A number of pieces of in vitro
data suggest that the NG2 proteoglycan promotes the proliferation and motility/recruitment of immature cells and tumor cells, acting via potentiation of signaling through β1 integrins and growth factor receptors 
. This function of NG2 is borne out by in vivo
studies on the NG2 null mouse that reveal deficits in the development of cell types that are normally positive for NG2. Studies of pathological microvessels in the NG2 null mouse demonstrate reduced recruitment of pericytes and reduced pericyte ensheathment of endothelial cells 
. In the central nervous system, oligodendrocyte progenitors in the NG2 null mouse do not proliferate normally, resulting in production of reduced numbers of mature oligodendrocytes and deficits in myelination 
. In early postnatal skin, generation of both keratinocytes and white adipocytes is sub-normal in the NG2 null mouse, leading to transient deficits in skin development 
In light of these developmental deficits, it has been surprising to discover that NG2 null mice develop adult-onset obesity, with adult males and females weighing as much as 20% more than wild-type counterparts due to white adipose deposits that can be up to twice as large as corresponding wild-type deposits. The increased fat pad size is due at least in part to a 20% increase in the diameter of white fat cells, indicating an increase in lipid storage in NG2 null mice. This increased storage may be linked to increased serum lipid levels in the NG2 null mouse, a factor that may also explain the occurrence of severe liver steatosis in older males. Male NG2 null mice also exhibit mild diabetic symptoms, including glucose intolerance and insulin resistance. The hypertrophy of white adipose tissue in NG2 null mice stands in vivid contrast to the hypotrophic phenotypes of other tissues studied in these mice, including early postnatal white adipose tissue 
. This paradox suggested the possibility that the unexpected effect of NG2 ablation on white fat might be an indirect one caused by NG2 ablation in some other cell type.
One scenario was that NG2 ablation in OPCs in the CNS might affect hypothalamic control of metabolism due to deficits in neuronal impulse conduction resulting from hypomyelination of axons. This possibility was ruled out by the finding that OPC-specific ablation of NG2 yielded a phenotype opposite to that seen in the global NG2 null mouse; namely, the OPC-specific NG2 null mice were leaner than control mice with two-fold smaller epididymal fat pads. An alternative clue regarding the basis of the global NG2 null phenotype was provided by metabolic studies that revealed decreased production of CO2 and decreased consumption of O2 in NG2 null mice, in spite of food intake and activity levels that matched those of wild-type mice. These findings suggested an alteration in the balance between energy expenditure and energy storage in NG2 null mice. Since energy homeostasis depends heavily on the function of brown adipose tissue, we looked for evidence of impaired brown fat function in NG2 null mice. Indirect evidence for such a defect was seen in the performance of NG2 null mice when challenged with cold exposure or high fat diet feeding. In both types of tests, NG2 null mice failed to maintain normal body temperature, diagnostic of impaired adaptive thermogenesis, a process that depends on efficient brown fat function.
More direct evidence for deficits in brown fat function in the cold challenge test was obtained by examining levels of transcripts for the uncoupling protein UCP1 and the cold-inducible transcriptional activator PGC1-α, both of which are normally highly up-regulated in response to cold exposure 
. Up-regulation of both UCP1 and PGC1-α were reduced in NG2 null mice following cold exposure. Changes in levels of transcripts for the β3 adrenergic receptor were not affected by NG2 ablation, showing that effects of NG2 ablation on UCP1 and PGC1-α are not due to NG2-dependent alterations in adrenergic receptor levels. From a developmental standpoint, interscapular brown fat from postnatal day 5 NG2 null mice exhibited reduced lipid content, although BAT mass was similar to wild-type controls. More importantly, NG2 null IBAT exhibited reduced levels of transcripts for both PGC1-α and PRDM16, factors that are required for brown fat development 
. In parallel, we were able to demonstrate a large deficit in the ability of NG2 null brown pre-adipocytes to undergo differentiation in cell culture. As seen in vivo, NG2 null brown pre-adipocytes also exhibited great reduced levels of PGC1-α and PRDM16.
A recurring theme in our data is that various deficits associated with brown fat function occur prior to the time at which NG2 null mice begin to exhibit excess weight gain. This is especially true of the adipogenesis assays performed with brown adipocytes isolated from postnatal day 5 mice and of the PGC1-α and PRDM16 transcription assays performed with both whole tissue and cultured adipocytes at this same early time point. It is also true of the metabolic and cold challenge tests performed with 13-week old mice. Taken together, these findings provide a firm basis for concluding that excess weight gain is unlikely to be responsible for the brown fat dysfunction observed in NG2 null mice. Instead, it is much more likely that impaired brown fat development and/or function underlies the obese phenotype of the NG2 null mouse. Impaired energy expenditure in the NG2 null mouse likely leads to increased storage of excess lipids by white adipocytes, resulting in the observed increase in adipocyte size and fat pad size. This NG2-dependent deficit in brown fat development is more in line with our general “rule” that NG2 is normally involved in expansion of immature cell populations and that its ablation results in subnormal development. In this case, deficits in BAT development/function have indirect hypertrophic effects on white adipocytes that appear to override the direct early hypotrophic effect of NG2 ablation on this population. In light of recent evidence concerning the importance of BAT in adult humans, this interplay between BAT and WAT is of significant interest.
Additional work is required to understand the mechanism by which NG2 ablation affects both adaptive thermogenesis and brown adipocyte development. Both processes are dependent on activation of β3 adrenergic receptor, leading to cAMP-mediated activation of protein kinase A (PKA) 
. In the former case, PKA activation stimulates lipolysis and UCP1 activation to produce heat. In the latter case, PKA activation mediates phosphorylation of the cAMP responsive element binding protein (CREB) to induce differentiation of brown pre-adipocytes 
. It is therefore tempting to speculate that, as a membrane-spanning protein, NG2 could be involved in potentiating the activity of either the G protein-coupled β3 adrenergic receptor or adenylate cyclase itself. However, no evidence currently exists for involvement of the proteoglycan in adrenergic signaling. More realistic possibilities are based on our findings that NG2 promotes activation of signaling through β1 integrins 
and through growth factor receptors for FGF and PDGF 
. While neither integrins nor growth factor receptors represent traditional G protein-coupled receptors, there are nevertheless numerous reports of G protein-dependent and G protein-independent activation of PKA by β1 integrin 
and receptors for FGF or PDGF 
. Future work will examine the ability of NG2 to activate PKA signaling via β1 integrin and growth factor receptor-dependent mechanisms. New research efforts will also be directed toward understanding the basis of the lean phenotype of the OPC-specific NG2 null mouse. NG2 clearly has the potential to affect metabolism via its actions in several different tissues, making the proteoglycan a subject of substantial interest in the field of metabolism.