We have shown that central administration of GM-CSF reduced food intake and decreased body weight in rats. Body weight loss was greater in GM-CSF–treated than vehicle-treated animals, even when food was withheld for 24 hours following treatment, suggesting that energy expenditure increased as a result of central GM-CSF signaling. Correspondingly, loss of GM-CSF expression caused late-onset obesity in mice, with a nearly 3-fold increase in body fat and decreased energy expenditure. Taken together, these studies indicate that GM-CSF can influence energy balance. Central administration of GM-CSF decreased energy intake and increased energy expenditure, while loss of GM-CSF signaling increased energy intake and decreased energy expenditure.
Like leptin, central administration of GM-CSF produces weight loss via potent effects on food intake that are not secondary to motor impairment or illness (25
). In fact, GM-CSF is considerably more potent than leptin on a molar basis in this regard, with a threshold dose approximately 8-fold lower than that of leptin administered in a similar paradigm (26
). As is the case with leptin, GM-CSF receptors are found in the CNS in neurons in the ARC of the hypothalamus (27
). However, unlike the LepR, significant GM-CSF receptor immunoreactivity is also found in the PVN. Although milder than those occurring with leptin deficiency, GM–/–
mice have pronounced increases in adiposity, resulting from increased food intake and decreased energy expenditure.
Central administration of GM-CSF significantly reduced NPY and AgRP expression in rats. Plasma leptin levels were decreased 24 hours after receiving central GM-CSF administration, even in ad libitum–fed animals. This time point coincides with maximal weight loss after injection and is consistent with the hypothesis that a significant part of the weight loss is adipose.
Several lines of evidence indicate that while GM-CSF does circulate, many of its functions are the result of paracrine actions. For example, GM-CSF promotes maturation of myeloid cells within the bone marrow, stimulates macrophage chemotaxis and phagocytosis of bacteria at the site of infection, and regulates pulmonary surfactant catabolism within cells of the alveolar lumen (4
). Expression in endothelial cells is upregulated in response to local signals and attracts leukocytes to migrate through vessel walls and infiltrate damaged or inflamed tissues (33
). In the present studies, we found that even very high doses of GM-CSF delivered peripherally did not produce any reductions in food intake. This is consistent with the hypothesis that, like GM-CSF’s other functions, its role in energy balance may also be paracrine. One possibility is that GM-CSF may be made in the CNS by neurons or other CNS cell types to act upon hypothalamic GM-CSF receptors.
Leptin enters the CNS via an active receptor-mediated uptake system (34
), and while some evidence indicates that peripherally derived GM-CSF enters the CNS (35
), our data do not support a role for peripherally derived GM-CSF in energy balance. In both fed and fasted conditions, circulating GM-CSF did not reach detectable levels, and peripheral administration that resulted in plasma levels greater than those associated with inflammation did not influence food intake or body weight (36
). Rather, our findings suggest that cells of the CNS are more likely the critical source of the GM-CSF that interacts with the CNS receptors we have identified.
Our immunohistochemistry and in situ findings identified neuronal expression of GM-CSF receptors in the PVN and ARC. These findings are consistent with those demonstrating immunofluorescence in neurons of the PVN and suggest that GM-CSF can act directly on neurons. GM-CSF receptor expression in neural tissues has been well documented in several studies of fetal neurons, neuronal cell lines, and isolated microglial cells and astrocytes (13
). Since GM–/–
mice are completely lacking in GM-CSF expression during development, as well as adulthood, it is possible that a subset of GM-CSF–expressing CNS cell type is absent in these mice. An important goal will be to completely map expression of GM-CSF receptors in the CNS, including identifying the critical cell types that produce the receptors in hypothalamus.Future studies will include in situ hybridization and immunohistochemical staining of other regions known to project to the hypothalamus, including nucleus accumbens and regions of the brain stem.
One of the key questions raised by our studies is the source of CNS GM-CSF expression. The present study included amplification of GM-CSF mRNA in brain tissues and indicates that GM-CSF expression is widespread throughout the CNS. However, the precise source of endogenous GM-CSF that would influence energy balance remains unknown. GM-CSF expression has been reported in oligodendrocytes, microglial cells, and macrophages in the brain, as well as endothelial cells of the vessels in neural tissues. There is also evidence that GM-CSF levels in spinal fluid change in Alzheimer disease, chronic fatigue syndrome, and spinal cord injury (38
). Induction of GM-CSF expression is thought to be vital to initiation of the proinflammatory cytokine cascade that follows spinal cord injury (40
). Our original hypothesis was that GM-CSF is produced as part of the inflammatory state associated with adipose tissues in obese individuals, and we predicted that GM-CSF would be another cytokine link between peripheral inflammation and central energy regulation. While large doses of peripheral GM-CSF had no effect on food intake or body weight in these studies, this does not rule out a peripheral mechanism for GM-CSF in regulating adipose mass.
In this study, we found that GM–/– mice show a pronounced body fat phenotype. The visceral fat pads were grossly larger and heavier than those from wild-type mice. In addition, the s.c. fat depot was visibly enlarged in the GM–/– mice. This suggests that while lipid storage in the GM–/– mice was increased, the fat distribution pattern remained unchanged. We measured M-CSF expression in mesenteric fat, since it is known to stimulate adipogenesis. However, M-CSF was expressed at a lower level in GM–/– mice than it was in GM+/+ mice. Thus, while M-CSF may contribute to adipogenesis in wild-type animals, it is unlikely to be involved in the mechanism that increases fat expansion in the absence of GM-CSF signaling. The current data, however, cannot rule out the possibility that some other intermediary molecule compensates for GM-CSF signaling and stimulates adipogenesis.
Gene targeting has produced 2 distinct strains of GM-CSF–null mice and 2 of GM-CSF βc
receptor–deficient mice, yet this is the first study we know of describing alterations in energy homeostasis in association with disrupted GM-CSF signaling. Cellular substrates of GM-CSF–activated kinase activity are components of 3 distinct signaling pathways, including the JAK/STAT, PI3K/AKT, and Ras/MAPK cascades (41
). In the JAK/STAT pathway, ligand binding induces phosphorylation of JAK2, which in turn induces phosphorylation of the βc
subunit. STAT5A and STAT5B Src homology 2 domains bind to the phosphorylated sites on the βc
cytoplasmic tail. The STAT proteins then are phosphorylated by JAK2 and dimerize. Studies by Rosen et al. demonstrated that STAT5A homodimers and STAT5A–5B heterodimers form in monocytes in response to GM-CSF (42
). In addition, STAT3 is activated by GM-CSF in hepatocytes and polymorphonuclear leukocytes (43
). Since the STAT family has also been linked to the effects of leptin and insulin in regulating energy balance (45
), one possibility is that GM-CSF influences food intake and energy expenditure via similar intracellular pathways.
Obesity remains among the most daunting public health problems facing the developed and developing worlds. Increased obesity will inevitably result in increased rates of type 2 diabetes, heart disease, and some cancers. The current rise in obesity rates is particularly troubling since existing therapies are at best moderately successful. Thus, there is pressing need for more understanding of how energy balance is regulated and how it might be dysregulated to produce obesity. The current work points to a possible role for GM-CSF acting in the CNS to regulate energy balance. This opens up possibilities for new insights about how body adipose stores are regulated by proinflammatory cytokines and how we might produce biologically meaningful treatments that result in significant and sustained weight loss.