Leptin integrates the status of peripheral fat stores with the central control of energy expenditure and food intake for the homeostatic control of body weight. In normal-weight individuals, complex biological mechanisms protect against both weight gain and weight loss to stabilise fat stores and ensure survival and reproductive fitness. Specific feedback mechanisms exist to protect against excessive weight loss that may otherwise be associated with prolonged leptin signaling (Myers et al., 2008
). In this study, we demonstrate that the tyrosine-specific phosphatase TCPTP serves in a negative feedback loop for the inhibition of leptin signaling. We report that hypothalamic TCPTP expression is induced by leptin and that elevated leptin levels in obesity result in increased TCPTP to exacerbate the development of cellular resistance and progression towards morbid obesity. Therefore, our studies identify TCPTP as a member of regulatory triad that includes PTP1B and SOCS3 and functions to comprehensively attenuate the leptin response in obesity by inhibiting signaling at the receptor, JAK2 and STAT3.
Leptin exerts its effects on body weight via the activation of the tyrosine kinase JAK2 that phosphorylates STAT3 and in parallel activates the Ras/MAPK and PI3K/Akt pathways (Myers et al., 2008
). Importantly, STAT3 can elicit changes in Pomc
, but not Npy
expression (Bates et al., 2003
). In this study, we found that TCPTP attenuated leptin-induced STAT3, but not Akt or ERK1/2 signaling in cell culture systems in vitro
, and that TCPTP dephosphorylated STAT3 in the nucleus. Moreover, we found that leptin-induced hypothalamic STAT3 signaling and responses were elevated in neuronal cell-specific TCPTP-deficient mice in the ARC and VMH and that this was associated with changes in Pomc
, but not Npy
expression/secretion. Hence, our studies are consistent with TCPTP selectively regulating leptin-induced STAT3 signaling in vivo
. Previous studies have established that PTP1B, but not TCPTP dephosphorylates and inactivates JAK2 (Myers et al., 2001
; Simoncic et al., 2002
). Moreover, PTP1B and TCPTP can function cooperatively in cells in vitro
to regulate the phosphorylation of JAKs and STATs in the cytoplasm & nucleus respectively (Lu et al., 2007
; Lu et al., 2008
; Sharma et al., 2008
; Simoncic et al., 2006
). Accordingly, we propose that the two phosphatases act to co-ordinately regulate leptin signaling in vivo
: PTP1B attenuating JAK2 phosphorylation and possibly STAT3 in the cytoplasm, and TCPTP dephosphorylating STAT3 in the nucleus.
As reported previously for Nes
mice (Bence et al., 2006
), neuronal cell-specific TCPTP-deficient mice were proportionately smaller than their floxed littermates. Although recent studies by Briancon et al.
(Briancon et al., 2010
) have reported that some strains of Nes
-Cre mice exhibit a phenotype (being smaller than wild type mice), this was not evident in our studies. We noted no overt difference in body weight, leptin sensitivity or glucose homeostasis in hemizygous Nes
mice or Nes
mice, consistent with previous studies using the same Nes
-Cre strain (Bence et al., 2006
). Moreover, we found that the combined deficiency of PTP1B and TCPTP had additive effects on body weight and size. Our studies suggest that the size difference in Nes
mice might be due to perturbations in the GH/IGF-1 axis. Previous studies have shown that, PTP1B can dephosphorylate JAK2, STAT5 and the IGF-1 receptor (IGF-1R) in cells to attenuate GH and IGF-1 signaling (Aoki and Matsuda, 2000
; Gu et al., 2003
; Myers et al., 2001
), whereas TCPTP has the capacity to dephosphorylate STAT5 (but not JAK2 or IGF-1R) (Aoki and Matsuda, 2002
; Buckley et al., 2002
; Simoncic et al., 2002
). We found that GH and IGF-1 levels were decreased in TCPTP knockout (Nes
) mice and that hypothalamic STAT5 phosphorylation was enhanced in response to bolus GH administration. Increased STAT5 phosphorylation in the hypothalamus would inhibit GH release from the pituitary, thus perturbing the GH/IGF-1 axis (Becker et al., 1995
; Burton et al., 1992
; Minami et al., 1993
; Romero et al., 2010
). We suggest that the lower circulating GH and IGF-1 levels account for the decreased size and the increased relative adiposity in chow-fed Nes
mice, as GH promotes postnatal skeletal growth and lipolysis (Lichanska and Waters, 2008
). Recent studies have also reported that neuronal cell-specific PTP1B knockout mice have reduced circulating IGF-1 (Briancon et al., 2010
). We expect that the combined deficiency of PTP1B and TCPTP (in DKO mice) probably further decreases GH and IGF-1 levels, compounding the effects on body size.
