R; Tartaglia et al . 1995 ), which is a product of the db gene ( Chen et al . 1996 , Chua et al