Leptin resistance is the hallmark of DIO. The underlying mechanisms are not clearly delineated, and several defects likely contribute to leptin resistance (Morrison et al., 2005
; Myers et al., 2008
). One consequence of HF feeding is a resistance to central administration of leptin, and this cellular leptin resistance has been identified to be associated with impaired leptin signaling events within in specific subpopulations of hypothalamic neurons in mice (Munzberg et al., 2004
; Metlakunta et al., 2008
). One study in C57BL/6J mice indicated that in as early as 4 days of HF feeding, leptin-mediated STAT3 phosphorylation is impaired in the ARC (Munzberg et al., 2004
), yet with no evidence of leptin resistance in other hypothalamic brain regions with up to 16 weeks of HF feeding. In contrast, another study in FVB/N mice reported no decline in leptin-mediated STAT3 signaling after 4 weeks of HF feeding, but leptin resistance in four hypothalamic regions after 19 weeks (Metlakunta et al., 2008
). Neither study examined regions outside the hypothalamus. Recently, leptin function in energy homeostasis has been linked to regions outside the hypothalamus, in particular, the mesolimbic dopamine system in the ventral tegmental area (VTA) of the midbrain reward circuitry (Fulton et al., 2000
; Hommel et al., 2006
). Leptin function in this brain region is associated with sweet preference (Hommel et al., 2006
) and has a potential role in the dietary preference for HF food (Scarpace et al., 2010
). These observations predict that this region may be subject to dietary-induced leptin resistance. The present study revisits dietary-induced selective leptin resistance by examination of leptin-mediated STAT3 signaling both in specific hypothalamic regions as well as the VTA. Leptin signaling was assessed following a single icv injection of a pharmacological dose of leptin. Previous studies detailing the dose-response leptin-stimulation of hypothalamic STAT3 phosphorylation indicated that the Kact (concentration that results in half-maximal activation) for leptin is 41ng with a maximum stimulation achieved at 100 ng following i.c.v. injection (Scarpace et al., 2001
). The present study used a dose 10 times this level thus likely achieving maximal stimulation of leptin receptor across the brain areas examined. Our findings in rats support earlier evidence in C57BL/6J mice that within the hypothalamus, HF induced-leptin resistance is limited to the ARC region of the hypothalamus. In addition, we identified dietary induced cellular leptin resistance within the VTA. The previous study in FVB/N mice found that occurrence of leptin resistance in other hypothalamic regions that was depended on the length of the HF feeding (Metlakunta et al., 2008
). In our study, it is unlikely that the lack of leptin resistance in other medial basal hypothalamic regions examined was due to the length of exposure to the HF diet. These rats were HF fed for greater than 6 months, a feeding period longer than both of the previous studies in mice.
Leptin resistance is generally believed to have a causative role in obesity and is demonstrated to predispose rodents to subsequent HF-induced obesity (Scarpace and Zhang, 2009
). For instance, in rats with pre-existing leptin resistance due to chronic overexpression of leptin or age-related leptin resistance, subsequent exposure to a HF diet exacerbates food consumption and weight gain compared with HF-fed leptin responsive counterparts (Scarpace et al., 2005
; Judge et al., 2008
). The known role of VTA leptin receptor activity in consumption of sugar (Hommel et al., 2006
) and the link between HF-dietary preference and VTA leptin signaling (Scarpace et al., 2010
) indicates a role for VTA leptin function in tempering the consumption of palatable foods. Disruption in leptin function in the VTA would then predict increased vulnerability to HF-induced weight gain. Consistent with this idea is the observation in the present study that the preference for a 60% HF/7% sucrose diet diminished by the third day in the control group but not in the rats with leptin-induced leptin resistance. The identification of HF-induced leptin resistance in the VTA, in addition to the leptin resistance in the hypothalamus, provides one potential mechanism, underlying the increased susceptibility of leptin resistant rats to HF-induced obesity.
Interestingly, in this study, the degree of HF feeding induced leptin resistance is rather mild. Even in the ARC and VTA, the two regions in which leptin resistance was identified, there was still considerable leptin induced phosphorylated STAT3 in both the chow and HF fed rats. The extent of obesity in rodents lacking leptin or leptin receptor function exceeds that in rats with the dietary induced leptin resistance. Indeed, this observation first led to the hypothesis that HF-induced leptin resistance may be selective, and that some signaling must remains active in DIO rodents (Munzberg et al., 2004
). The present data indicates that not only is HF-induced leptin resistance selective, but in those areas effected, it is incomplete, at least with respect to leptin-mediated STAT3 signaling. These findings are consistent with discrepancy in the degree of obesity with HF feeding and genetic models of obesity.
