Previous electrophysiologic studies have painted an intricate but complex picture of hypothalamic energy balance circuitry. POMC neurons marked with an EGFP transgene are depolarized in response to both leptin (7
) and elevated extracellular glucose (20
); however, a subset of unmarked hypothalamic neurons selected for the same type of glucose responsiveness was found to be hyperpolarized by leptin (26
). Our findings provide a new perspective with respect to the nature of both the response and the circuitry, in which PI3K may contribute to the activation of both Agrp and POMC neurons, but the direction of its regulation by leptin depends on whether the action is mediated directly or indirectly (Figure C). Thus, reciprocal regulation of PI3K by leptin provides a unifying hypothesis to explain how this hormone exerts opposing effects on the activity of these 2 key subsets of neurons.
An important implication of our findings has to do with the notion that insulin and leptin play similar roles as peripheral indicators of energy balance. This idea was initially based on observations that insulin circulates and enters the brain at a level proportional to the body fat stores and has been supported by observations that central administration of insulin inhibits food intake and reduces body weight (reviewed in refs. 1
) and that neuron-selective insulin receptor knockout mice develop moderate diet-induced obesity (27
Our results suggest that parallel effects of leptin and insulin on energy balance could be integrated at the level of POMC neurons, where both hormones activate PI3K, and leptin has been shown to cause membrane depolarization and an increased firing rate. Although previous studies by Ashford and colleagues (11
) identified a subset of hypothalamic neurons in which the effects of leptin or insulin on membrane hyperpolarization could be blocked by PI3K inhibitors, our results do not distinguish whether changes in POMC PI3K are independent of or causally related to changes in POMC electrical activity. Both phenomenon occur rapidly and may contribute to acute responses such as neuropeptide release and rapid changes in feeding behavior.
However, our observation that insulin and leptin have opposite effects on PI3K activity in Agrp neurons suggests that the functions of these 2 hormones as energy balance signals do not overlap completely. Indeed, differential effects of leptin and insulin on Agrp expression have been described previously in the setting of streptozotocin-induced diabetes, in which leptin, but not insulin administration, normalizes Agrp expression (28
). Furthermore, the effects of insulin on neuronal growth and survival exceed what might be expected for a simple adiposity signal, with widespread expression of insulin receptors in many brain regions (30
) and a connection between insulin action and neurodegeneration (31
). Finally, leptin and the melanocortin system are specific to vertebrates while the effects of insulin on the brain are conserved across all metazoans, with genetic studies in invertebrates pointing to an ancient role for insulin in energy balance as well as aging and growth (33
). From this perspective, leptin may serve as a specialized energy balance signal invented during vertebrate evolution whose effects partially overlap with a more ancient role for insulin.
In addition to the differential effects of leptin on PI3K activity in POMC and Agrp neurons, our results indicate that the circuits underlying these effects also differ, since the inhibitory effect of leptin on PI3K in the majority of Agrp neurons requires synaptic transmission. However, previous neuroanatomical studies by us and by others have demonstrated that a subset of Agrp neurons (or Npy neurons in the arcuate nucleus) expresses the long form of the leptin receptor (35
). This apparent paradox — an indirect response to leptin despite the presence of leptin receptors on some Agrp neurons — could be explained if Agrp neurons require multiple signals to activate PI3K in response to leptin withdrawal, some that are mediated autonomously via a leptin receptor and others that are mediated via synaptic transmission. Alternatively, there may be functional heterogeneity within Agrp neurons if, for example, the subset of Agrp neurons that express the leptin receptor are distinct from those that activate PI3K in response to leptin withdrawal. If so, this latter group of neurons might receive leptin-mediated inhibitory signals from POMC or other neurons via γ-aminobutyric acid (GABA) (40
) or other inhibitory neurotransmitters (Figure C).
Heterogeneity of POMC neurons might also help to explain the observation that leptin activation of PI3K in POMC neurons does not require Stat3 phosphorylation. A recent neuroanatomical study reported that 37% of POMC cells were immunopositive for phospho-Stat3 after leptin treatment (41
), and in our imaging system, leptin activated PI3K in approximately 60% of the POMC neurons. Thus, leptin receptor engagement on distinct subsets of POMC neurons could activate different downstream effectors. Regardless, these results together with earlier work (7
) suggest a view of leptin signaling in which there are 2 types of direct intracellular effectors with different actions and possibly different mechanisms of activation. One type, exemplified by Stat3, is likely to mediate responses that require changes in gene expression, whereas a second type, exemplified by the direct effects of leptin on PI3K activation and electrical activity in POMC neurons, is likely to mediate acute responses such as neuropeptide release and rapid changes in feeding behavior. In contrast to Stat3, whose mechanism of activation by leptin receptor engagement is well characterized (21
), the biochemical mechanisms by which leptin activates PI3K and modulates membrane potential in POMC neurons are not yet clear. However, the two may be causally related, since depolarization of POMC neurons by leptin involves a nonselective cation channel (7
) and PIP3 has recently been shown to be capable of modulating ion channels (44
). In addition, work of Myers and colleagues (6
) has shown that animals carrying a modified leptin receptor that cannot activate Stat3, Lepr1138Ser
, develop a subset of the same phenotypic abnormalities caused by a null Lepr
allele; thus, it will be interesting to examine the effects of leptin on PI3K activation and electrical activity in POMC neurons that carry the Lepr1138Ser
We chose to focus on POMC and Agrp neurons because of previous work suggesting they serve as primary sensors for circulating adiposity signals. Balthasar et al. (46
) recently demonstrated that animals with POMC-specific Lepr
deletion develop a mild obesity and metabolic derangement. To the extent that results from different studies can be compared, the effect of the POMC-specific Lepr
deletion appears to be less dramatic than that of a complete Pomc
, or Lepr
). These observations suggest that POMC neurons use mediators and/or signaling mechanisms in addition to leptin to control body weight and, furthermore, that the effect of leptin on CNS control of body weight involves neurons in addition to those that express Pomc
. In this regard, comparison of POMC-specific Lepr
deletion mice with the POMC-specific Stat3
deletion mice could reveal the extent to which leptin signaling in POMC neurons is mediated by Stat3 or other effectors such as PI3K.
The approach introduced here — a combination of transgenic biology and gene therapy that enables dynamic histochemical measurements of neuronal cell signaling recorded by multiphoton microscopy of brain slices — can be applied to additional signaling events and to diverse pathophysiologic problems in which the brain measures and responds to changes in environmental parameters including pH, temperature, glucose concentration, and cytokine levels. Advances in image processing and miniaturization should allow events from multiple neurons to be recorded simultaneously from living animals, an advance that could help in the dissection of molecular and cellular events that underlie complex behaviors.