| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 93 === Diabetes is characterized by mild to moderate hyperglycemia, glucosuria, polyphagia, hypoinsulinemia, hyperlipidemia, and weight loss. All forms of diabetes are characterized by chronic hyperglycemia and the development of many serious complications, for example, heart disease (cardiovascular disease), blindness (retinopathy), nerve damage (neuropathy), and kidney damage (nephropathy). Diabetes has also been reported with a net loss of bone. However, the effect of hyperglycemia on bone loss remains unclear. Bone loss in aging and osteoporosis are associated with a decrease in the number and activity of osteoblasts and a parallel increase in the number of adipocytes. Here we have demonstrated that hyperglycemia affected the mesenchymal stem cells (MSCs) differentiation by enhancing adipocyte differentiation (adipogenesis). We used high glucose (HG, 25.5 mM) to mimic the hyperglycemia condition.
To determine the effects of hyperglycemia on adipogenesis, we cultured mouse MSCs in an adipogenic hormonal cocktail, and adipogenesis was strong enhancement by supplementation of HG and 15-deoxy-∆12,14-PGJ2 (15d-PGJ2), which has been identified as an endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), inducing adipogenesis in vitro. This ligand improves insulin sensitivity through the activation of the transcription factor, PPARγ. In addition to sensitizing cells to insulin, the PPARγ2 isoform appears to be critical for the regulation of osteoblast differentiation (osteoblastogenesis) and adipocyte differentiation (adipogenesis) of MSCs in bone marrow. In this HG cultures, the expression of PPARγ2 was up-regulated even prior to adipogenic induction. Moreover, treatment with PPARγ agonists, GW 9662 (20 μM) or inhibitors of phosphatidylinositol 3-kinase (PI3K), LY 294002 (7.5 μM), leads to the complete blockade of HG-enhanced adipogenesis of MSCs by inhibited the PPARγ expression. HG-activated Akt on adipogenesis of MSCs was also inhibited by LY 294002. Likewise, blocking the PI3K or Akt activity with the dominant-negative vectors DN-p85 or DN-Akt, respectively, also greatly inhibited the HG-enhanced the expression of PPARγ. These suggesting that HG enhanced adipogenesis of MSCs in the adipogenic medium may through a PI3K/Akt regulated PPARγ pathway.
Another intriguing finding was that 15d-PGJ2 (1 μM) enhanced adipogenesis by increasing the PPARγ expression which was inhibited by LY 294002, DN-p85 and DN-Akt. Collectively, these data provide a new insight into the PI3K/Akt pathway on MSCs differentiation. So, next we examine the mechanisms of osteoblastogenesis of MSCs. First, we found that 15d-PGJ2 and HG decreased alkaline phosphatase activity, which was used as early differentiation markers of osteoblastogenesis from MSCs in osteoblastogenic medium, and treatment with GW 9662 or LY294002 was significantly increasing the alkaline phosphatase activity. Second, the 15d-PGJ2 inhibited the expression of the osteocalcin, which gene marked the late stages of osteoblastogenesis, and treatment with GW 9662 or LY294002 was significantly increasing the osteocalcin gene expression. But HG was no effect on osteocalcin expression. Third, mineralization was reduced by treatment of MSCs with 15d-PGJ2 in osteoblastogenic medium and dominant-negative vectors DN-p85 or DN-Akt greatly increased mineralization. HG was also no effect on mineralization. Mineralization correlated closely with osteocalcin gene expression. Taken together, these results indicate that activation of PI3K/Akt pathway in MSCs may increase adipogenesis and decrease osteoblastogenesis of MSCs.
On the other hand, in in vivo study, mice were made diabetic by multiple low-dose streptozotocin (STZ) treatment, and controls were treated with vehicle alone. After 3 weeks, chose the diabetic mice which had ≧400 mg/dl blood sugar. We cultured diabetic MSCs and control MSCs in adipogenic medium combined with HG or 15d-PGJ2 and compared their adipogenesis of MSCs. We found that the level of diabetic adipogenesis of MSCs was higher than the level of control mice. Moreover, the tibial bone from the proximal metaphysis to the tibiofibular junction was snap frozen in liquid nitrogen, pulverized, and we found that the expression of PPARγ and triglyceride amounts were significantly increased in the diabetic bone whereas alkaline phosphatase activity was reduced in the diabetic bone.
These findings support a reciprocal relationship between the development of bone and fat under hyperglycemia, and may prompt further exploration of the PI3K/Akt regulated MSCs differentiation as a potential target for intervention in diabetic osteoporosis.
|
|---|
