Adipocyte differentiation requires integration of a multitude of stimuli and coordinated regulation of cellular responses 
. Despite recent confluence of discoveries on brown fat adipogenesis, molecular regulators of this complex mechanism are not fully identified. PTP1B is an established physiological regulator of systemic insulin sensitivity and energy balance, but its role in brown fat adipogenesis warrants additional investigation. In this study we utilized immortalized brown preadipocytes from wild type and PTP1B KO mice and reconstituted KO preadipocytes to address the role of PTP1B in adipogenesis. These cells provide a useful platform since preadipocytes can differentiate into mature brown adipocytes with accumulation of multilocular fat droplets and expression of adipogenic and differentiation markers 
. In addition, these cells are an established model for dissecting the contribution of components in insulin signaling to brown fat adipogenesis and glucose uptake 
. Moreover, PTP1B-reconstituted cells help identify if observed alterations in KO cells are directly caused by PTP1B deletion.
Using standard differentiation protocols, KO and D/A preadipocytes exhibited a trend (that did not reach statistical significance) for increased differentiation and accumulation of fat droplets compared with WT cells. These findings are in line with those of Miranda et al.
who report beneficial effects of PTP1B deficiency on brown fat adipogenesis 
. Conversely, differentiation of preadipocytes expressing a sumoylation-resistant PTP1B mutant (K/R) was dramatically reduced compared with controls. The underlying reason(s) for attenuated K/R preadipocyte differentiation is not clear. One scenario involves attenuated insulin signaling in these cells (see next paragraph). Another possibility involves alteration of signaling in distinct cellular compartment(s). The bulk of sumoylated PTP1B localizes to the perinuclear region 
, and since K/R is resistant to insulin-induced downregulation, then presumably K/R cells manifest increased and/or prolonged PTP1B activation at this region. Therefore, K/R could modulate insulin (and potentially other) signaling amplitude and/or duration to attenuate differentiation. In addition, we cannot rule out that K/R regulates a distinct set of cellular substrate(s), and this warrants additional investigation. At any rate, treatment of K/R cells with the PPARγ agonist troglitazone fully recovers the differentiation blockade in these cells. This could be due to direct activation of PPARγ, and/or indirectly caused by the insulin-sensitizing effects of thiazolidinedione. Of note, troglitazone treatment of IR-deficient brown preadipocytes slightly improves their differentiation profile 
, and in IRS1-deficient preadipocytes reverses some of their differentiation deficits (Glut4 expression) without affecting others (fat accumulation) 
. Although the exact mechanism of impaired differentiation of K/R cells requires additional investigation, our results demonstrate that PTP1B regulates brown fat adipogenesis.
PTP1B can regulate brown fat adipogenesis through insulin-dependent and insulin-independent signaling pathways (please see schematic in ). Numerous studies establish insulin signaling as a critical regulator of brown fat adipogenesis 
. Brown preadipocyte cell lines from IR KO mice exhibit dramatically impaired differentiation 
. Similarly, preadipocytes from IRS1 KO mice exhibit a marked decrease in differentiation and lipid accumulation 
. In addition, expression of adipogenic makers and transcription factors (such as PPARγ, C/EBPα, PGC1α, Glut4, and fatty acid synthase) is attenuated in preadipocytes from IR and IRS1 KO mice 
. Our studies clearly established PTP1B as a regulator of IR and IRS1 signaling in differentiated brown adipocytes and demonstrated that IRS1 is a substrate of PTP1B in these cells. Insulin-induced IR and IRS1 tyrosyl phosphorylation was elevated in cells with abolished/minimal PTP1B activity (KO and D/A, respectively) and attenuated in those with increased/prolonged PTP1B activity (K/R). Similarly, PPARγ, C/EBPα, and PGC1α mRNA was increased in WT, K/O and D/A cells compared with K/R. Notably, our studies demonstrated regulation of insulin-stimulated glucose uptake in adipocytes by PTP1B, but that did not fully correlate with alterations in IR and IRS1 tyrosyl phosphorylation and Glut4 expression. This in line with previous studies 
that suggest that a multitude of factors are required to evoke maximal insulin-stimulated glucose transport including, but not limited to, modulation of signaling at specific intracellular compartments. Collectively, our findings indicate that alteration of insulin signaling via modulation of PTP1B activity accounts, at least in part, for the observed differentiation effects.
Proposed model of regulation of brown fat adipogenesis by PTP1B.
PTP1B also can regulate brown fat adipogenesis through insulin-independent signaling pathways (). AMPK has been implicated in the regulation of brown fat adipogenesis. Inhibition of AMPK blocks brown but not white adipocyte differentiation, and chronic activation of AMPK in vivo
increases brown adipocytes within WAT depots 
. Notably, AMPK activity is elevated, and AMPK target genes that regulate mitochondrial biogenesis are induced in BAT of PTP1B KO mice 
. In line with this, our data demonstrated regulation of AMPK signaling by PTP1B in brown adipocytes. Collectively, these findings indicate that PTP1B can regulate brown adipose differentiation, at least in part, through AMPK signaling. Finally, we cannot rule out modulation of additional affectors(s) of brown adipose differentiation by PTP1B (). Adipogenic differentiation is associated with downregulation of Wnt/β-catenin signaling 
. Since PTP1B has been implicated in regulating β-catenin signaling 
it is tempting to speculate that it could influence adipogenesis through this signaling pathway. Proper execution of adipogenesis requires integration of a wide array of stimuli and regulated expression of numerous genes, so it is not surprising that PTP1B participates in this complex process through regulating various signaling pathways.
In summary, our studies identify PTP1B as a modulator of brown fat adipogenesis, and suggest that adipocyte differentiation requires regulated expression of PTP1B. These findings are of direct relevance to obesity and diabetes given the contribution of brown fat to energy homeostasis, and considering that PTP1B is a target that is being harnessed as a potential therapeutic.