The current study explored the role of ROCK1 in the regulation of food intake and energy metabolism, focusing on the possibility that hypothalamic ROCK1 activation is required for normal body-weight regulation. Although ROCK1 has not previously been suspected to play a role in control of energy balance, our data demonstrate the necessity of hypothalamic ROCK1 in the regulation of food intake and normal body-weight homeostasis, by phosphorylation and activation of leptin receptor-associated JAK2. Thus, we identify ROCK1 as a key regulator of adiposity, and a potentially novel therapeutic target for the prevention and treatment of obesity.
Our experimental evidence demonstrates that leptin-dependent JAK2 tyrosine phosphorylation is markedly reduced by inactivation of ROCK1, suggesting that ROCK1 acts at a proximal step in the LepRb signaling pathway., We also show that upon leptin stimulation, ROCK1 physically interacts with JAK2, but not LepRb, the initial crucial component in intracellular LepRb signal transduction. Tyrosine phosphorylation of JAK2 proteins in ROCK1-JAK2 complexes is greatly elevated in samples from leptin-treated cells or hypothalamus tissues indicating enhanced JAK2 enzymatic activity. It is therefore predicted that all LepRb-pathways downstream of JAK2 are influenced by ROCK1, including Stat3 and FOXO1 regulation, both of which are impaired in cells lacking ROCK1 activity during leptin stimulation. In this regard, it should be noted that leptin-mediated Stat3 tyrosine phosphorylation in ROCK1 knockdown GT1-7 cells and in POMC neurons lacking ROCK1 is impaired by ~50% when JAK2 phosphorylation is nearly abolished. The discrepancy may be due to the fact that leptin-induced Stat3 phosphorylation can occur in the absence of JAK2, presumably via Src and Fyn signaling31
. Thus, it is likely that the partial inhibition of leptin-stimulated Stat3 phosphorylation by ROCK1 loss is due to JAK2-independent leptin signaling.
A crucial metabolic signal is the fat-derived anorexigenic hormone leptin, which directly activates hypothalamic POMC neurons, an important site for the regulation of energy homeostasis13,30
. We thus investigated the role of ROCK1 in POMC-expressing neurons. Interestingly, the metabolic phenotypes of POMC neuron-specific ROCK1-deficient mice are nearly identical to those of POMC neuron-specific LepRb-deficient mice13
. For example, the degree of obesity (~10%) seen in POMC-Cre, ROCK1flox/flox
mice is similar to that seen in POMC-Cre, LepRbflox/flox
mice, and also consistent with the recent findings that restoration of LepRb in only POMC neurons in LepRb-deficient mice causes a ~10% decrease in body weight relative to male Leprdb/db
control mice of same age29
. Thus, ROCK1 signaling in POMC neurons is a key downstream regulator of LepRb signaling that is necessary for the homeostatic control of body weight. Furthermore, the degree of obesity caused by ROCK1 deficiency is consistent with the possibility that ROCK1 is a key cellular mediator of LepRb signaling in POMC neurons.
An important function of leptin is to modulate the function of specific neuronal subtypes by altering neuronal electrical activity in hypothalamic neurons23,30
. Electrophysiological studies have shown that leptin depolarizes and increases the firing rate of POMC neurons31
and inhibits the tone of AgRP neurons9,30
. Specifically, leptin’s effect on POMC neuronal activity is thought to be mediated via the PI3K signaling pathway22
. In addition to this current dogma, our data suggest the requirement of ROCK1 in leptin-induced neuronal activity of POMC neurons, as revealed by the ablation of leptin’s effects on membrane potential and firing rate in the absence of ROCK1. These results are similar to findings in mice lacking PI3K in POMC neurons, raising the possibility that both ROCK1 and PI3K activate the same signaling node to regulate neuronal activity. This notion is further supported by our findings showing that the PI3K-FOXO1 pathway is regulated via a JAK2/ROCK1-dependent signaling mechanism. Thus, we propose that ROCK1 is a key upstream regulator of PI3K and that ROCK1-PI3K activation is necessary for the control of leptin-dependent electrical activity in the hypothalamic POMC neurons, establishing a new ROCK1-PI3K signaling axis in neuronal leptin action.
