SummaryAging or glucocorticoid excess decrease bone strength more than bone mass in humans and mice, but an explanation for this mismatch remains elusive. We report that aging in C57BL ⁄ 6 mice was associated with an increase in adrenal production of glucocorticoids as well as bone expression of 11b-hydroxysteroid dehydrogenase (11b-HSD) type 1, the enzyme that activates glucocorticoids. Aging also decreased the volume of the bone vasculature and solute transport from the peripheral circulation to the lacunar-canalicular system. The same changes were reproduced by pharmacologic hyperglucocorticoidism. Furthermore, mice in which osteoblasts and osteocytes were shielded from glucocorticoids via cell-specific transgenic expression of 11b-HSD type 2, the enzyme that inactivates glucocorticoids, were protected from the adverse effects of aging on osteoblast and osteocyte apoptosis, bone formation rate and microarchitecture, crystallinity, vasculature volume, interstitial fluid, and strength. In addition, glucocorticoids suppressed angiogenesis in fetal metatarsals and hypoxia inducible factor-1a transcription and vascular endothelial growth factor production in osteoblasts and osteocytes. These results, together with the evidence that dehydration of bone decreases strength, reveal that endogenous glucocorticoids increase skeletal fragility in old age as a result of cell autonomous effects on osteoblasts and osteocytes leading to interconnected decrements in bone angiogenesis, vasculature volume, and osteocyte-lacunar-canalicular fluid.

In the current study, we investigated the role of tribbles homolog 3 (TRIB3) in glucose-induced insulin resistance and whether the induction of TRIB3 by glucose is dependent on the nutrient-sensing hexosamine biosynthetic pathway (HBP) known to mediate glucose toxicity in diabetes. In diabetic rats, TRIB3 expression in skeletal muscle was increased after 10 days of hyperglycemia, and glycemia and muscle TRIB3 were both restored toward normal by insulin therapy. In L6 myocytes, the induction of TRIB3 by high glucose or glucosamine was reversible upon removal of these substrates. To assess the role of HBP in the induction of TRIB3, we demonstrated that the ability of high glucose to augment TRIB3 expression was prevented by azaserine, an inhibitor of glutamine: fructose-6-phosphate amidotransferase (GFAT), which is the rate-limiting enzyme in the HBP pathway. TRIB3 expression was also substantially stimulated by glucosamine, which bypasses GFAT, accompanied by a decrease in the insulin-stimulated glucose transport rate, and neither response was affected by azaserine. Further, knockdown of TRIB3 inhibited, and TRIB3 overexpression enhanced, the ability of both high glucose and glucosamine to induce insulin resistance. These data provide the mechanistic link between the HBP flux and insulin resistance and point to TRIB3 as a novel target for treatment of glucose-induced insulin resistance.

The purine anti-metabolite 6-mercaptopurine (6-MP) is widely used for the treatment of leukemia and inflammatory diseases. The cellular effects of 6-MP on metabolism remain unknown; however, 6-MP was recently found to activate the orphan nuclear receptor NR4A3 in skeletal muscle cell lines. We have reported previously that NR4A3 (also known as NOR-1, MINOR) is a positive regulator of insulin sensitivity in adipocytes. To further explore the role of NR4A3 activation in insulin action, we explored whether 6-MP activation of NR4A3 could modulate glucose transport system activity in L6 skeletal muscle cells. We found that 6-MP increased both NR4A3 expression and NR4A3 transcriptional activity and enhanced glucose transport activity via increasing GLUT4 translocation in both basal and insulin-stimulated L6 cells in an NR4A3-dependent manner. Furthermore, 6-MP increased levels of phospho-AS160, although this effect was not modulated by NR4A3 overexpression or knockdown. These primary findings provide a novel proof of principle that 6-MP, a small molecule NR4A3 agonist, can augment glucose uptake in insulin target cells, although this occurs via both NR4A3-dependent and -independent actions; the latter is related to an increase in phospho-AS160. These results establish a novel target for development of new treatments for insulin resistance.

Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.