To determine the potential roles for Jak2-autonomous LepRb signals in leptin action in vivo, we generated a mouse model in which LepRb is replaced by a truncation mutant (LepRbΔ65) that contains within its intracellular domain only the sequences required to associate with and activate Jak2. We found that the hyperphagia, obesity, linear growth, ARC physiology, and immune function of these Δ/Δ mice closely resembled that of entirely LepRb-deficient db/db mice. Δ/Δ and db/db animals did demonstrate some modest differences in glucose homeostasis; however, both male and female Δ/Δ mice exhibited a delayed progression to frank hyperglycemia compared with db/db mice. Taken together, these findings demonstrate that Jak2-autonomous LepRb signals may contribute modestly to the modulation of glucose homeostasis by leptin, but emphasize the necessity of signals emanating from the COOH-terminus of LepRb (beyond the Jak2-associating Box1 and Box2 motifs) for most leptin action.
The finding that 4-week-old Δ/Δ mice display similar insulin and C-peptide levels as db/db
animals, but exhibit improved blood glucose levels, suggests improved glucose disposal or decreased glucose production in the Δ/Δ mice independent of insulin production. This is consistent with data suggesting that central nervous system leptin action suppresses hepatic glucose production, and with our previous finding that some portion of this is mediated independently of Tyr1138
→STAT3 signaling (2
). The Δ/Δ mice progress rapidly (by 6 weeks of age) to dramatic hyperglycemia and parameters of glucose homeostasis indistinguishable from db/db
animals, however. Indeed, even at 5 weeks of age, the response to a glucose bolus is comparably poor in male Δ/Δ and db/db
animals and barely improved in female Δ/Δ compared with db/db
animals. Furthermore, the increased insulin production of Δ/Δ relative to db/db
males at 6 weeks of age and beyond fails to ameliorate their hyperglycemia. Thus, the improvement in glucose homeostasis mediated by Jak2-autonomous LepRb signals in Δ/Δ mice compared with db/db
animals is very modest, as it is easily overwhelmed by a large glucose load and/or the increasing insulin resistance and diabetes of advancing age. No difference in β-cell mass was detected between Δ/Δ and db/db
males. The mechanism(s) mediating the increased insulin production of the Δ/Δ relative to db/db
males is unclear, but could represent an improvement in β-cell function due to the later time at which Δ/Δ animals become diabetic or to some residual leptin action in the Δ/Δ β-cell (35
Although the overall similarity of ARC gene expression and physiology between Δ/Δ and db/db mice indicates little role for Jak2-autonomous LepRb signals in ARC leptin action, the finding of decreased (worsened) Pomc mRNA expression in Δ/Δ compared with db/db mice was surprising. One possible explanation for this observation is that some residual signal mediated by LepRbΔ65 modestly attenuates Pomc expression and that this attenuating signal is overwhelmed under normal circumstances by the LepRb signals that enhance Pomc expression. Unfortunately, the low Pomc content of db/db and Δ/Δ animals and low activity of these neurons at baseline rendered the examination of POMC c-fos uninformative.
Although the molecular mechanisms underlying Jak2-autonomous LepRb action remain unclear, several pathways could contribute. In cultured cells, the activation of Jak2 by LepRbΔ65
and similar receptor mutants mediates some activation of the extracellular signal–related kinase pathway (13
). Indeed, chemical inhibitor studies have suggested a role for extracellular signal–related kinase signaling in the regulation of autonomic nervous system function by leptin, and the autonomic nervous system underlies a major component of the leptin effect on glucose homeostasis (37
). Phosphatidylinositol 3-kinase (PI 3-kinase) also plays a role in the regulation of glucose homeostasis by leptin (38
). As we have been unable to observe the regulation of PI 3-kinase by leptin in cultured cells and the analysis of this pathway in the hypothalami of obese, hyperleptinemic mice remains problematic, the molecular mechanism by which LepRb engages this pathway remains unclear. Although difficult to test directly, it is thus possible that Jak2-autonomous LepRb signals might modulate this pathway in vivo. We have previously demonstrated that the major regulation of hypothalamic mammalian target of rapamycin (mTOR), including in response to nutritional alteration, occurs indirectly, via neuronal activation (42
). We examined this pathway (along with the phosphorylation of STAT3) in the hypothalamus of Δ/Δ animals (supplementary Fig. 4), revealing the expected absence of STAT3 signaling and increased mTOR activity (secondary to the activation of orexigenic neurons) in the mediobasal hypothalamus of db/db
and Δ/Δ animals. Thus, the regulation of mTOR is similar in these mouse models.
A potential intermediate linking Jak2 activation to PI 3-kinase activation is SH2B1, a SH2 domain–containing protein that binds phosphorylated Tyr813
on Jak2 (43
). Cell culture studies show that SH2B1 binds directly to Jak2, augments its kinase activity, and couples leptin stimulation to insulin receptor substrate activation, a well-known activator of PI 3-kinase (44
). Indeed, SH2B1-null mice display hyperphagia, obesity, and diabetes (45
). SH2B1 could also mediate other, unknown Jak2-autonomous LepRb signals.
Importantly, the phenotype of mice expressing LepRbΔ65
differs significantly from a mouse model in which LepRbY123F
(mutated for the three tyrosine phosphorylation sites, but with an otherwise intact intracellular domain) replaces LepRb (22
). Unlike Δ/Δ animals, these LeprY123F
mice display improved energy homeostasis and more dramatic improvements in glucose homeostasis (both of which are sustained into adulthood), compared with db/db
animals. Unfortunately, the C57 genetic background strain used to study the LeprY123F
mice not only differs compared with the C57BL/6J background that we used to study our Δ/Δ and db/db
animals, but also diverges from that of the db/db
animals used as comparators for the LeprY123F
). Similarly, it is also possible that minor differences between the incipient C57BL/6J backgrounds of db/db
and Δ/Δ mice could contribute to the modest distinctions observed between these two models.
Aside from the background strain, the intriguing possibility arises that the intracellular domain of LepRb may mediate heretofore unsuspected signals independently from tyrosine phosphorylation. As it is, future studies will be needed to carefully compare the phenotype of LeprY123F, Δ/Δ, and db/db animals within the same facility and on the same genetic background; should these differences remain, further work will be necessary to confirm the importance and determine the identity of the underlying signaling.
In summary, our findings reveal the insufficiency of Jak2-autonomous LepRb signals for the bulk of leptin action. These finding do not rule out the possibility that Jak2-autonomous signals may be required to support the action of LepRb phosphorylation, however. Indeed, our present findings suggest a modest role for Jak2-autonomous LepRb signals in the regulation of glucose homeostasis by leptin. Understanding collaborative roles for Jak2-autonomous signals in leptin action and deciphering the mechanisms underlying these signals and potential tyrosine phosphorylation–independent signals mediated by the COOH-terminus of LepRb will represent important directions for future research.