Leptin and its central signaling have many roles beyond its lipostatic function. A wide range of data links leptin to both the regulation of energy homeostasis and reproduction. One key population of Lepr is found on POMC neurons in the arcuate nucleus, and evidence links the anorexigenic effects of estrogen as being mediated through these POMC neurons as well (19
mice lacking leptin receptors specifically on POMC neurons and control Leprflox/flox
mice with normal leptin receptors were used in the current studies to determine whether or not leptin receptors on POMC neurons are critical for reproductive capacity and/or for estrogens’ influence on adiposity and glucose homeostasis. Our data suggest that lack of Lepr on POMC neurons has no obvious influence on reproductive capacity. ERα has been suggested to be the main nuclear estrogen receptor that mediates estradiol’s catabolic effect in both sexes (25
). We found that the hypothalamic ERα mRNA level was lower in sham-operated female Pomc
mice compared to Leprflox/flox
females, and it was comparable between male Pomc
mice, suggesting that central leptin signaling dysfunction due to lack of Lepr on POMC neurons modulates hypothalamic ERα gene expression in cycling females. It is not clear how lacking leptin signaling on POMC neurons would result in decreased hypothalamic ERα gene expression, and is worth further investigation.
Pomc-Cre, Leprflox/flox males had greater adiposity and were glucose intolerant and insulin insensitive compared with control males. The impaired glucose homeostasis in the male Pomc-Cre, Leprflox/flox mice was likely a secondary effect of visceral obesity. In contrast, control and Pomc-Cre, Lepr flox/flox females had similar glucose tolerance although Pomc-Cre, Leprflox/flox females had greater carcass adiposity than Leprflox/flox females. We further investigated the interaction between estrogens and Lepr on POMC neurons on body fat accumulation and glucose tolerance using OVX mice. OVX Pomc-Cre, Leprflox/flox and Leprflox/flox females did not cycle and had significantly lower circulating estradiol levels than intact females, indicating successful OVX surgeries. OVX increased adiposity in both Leprflox/flox and Pomc-Cre, Leprflox/flox females. Specifically, OVX increased both subcutaneous and carcass adiposity in female Leprflox/flox mice, whereas majority of the increased adiposity was allocated in the subcutaneous compartment of Pomc-Cre, Leprflox/flox females (). The fat distribution data were consistent with the finding that OVX significantly impaired glucose tolerance in Leprflox/flox females but only had a trend to do so in Pomc-Cre, Leprflox/flox females (). The two OVX female groups had comparable glucose levels during ipGTT, suggesting that the sex difference in leptin-POMC signaling on glucose tolerance was independent of ovarian hormones. OVX Pomc-Cre, Leprflox/flox females secreted significantly more insulin than OVX Leprflox/flox females during the ipGTT (); thus, although OVX Pomc-Cre, Leprflox/flox did not develop glucose intolerance, they were insulin resistant compared to OVX Leprflox/flox females.
The long form of Lepr has been reported to be expressed in several peripheral tissues. Several lines of evidence suggest that it is the central Lepr that is critical in the regulation of energy and glucose homeostasis. First, mice with deletion of Lepr in the brain are obese and insulin resistant, whereas mice with deletion of Lepr in the liver are normal (26
), suggesting that direct leptin signaling in the liver alone does not affect energy balance and glucose homeostasis. Furthermore, removal of the signaling domain of the leptin receptor in most peripheral tissues including liver, adipose tissues, and small intestine, but not in the brain, causes no changes in energy balance or glucose homeostasis (27
). Second, a neuron-specific Lepr transgene (28
) or expressing Lepr exclusively in POMC neurons (29
) completely rescues the obesity, diabetes, and infertility in db/db
mice, supporting the hypothesis that leptin’s effects on energy balance, glucose homeostasis, and fertility are centrally mediated . Pomc
mice, with Lepr removed only from the POMC neurons, exhibit an obesity phenotype in both sexes and a diabetes phenotype in males. Taken together, these data suggest that Lepr signaling in the CNS, other than in peripheral leptin receptor-expressing cells, plays a critical role in the regulation of energy balance and glucose homeostasis.
