Bone formation is carried out by the osteoblast, a mesenchymal cell whose lifespan and activity are regulated by growth factor signaling networks. Growth factors activate phosphatidylinositol 3-kinase (PI3K), which enhances cell survival and antagonizes apoptosis through activation of Akt/PKB. This process is negatively regulated by the Pten phosphatase, which inhibits the activity of PI3K. In this study, we investigated the effects of Akt activation in bone in vivo by conditionally disrupting the Pten gene in osteoblasts by using Cre-mediated recombination. Mice deficient in Pten in osteoblasts were of normal size but demonstrated a dramatic and progressively increasing bone mineral density throughout life. In vitro osteoblasts lacking Pten differentiated more rapidly than controls and exhibited greatly reduced apoptosis in association with markedly increased levels of phosphorylated Akt and activation of signaling pathways downstream of activated Akt. These findings support a critical role for this tumor-suppressor gene in regulating osteoblast lifespan and likely explain the skeletal abnormalities in patients carrying germ-line mutations of PTEN.Akt ͉ bone acquisition ͉ osteoblast survival ͉ high bone mass T he development and maintenance of the mammalian skeleton are controlled by actions of morphogens and growth factors on bone cells. Bone formation is carried out by the osteoblast, a mesenchymal cell whose lifespan and activity are regulated by growth factor signaling networks (1, 2). Skeletal growth factors such as insulin-like growth factor-1 (IGF-1) affect osteoblast proliferation and lifespan by activating anti-apoptotic pathways, increasing cell proliferation, and influencing differentiation (3). A key control point in many anti-apoptotic pathways is the lipid kinase phosphatidylinositol (PI) 3-kinase (PI3K), which is activated in response to various extracellular signals (4, 5). On activation of growth factor receptor tyrosine kinases, a p85 regulatory subunit of PI3K is recruited to phosphorylated tyrosine residues. This action engages and activates a catalytic subunit (p110) and induces phosphorylation of the inositol ring of PI(4)P or PI(4,5)P 2 at the D position to generate the second messengers PI(3,4)P 2 and PI(3,4,5)P 3 (4, 5). A key downstream target of PI3K and PIP 3 is the serine-threonine Akt kinase family (also known as protein kinase B). PIP 3 generated in the plasma membrane recruits Akt and PI-dependent kinase 1 (PDK1) through an interaction between the PI and the Akt or PDK1 pleckstrin homology (PH) domains. Once recruited to the plasma membrane, Akt is phosphorylated and activated by PDK1.

Tribbles homolog 3 (TRIB3) was found to inhibit insulin-stimulated Akt phosphorylation and modulate gluconeogenesis in rodent liver. Currently, we examined a role for TRIB3 in skeletal muscle insulin resistance. Ten insulin-sensitive, ten insulin-resistant, and ten untreated type 2 diabetic (T2DM) patients were metabolically characterized by hyperinsulinemic euglycemic glucose clamps, and biopsies of vastus lateralis were obtained. Skeletal muscle samples were also collected from rodent models including streptozotocin (STZ)-induced diabetic rats, db/db mice, and Zucker fatty rats. Finally, L6 muscle cells were used to examine regulation of TRIB3 by glucose, and stable cell lines hyperexpressing TRIB3 were generated to identify mechanisms underlying TRIB3-induced insulin resistance. We found that 1) skeletal muscle TRIB3 protein levels are significantly elevated in T2DM patients; 2) muscle TRIB3 protein content is inversely correlated with glucose disposal rates and positively correlated with fasting glucose; 3) skeletal muscle TRIB3 protein levels are increased in STZ-diabetic rats, db/db mice, and Zucker fatty rats; 4) stable TRIB3 hyperexpression in muscle cells blocks insulin-stimulated glucose transport and glucose transporter 4 (GLUT4) translocation and impairs phosphorylation of Akt, ERK, and insulin receptor substrate-1 in insulin signal transduction; and 5) TRIB3 mRNA and protein levels are increased by high glucose concentrations, as well as by glucose deprivation in muscle cells. These data identify TRIB3 induction as a novel molecular mechanism in human insulin resistance and diabetes. TRIB3 acts as a nutrient sensor and could mediate the component of insulin resistance attributable to hyperglycemia (i.e., glucose toxicity) in diabetes. glucose toxicity; type 2 diabetes; insulin signaling THE PREVALENCE OF TYPE 2 DIABETES MELLITUS (T2DM) is rapidly increasing in Westernized nations. Although it likely results from both genetic and environment factors, a key pathogenic characteristic of T2DM is insulin resistance, due to impaired stimulation of glucose uptake in skeletal muscle.To obtain a more comprehensive understanding of insulin resistance, we have performed cDNA microarray studies to systematically assess differential gene expression in skeletal muscle from insulin-sensitive (IS) vs. insulin-resistant (IR) humans (59, 60). These analyses identified Tribbles homolog 3 (TRIB3) as a gene with increased expression in patients with T2DM. Tribbles was first identified by Mata et al. (31) in 2000 as a regulator of germ-cell development in Drosophila (31). Tribbles inhibits mitosis and regulates DNA damage repair by promoting ubiquitination and proteasome-mediated degradation of specific cell cycle regulators early in development (17,31,46,49). Mammals express a family of three genes, TRIB1, TRIB2, and TRIB3, that are homologous to Tribbles. These family members are characterized by a variant kinase domain in the center of molecule with a high homology to serine/ threonine kinases (22). However, they app...

Cultivated (Citrullus lanatus var. lanatus) and citron type (var. citroides) watermelon collected from different areas on the African continent are remarkably diverse in fruit and seed morphology. Chloroplast DNA investigations using PCR-RFLP and sequencing analysis of several non-coding regions were conducted to infer their biogeographic and evolutionary relationships, origin and domestication history. Variability within C. lanatus was observed at regions of high A + T content, resulting in indels and transversions mainly. Distinct chlorotype lineages were identified separating the cultivated and egusi-type watermelon from var. citroides accessions. This suggests an ancient split from a common ancestor and haplotype fixation. Three haplotypes as a result of relatively recent indel events were detected within var. citroides. The geographical range of two of the main citroides haplotypes is relatively similar across southern Africa. Accessions with the most ancient citroides haplotype originated in Swaziland and South Africa resulting in colonization routes from this area all over the world. Chloroplast divergence is not associated with morphological divergence. The cultivated and wild watermelon appear to have diverged independently from a common ancestor, possibly C. ecirrhosus from Namibia.

Both human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been explored as attractive mesenchymal stem cells (MSCs) sources, but very few parallel comparative studies of these two cell types have been made. We designed a side-by-side comparative study by isolating MSCs from the adipose tissue and umbilical cords from mothers delivering full-term babies and thus compared the various biological aspects of ASCs and UC-MSCs derived from the same individual, in one study. Both types of cells expressed cell surface markers characteristic of MSCs. ASCs and UC-MSCs both could be efficiently induced into adipocytes, osteoblasts, and neuronal phenotypes. While there were no significant differences in their osteogenic differentiation, the adipogenesis of ASCs was more prominent and efficient than UC-MSCs. In the meanwhile, ASCs responded better to neuronal induction methods, exhibiting the higher differentiation rate in a relatively shorter time. In addition, UC-MSCs exhibited a more prominent secretion profile of cytokines than ASCs. These results indicate that although ASCs and UC-MSCs share considerable similarities in their immunological phenotype and pluripotentiality, certain biological differences do exist, which might have different implications for future cell-based therapy.