Several lines of evidence support the conclusion that TCPTP acts as a key regulator of hypothalamic leptin signaling. First, TCPTP expression was increased transiently in the hypothalami of C57BL/6 mice in response bolus leptin administration and in LEPR-B expressing cells treated with leptin, consistent with TCPTP serving in a feedback loop. Second, despite the decreased ambulatory activity and increased relative adiposity, Nes-Cre;Ptpn2lox/lox mice had significantly lower circulating leptin levels and lower fasting blood glucose and insulin levels, consistent with enhanced leptin and insulin sensitivity. This is particularly striking and suggests that the impact of TCPTP deficiency on leptin sensitivity and the central control of glucose homeostasis overrides the increase in leptin production and the development of peripheral insulin resistance that would otherwise be expected with increased adiposity. Third, despite the decreased ambulatory activity, Nes-Cre;Ptpn2lox/lox mice exhibited increased oxygen consumption and energy expenditure (corrected for body weight) and decreased food intake as might be expected for enhanced leptin sensitivity. Fourth, the reduction in food intake and body weight after leptin administration was significantly enhanced by TCPTP deficiency in both chow and HFF mice. Fifth, hypothalamic STAT3 phosphorylation was significantly enhanced in the ARC and VMH after leptin administration coinciding with significant changes in Pomc and Agrp expression in Nes-Cre;Ptpn2lox/lox mice. The increase in leptin-induced signaling could be recapitulated in vitro in cells of neuronal origin after TCPTP knockdown. Sixth, TCPTP deficiency enhanced the leptin-induced α-MSH secretion from hypothalamic slices ex vivo, making any extrinsic influences that may be associated with the differences in body weight in the whole animals unlikely. Finally, the effects of TCPTP deficiency could be recapitulated by the icv administration of a specific TCPTP inhibitor. Inhibition of TCPTP in C57BL/6 mice enhanced leptin-induced hypothalamic STAT3 signaling and increased the effects of leptin on body weight and energy expenditure. Importantly, the inhibitor did not have any overt effect on leptin-induced responses in Nes-Cre;Ptpn2lox/lox mice, consistent with the inhibitor being specific for TCPTP. Taken together, these studies provide compelling evidence for TCPTP being a key regulator of central leptin sensitivity.
Our studies indicate that increased hypothalamic TCPTP contributes to cellular leptin resistance in obesity. In HFF C57BL/6 mice, increased hypothalamic TCPTP coincided with increased circulating leptin. The increase in TCPTP was preceded by increases in hypothalamic PTP1B and SOCS3 that coincided with elevated circulating TNF and IL-6. Zabolotny et al.
(Zabolotny et al., 2008
) have reported that hypothalamic PTP1B expression can be driven by TNF in vivo
, whereas hypothalamic SOCS3 expression can be induced by IKKβ/NFκB signaling (Zhang et al., 2008
), an effector pathway of TNF. Accordingly, we suggest that cellular leptin resistance and obesity develop along a continuum, with inflammation associated with high fat feeding at first promoting hypothalamic PTP1B and SOCS3 to attenuate JAK2 and reduce leptin sensitivity, to consequently increase adiposity and circulating leptin to promote hypothalamic TCPTP expression. The increased TCPTP would in turn dephosphorylate STAT3 to further attenuate the leptin response and contribute to the development of overt cellular leptin resistance and progression to morbid obesity. Although one might expect that the hyperleptinemia should solely compensate for the developing leptin resistance, it is possible that leptin signaling pathways and/or leptin responsive neurons may be differentially sensitive to leptin. In keeping with this possibility, some leptin functions, such as leptin’s cardiovascular effects, remain intact in the leptin-resistant state (Correia et al., 2002
; Rahmouni et al., 2005
). In the case of TCPTP, it is important to note that TCPTP has a long protein half-life (Bukczynska et al., 2004
) so that the sustained hyperleptinemia in obesity may be sufficient to promote and maintain TCPTP levels. However, we cannot exclude the possibility that in the obese state additional molecular factors, which themselves have no effect, contribute to the promotion of TCPTP expression. Nevertheless, consistent with increases in hypothalamic TCPTP and PTP1B coordinately contributing to the onset and progression of cellular leptin resistance, we found that the combined ablation of TCPTP and PTP1B had additive effects in protecting mice from high fat diet-induced weight gain. Briancon et al.
(Briancon et al., 2010
) have reported that the combined inactivation of PTP1B and SOCS3 in neuronal cells decreases adiposity and improves glucose tolerance, with minimal if any impact on high fat diet-induced, or age-associated weight gain. In our studies we found that the combined deletion of PTP1B and TCPTP had a pronounced effect on high fat diet-induced weight gain, but did not completely protect mice. The latter may be attributable to varied factors, among which include increases in SOCS3, the hedonistic attractiveness of a high fat diet, and/or gliosis and changes in the blood brain barrier that may impair leptin’s access to neurons in the ARC (El-Haschimi et al., 2000
; Horvath et al., 2010
The results of this study define the role of TCPTP in the central control of leptin signaling and delineate a negative feedback loop that functions together with PTP1B and SOCS3 for the attenuation of the leptin response. Importantly, our results indicate that increases in hypothalamic TCPTP may be causally linked to the attenuation of leptin sensitivity working in conjunction with PTP1B for the coordinated suppression of JAK2/STAT3 signaling and the promotion of cellular leptin resistance. Our studies underscore the highly specific nature of phosphatases such as PTP1B and TCPTP in vivo and highlight their capacity to work in concert for the comprehensive regulation of signaling networks and biological responses. Moreover, our findings indicate that the combined inhibition of PTP1B and TCPTP might be required for the effective alleviation of cellular leptin resistance in obesity, and therapeutic approaches currently aimed at targeting PTP1B should take this into consideration.