Leptin has significant effects on many aspects of reproduction, including an association with the onset of puberty as well as fertility for males and females. Male and female leptin-deficient obese mice are infertile (30
) and adding back leptin reverses the infertility and resulted in sexual maturity (31
). Balthasar et al. previously reported that “Pomc
-Cre, Lepr flox/flox
are fertile and able to lactate” (32
). Whether or not the leptin receptors on POMC neurons are critical in the regulation of reproductive capacity and influence processes such as puberty, sex hormone levels, and estrous cycle has been inconclusive until the current study. We compared their reproductive function, onset of puberty, vaginal cytology, and circulating estradiol levels of female control Leprflox/flox
mice. Our findings are in agreement with the previous observation that “Pomc
mice are fertile and able to lactate” (32
). In addition, we found that Pomc
mice displayed a similar onset of puberty and had comparable reproductive capacity, as indicated by number of litters produced and reproductive success rate, and also comparable circulating estradiol levels as control Leprflox/flox
The long form of Lepr is expressed in many extra-arcuate regions, including ventromedial hypothalamus, dorsomedial hypothalamus, premammillary ventral nucleus, lateral hypothalamus, ventral tegmental area, and nucleus of the solitary tract (33
). Although significant attention has been focused on the arcuate nucleus in terms of the regulation of energy and glucose homeostasis, other leptin receptor expressing neurons may have key roles in the regulation of reproduction. The dorsomedial hypothalamus and premammillary ventral nucleus innervate areas related to reproductive control including the anteroventral periventricular nucleus and the medial preoptic area (36
). The fact that reproductive function was not altered in the Pomc
mice suggests that Lepr in leptin target neurons other than arcuate POMC neurons may play an important role in mediating leptin’s effect on reproduction. Indeed, recent studies indicated that Lepr expressing neurons on hypothalamic premammilary nucleus regulate luteinizing hormone secretion (37
) and project to the rostral hypothalamus to directly innervate GnRH neurons (38
) to regulate reproduction.
Visceral fat varies inversely with circulating estrogens; as levels of estrogens decrease, visceral adiposity increases. An increase in visceral adiposity is often associated with a reduction in estrogens in females, but this was not the case in these female Pomc-Cre, Leprflox/flox mice which had similar circulating estradiol level as their controls () but accumulated carcass adiposity (). There are several possibilities for this. First, the increased visceral adiposity could be due to reduced central and peripheral estrogen action, as indicated by reduced hypothalamic ERα gene expression. Second, the combination of estrogen and functional leptin signaling may be required for sex-specific fat distribution. Sex differences in total body fat and fat distribution were detected in sham-operated male and female control Leprflox/flox mice but not in Pomc-Cre, Leprflox/flox mice with interrupted central leptin signaling; such sex differences between male and female Leprflox/flox mice were abolished by estrogen deficiency via OVX (). Lack of either estrogen or functional leptin signaling would consequently lead to visceral obesity in females. It is possible that although circulating estradiol is maintained at normal levels in Pomc-Cre, Leprflox/flox females, it fails to enhance leptin’s ability to stimulate sympathetic activity of visceral fat, the most highly innervated fat depot, in the absence of leptin POMC signaling, and thus leads to accumulation of visceral adiposity.
There is a complex interaction between estrogens and leptin signaling in the regulation of energy balance and adiposity. Estrogen may regulate circulating leptin level and ob
gene expression. A significant increase in circulating leptin level has been observed following the weight gain associated with OVX in female rats (13
). These results are in contrast with one study that observed no change in plasma leptin concentration in OVX females, despite observing that the OVX rats weighed significantly more than sham-operated females (18
). Estradiol treatment increased ob
mRNA levels in white adipose tissue of OVX rats (40
). Using ovary-intact instead of OVX rats, Rocha et al. reported that estradiol treatment reduced body fat mass but did not change plasma leptin concentration or ob
gene expression in white adipose tissue (42
). These varying results indicate that the animal models (ovary-intact vs. OVX) and assay time may be critical for studying the physiological roles of estrogen and leptin in regulating energy balance.
Whether or not estrogens modulate leptin concentration or ob
gene expression does not exclude the estrogen interacting with hypothalamic leptin signaling as a mechanism to regulate energy balance. Previous studies investigated leptin sensitivity using either peripheral or central leptin administration in estrogen-treated or estrogen-deficient OVX rodents. OVX did not alter subcutaneously administered leptin’s ability to reduce food intake or body fat in rats (43
) or mice (44
), suggesting that estrogen might not directly mediate leptin’s effect. On the contrary, the ability of central leptin administration to reduce food intake was greater in ovary-intact females than in males (13
) or in OVX females (13
). Conversely, administration of estradiol to OVX females restored their central leptin sensitivity and changed their body fat distribution to mirror that of intact females (13
). Additionally, altering the sex hormone milieu in males with estradiol administration increased sensitivity to central leptin administration and increased subcutaneous fat deposition (13
). Estrogens influences hypothalamic Lepr expression. OVX caused a significant (17
) or a 50% but statistically non-significant (39
) reduction in expression of the long form of Lepr in the hypothalamus, and estradiol replacement restored its expression (17
). Since only Lepr mRNA expression has been measured, it is unknown how estrogen may impact the Lepr protein or signaling. The differences in leptin sensitivity caused by the presence or absence of estrogen may occur downstream of Lepr transcription and translation.
In summary, we have provided evidence that Lepr in POMC neurons, although important for leptin’s effect on the regulation of body fat accumulation and glucose homeostasis, is not required for leptin’s effect on reproduction. In addition, the sex difference in leptin signaling in POMC neurons on glucose homeostasis appears independent of ovarian hormones.