It is yet unclear to what extent POMC neuronal activity is required for the anti-obesity actions of leptin. Hill et al reports that PI3K signaling in POMC neurons is not a key contributor of long-term body-weight regulation despite its critical role in acute control of POMC neuronal activity22
. Our study demonstrates that modulation of ROCK1 in POMC neurons has a significant impact on both long-term body-weight homeostasis and on neuronal activity. If indeed it is the case that increased firing of POMC neurons by leptin does not influence long-term energy balance as surprisingly reported by Hill et al, it has to be concluded that the increased adiposity of POMC-Cre, ROCK1flox/flox
mice is caused by dysregulation of signaling pathways other than the PI3K pathway. For example, it is possible that the observed impaired leptin-activated Stat3 signaling in POMC-Cre, ROCK1flox/flox
mice is responsible for the obesity of these animals, since global deletion of the Stat3 activation site on LepRb causes massive obesity32
. On the other hand, selective deletion of Stat3 in POMC neurons only causes mild obesity in female mice33
, whereas we show obesity in both males and females. This might suggest that the deletion of ROCK1 affects several intracellular pathways in addition to Stat3. This possibility is consistent with our data showing that ROCK1 acts at the level of JAK2, the key upstream mediator of most if not all LepRb signaling pathways.
Given that inhibition of ROCK1 expression or activity impairs leptin signaling, it is hypothesized that ROCK1 deficiency would block leptin signaling in AgRP neurons, thereby leading to the impaired metabolic action of leptin. As a result, mice lacking ROCK1 in AgRP-expressing neurons would be expected to be obese, as our data show. Taken together with our data showing ROCK1 deficiency in POMC neurons impairs leptin but not insulin stimulated neuronal activity, these data suggest that ROCK1 regulation of AgRP (and POMC) neuron functions in body weight homeostasis is leptin-specific, rather than a general perturbation of AgRP neuronal physiology.
Considering that other neurons and sites in the hypothalamus are also involved in leptin’s regulation of normal body weight6
, it is hypothesized that ROCK1 deletion in wider regions of the hypothalamus can further accelerate the development of adiposity compared to deletion of ROCK1 in arcuate POMC or AgRP/NPY neurons. Consistent with this, our data show that a dramatic increase (~30%) in body weight is observed when ROCK1 is deleted in the hypothalamic regions that include ARC (possibly containing POMC, AgRP/NPY, and other neurons), VMH and DMH. The relative magnitude of increased body weight is ~3-fold compared to that of POMC neuron-specific ROCK1-deficient mice at the age of ~20 weeks, highlighting the importance of ROCK1 in other regions of the hypothalamus for energy balance. Supporting this, it has been shown that LepRb deficiency in both POMC and AgRP neurons (within ARC region) or in both POMC and SF-1 neurons (ARC and non-ARC region) has additive effects on body weight14,34
. Whether deficiency of ROCK1 on these neurons has additive or synergistic effects on body-weight gain is yet to be determined.
It should be noted that the metabolic phenotypes resulting from hypothalamic ROCK1 knockout or knockdown may also involve changes in other intracellular signaling pathways such as insulin or BDNF, both of which stimulate PI3K signaling20,35
. Indeed, our previous work demonstrates that insulin’s ability to activate PI3K is impaired by ROCK1 deficiency in skeletal muscle20
. Our data also do not establish the exact role of ROCK1 activity in normal physiological or pathophysiological leptin action. While ROCK1 activity was increased in the hypothalamus upon supraphysiologic leptin stimulation, it was not changed by acute physiological fasting (24 hr) or refeeding (2 hr). These data imply that though hypothalamic ROCK1 is required for intact leptin signaling, dynamic alterations in ROCK1 level or activity may play a limited role in acute changes in energy balance under physiologic or pathophysiologic circumstances. Further investigations are therefore needed to clarify this important question.
In conclusion, this study demonstrates that ROCK1 is an important positive regulator of leptin receptor signaling in hypothalamic neurons. Upon leptin stimulation, ROCK1 is activated and rapidly phosphorylates JAK2 on serine residues, which in turn promotes downstream signaling pathways of leptin, including Stat3 and PI3K signaling, ultimately leading to control of energy balance (Supplementary Fig. 8c
). We thus propose that ROCK1 will need to be posted as a new component of the current leptin signaling paradigm in the hypothalamus. This model provides a new mechanism that advances our understanding of central leptin action and body weight regulation.