Limitation of adipose tissue by the number of embryonic progenitor cells.

Adipogenesis in adulthood replaces fat cells that turn over and can contribute to the development of obesity. However, the proliferative potential of adipocyte progenitors in vivo is unknown (Faust et al., 1976; Faust et al., 1977; Hirsch and Han, 1969; Johnson and Hirsch, 1972). We addressed this by injecting labeled wild-type embryonic stem cells into blastocysts derived from lipodystrophic A-ZIP transgenic mice, which have a genetic block in adipogenesis. In the resulting chimeric animals, wild-type ES cells are the only source of mature adipocytes. We found that when chimeric animals were fed a high-fat-diet, animals with low levels of chimerism showed a significantly lower adipose tissue mass than animals with high levels of chimerism. The difference in adipose tissue mass was attributed to variability in the amount of subcutaneous adipose tissue as the amount of visceral fat was independent of the level of chimerism. Our findings thus suggest that proliferative potential of adipocyte precursors is limited and can restrain the development of obesity.

Antidiabetic effects of IGFBP2, a leptin-regulated gene.

We tested whether leptin can ameliorate diabetes independent of weight loss by defining the lowest dose at which leptin treatment of ob/ob mice reduces plasma glucose and insulin concentration. We found that a leptin dose of 12.5 ng/hr significantly lowers blood glucose and that 25 ng/hr of leptin normalizes plasma glucose and insulin without significantly reducing body weight, establishing that leptin exerts its most potent effects on glucose metabolism. To find possible mediators of this effect, we profiled liver mRNA using microarrays and identified IGF Binding Protein 2 (IGFBP2) as being regulated by leptin with a similarly high potency. Overexpression of IGFBP2 by an adenovirus reversed diabetes in insulin-resistant ob/ob, Ay/a, and diet-induced obese mice, as well as insulin-deficient streptozotocin-treated mice. Hyperinsulinemic clamp studies showed a 3-fold improvement in hepatic insulin sensitivity following IGFBP2 treatment of ob/ob mice. These results show that IGFBP2 can regulate glucose metabolism, a finding with potential implications for the pathogenesis and treatment of diabetes.

SNF1/AMPK pathways in yeast.

The SNF1/AMPK family of protein kinases is highly conserved in eukaryotes and is required for energy homeostasis in mammals, plants, and fungi. SNF1 protein kinase was initially identified by genetic analysis in the budding yeast Saccharomyces cerevisiae. SNF1 is required primarily for the adaptation of yeast cells to glucose limitation and for growth on carbon sources that are less preferred than glucose, but is also involved in responses to other environmental stresses. SNF1 regulates transcription of a large set of genes, modifies the activity of metabolic enzymes, and controls various nutrient-responsive cellular developmental processes. Like AMPK, SNF1 protein kinase is heterotrimeric. It is phosphorylated and activated by the upstream kinases Sak1, Tos3, and Elm1 and is inactivated by the Reg1-Glc7 protein phosphatase 1. Further regulation of SNF1 is achieved through autoinhibition and through control of its subcellular localization. Here we review the current understanding of SNF1 protein kinase pathways in Saccharomyces cerevisiae and other yeasts.

Regulation of the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase.

Snf1 protein kinase containing the beta subunit Gal83 is localized in the cytoplasm during growth of Saccharomyces cerevisiae cells in abundant glucose and accumulates in the nucleus in response to glucose limitation. Nuclear localization of Snf1-Gal83 requires activation of the Snf1 catalytic subunit and depends on Gal83, but in the snf1Delta mutant, Gal83 exhibits glucose-regulated nuclear accumulation. We show here that the N terminus of Gal83, which is divergent from those of the other beta subunits, is necessary and sufficient for Snf1-independent, glucose-regulated localization. We identify a leucine-rich nuclear export signal in the N terminus and show that export depends on the Crm1 export receptor. We present evidence that catalytically inactive Snf1 promotes the cytoplasmic retention of Gal83 in glucose-grown cells through its interaction with the C terminus of Gal83; cytoplasmic localization of inactive Snf1-Gal83 maintains accessibility to the Snf1-activating kinases. Finally, we characterize the effects of glucose phosphorylation on localization. These studies define roles for Snf1 and Gal83 in determining the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase.

Pak1 protein kinase regulates activation and nuclear localization of Snf1-Gal83 protein kinase.

Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the beta-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Delta mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

Cyclic AMP-dependent protein kinase regulates the subcellular localization of Snf1-Sip1 protein kinase.

The Snf1/AMP-activated protein kinase family has diverse roles in cellular responses to metabolic stress. In Saccharomyces cerevisiae, Snf1 protein kinase has three isoforms of the beta subunit that confer versatility on the kinase and that exhibit distinct patterns of subcellular localization. The Sip1 beta subunit resides in the cytosol in glucose-grown cells and relocalizes to the vacuolar membrane in response to carbon stress. We show that translation of Sip1 initiates at the second ATG of the open reading frame, yielding a potential site for N myristoylation, and that mutation of the critical glycine abolishes relocalization. We further show that the cyclic AMP-dependent protein kinase (protein kinase A [PKA]) pathway maintains the cytoplasmic localization of Sip1 in glucose-grown cells. The Snf1 catalytic subunit also exhibits aberrant localization to the vacuolar membrane in PKA-deficient cells, indicating that PKA regulates the localization of Snf1-Sip1 protein kinase. These findings establish a novel mechanism of regulation of Snf1 protein kinase by the PKA pathway.