This study also examined regional signaling with leptin resistance induced by chronic overexpression of leptin. This form of leptin resistance develops gradually over time and is characterized by an absence of anorexic and weight reducing responses to centrally administered leptin (Scarpace et al., 2002b
) and diminished leptin-mediated STAT3 signaling in the hypothalamus (Scarpace et al., 2005
). The onset of leptin resistance in the present study is consistent with the absence of the increased WR activity over time with leptin overexpression. Surprisingly, the regional pattern of leptin resistance was distinctly different than that with HF-induced obesity. With chronic leptin overexpression, cellular leptin resistance was observed in every brain region examined including the VTA and in one region, the VMH, no leptin mediated signaling was detected in the rat with chronic leptin overexpression. Moreover, the degree of leptin resistance was substantially greater when compared with that observed with the 6-month HF feeding period. Possibly, the length of leptin overexpression (452 days) compared with 190 days of HF feeding accounts for the differences in degree and regional leptin resistance.
Worthy of note is the apparent increase in STAT3 phosphorylation in the ACSF injected rats with rAAV-leptin treatment compared with control vector counterparts. We generally observe this increase STAT3 signaling following rAAV-leptin administration and have confirmed this represents increased leptin receptor-mediated activation. The increased STAT3 phosphorylation with rAAV-leptin is completely reversed by subsequent infusion of a leptin receptor antagonist (Scarpace et al., 2007
). Thus, this increase in rAAV-leptin mediated STAT3 signaling is contributing evidence that the transgene-produced leptin is reaching the appropriate target sites in the brain. Moreover, this increased STAT3 phosphorylation with rAAV-leptin does not appear to be coupled to metabolic responses. We demonstrated that infusion of a leptin antagonist in rats with chronic leptin overexpression did not induce any increase in food consumption or body weight (Scarpace et al., 2007
). The implication is that downstream elements in the leptin receptor pathway may also have an important role in the mechanisms underlying leptin resistance.
Leptin receptor activation initiates a cascade of signaling events including specific phosphorylation of several tyrosine residues on the receptor. Of these, phosphorylation of Tyr 1138 recruits and promotes STAT3 phosphorylation, and this step is generally believed to be critical for the leptin mediation of feeding and energy regulation (Villanueva and Myers, 2008
). This would predict that the near complete absence of STAT3 phosphorylation in the leptin-induced leptin resistance rats would render them readily susceptible to obesity. In fact, leptin overexpression does not result in obesity as long as the rats are maintained on a chow diet, although such leptin resistant rats display exacerbated HF-induced obesity (Scarpace et al., 2005
). This is in contrast to that what is observed with a generalized sub-maximal central leptin receptor blockade. In a previous study, a partial blockade of leptin receptor activity in the brain was achieved by central overexpression of a leptin mutant, that acts as a dominate negative antagonist (Zhang et al., 2007
; Matheny et al., 2009
). This partial central receptor blockade was sufficient to elevate obesity on a chow diet under conditions both when food consumption was augmented or unchanged (Matheny et al., 2009
). This previous data suggest that unrestrained leptin receptor activity is critical for energy homeostasis, and that even a sub-maximal but global disruption of leptin receptor function is sufficient to induce obesity on a diet of standard chow. Thus, this global but partial leptin receptor disruption is more obesogenic than the multi-site and nearly complete loss of leptin-mediated STAT3 phosphorylation induced by leptin overexpression. Such data imply that additional leptin receptor containing sites are responsible for the ability of these rats to maintain normal energy homeostasis in the face of leptin overexpression and disruption of STAT3 signaling in the hypothalamus and VTA or that leptin receptor mediated signaling other than STAT3 is assuming that role in the examined rat model.
It is likely that HF- or leptin-mediated leptin resistance involves multiple defects, but none that matches the totality of leptin receptor blockade or genetic obesity such as the Ob/ob mice or obese fa/fa Zucker rats (Frederich et al., 1995
; da Silva et al., 1998
). In addition, the role of other leptin-stimulated pathways may not yet be fully delineated. Leptin stimulated PI3 kinase activity is diminished in the hypothalamus with chronic leptin treatment (Sahu and Metlakunta, 2005
; Metlakunta et al., 2008
) and HF feeding. In addition, the leptin decrease in AMP kinase activity in the hypothalamus is tempered with DIO (Martin et al., 2006
). However, at the present, there is no data on the consequences of HF feeding on region-specific leptin-mediated signaling involving these pathways. In addition, it is unknown whether downstream events in the STAT3 signaling pathway are also impaired in a region-specific manner with HF feeding.
In summary, this report demonstrates that HF feeding results in a regional specific decline in leptin-mediated STAT3 phosphorylation that is limited to the ARC and VTA. Despite this HF-induced leptin resistance, there remained robust residual leptin stimulated signaling in all regions. In contrast, with chronic leptin overexpression, leptin signaling was severely impaired in all areas examined and was extinguished in the VMH. The defective signaling in the ARC and VTA likely contribute to the increased susceptibility to HF-induced obesity in the leptin-resistance state, and suggest that these brain regions are favorable targets for restoration of leptin signaling to prevent or temper DIO.
- High fat feeding decreases leptin mediated STAT3 signaling in select brain regions.
- Diminished signaling was evident in arcuate nucleus and ventral tegmental area.
- Unchanged signaling in lateral, ventromedial, and dorsomedial hypothalamus.
- Leptin overexpression diminished leptin signaling in all brain regions examined.