In this laboratory study all test techniques left GP/sealer remnants within the root canal. The ProTaper Universal rotary retreatment system proved to be an efficient method of removing GP and sealer from maxillary anterior teeth.

Oxidative stress has been ascribed a role in the pathogenesis of diabetes and its complications, and stress proteins have been shown to protect organisms in vitro and in vivo against oxidative stress. To study the putative role of one of the most abundant cytoprotective stress proteins, inducible cytoplasmic 72-kDa-mass heat shock protein (Hsp-72), in the pathogenesis of diabetes, we measured its mRNA concentration in muscle biopsies from six type 2 diabetic patients and six healthy control subjects (protocol 1) as well as in 12 twin pairs discordant for type 2 diabetes and 12 control subjects undergoing a euglycemic-hyperinsulinemic clamp in combination with indirect calorimetry (protocol 2). The amount of Hsp-72 mRNA in muscle was significantly lower in type 2 diabetic patients than in healthy control subjects (in protocol 1: 5.2 ؎ 2.2 vs. 53 ؎ 32 million copies of Hsp-72 mRNA/g total RNA, n ‫؍‬ 6, P ‫؍‬ 0.0039; in protocol 2: 3.2 ؎ 3.3 vs. 43 ؎ 31 million copies of Hsp-72 mRNA/g total RNA, n ‫؍‬ 12, P ‫؍‬ 0.0001). Hsp-72 mRNA levels were also markedly reduced in the nondiabetic co-twins compared with healthy control subjects (5.8 ؎ 5.0 vs. 43 ؎ 31, n ‫؍‬ 12, P ‫؍‬ 0.0001), but they were also statistically significantly different from their diabetic co-twins when the difference between the pairs was compared (P ‫؍‬ 0.0280). Heat shock protein mRNA content in muscle of examined patients correlated with the rate of glucose uptake and other measures of insulin-stimulated carbohydrate and lipid metabolism. In conclusion, the finding of decreased levels of Hsp-72 mRNA in skeletal muscle of patients with type 2 diabetes and its relationship with insulin resistance raises the question of whether heat shock proteins are involved in the pathogenesis of skeletal muscle insulin resistance in type 2 diabetes.

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Insulin causes distinct cortical actin remodeling in muscle and fat cells, and interfering with actin dynamics halts glucose transporter 4 (GLUT4) translocation to the membrane. Phosphatidylinositol 3-kinase (PI3-K) and the small G protein Rac govern myocyte actin remodeling, whereas TC10 alpha contributes to adipocyte actin dynamics downstream of Cbl-associated protein (CAP) and Cbl, independently of PI3-K. Given the importance of insulin action in both cell types, it is paramount to determine whether signaling pathways and actin manifestations are cell type specific. We found CAP expression and insulin-mediated Cbl phosphorylation in differentiated myotubes but not in myoblasts. Unlike adipocytes, Cbl is phosphorylated on Y774 and Y731 in myotubes. TC10 alpha and beta-transcripts are amplified by RT-PCR in muscle cells, but the endogenous proteins are barely detectable using two unrelated antibodies. TC10 alpha transfected into myoblasts is activated by insulin despite the lack of CAP expression and Cbl phosphorylation. Moreover, dominant-negative TC10 alpha mutants do not prevent insulin-induced actin remodeling in either myoblasts or myotubes and do not interfere with insulin-mediated recruitment of c-myc epitope-tagged GLUT4 to the cell surface. In contrast to TC10 alpha, endogenous Rac is readily detectable in both muscle cells and adipocytes and binds GTP after insulin in a PI3-K-dependent manner. These data suggest that whereas individual components of the CAP to TC10 pathway are regulated by insulin, a functional TC10-dependent signaling pathway leading to actin remodeling and GLUT4 translocation may not operate in myocytes, as it does in adipocytes.

Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-à -vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (si-IRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70 -75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells. Insulin receptor substrates (IRSs)1 mediate diverse metabolic and mitogenic effects of insulin, and dysregulation of IRS expression and activation has been observed in both insulin resistance and diabetes (1-5). To date, six IRS proteins have been identified, but only IRS-1 and IRS-2 are thought to participate in regulation of glucose homeostasis (6 -9). Although IRS-1 and 2 have substantial amino acid similarity (10), there are significant structural differences between them. IRS-2 interacts with the insulin receptor via a phosphotyrosine-binding domain as well as a central domain located between amino acid 591 and 733 which is absent from IRS-1 (11, 12). Furthermore, IRS-1 and IRS-2 differ in their time course of insulin-stimulated tyrosine phosphorylation (13,14), interaction with the MAPK pathway (15), and intracellular localization (14).Defining the exact roles of IRS isoforms in insulin-stimulated glucose uptake is not without controversy. In rat adipocytes, overexpression of human IRS-1 increased, whereas elimination of IRS-1 via antisense ribozyme reduced, insulinstimulated GLUT4 translocation (16). However, other studies observed intact insulin-stimulated GLUT4 translocation when the interaction between insulin receptor and IRS was blocked, although insulin-mediated DNA synthesis and cell growth were reduced (17-19). Furthermore, IRS-1-null mice, although significan...

We examined the gene and protein expression of IRS 1 (insulin receptor substrate 1) in adipocytes from two groups of healthy individuals with an increased propensity for non-insulin-dependent diabetes mellitus (NIDDM): those with two first-degree relatives with diabetes and another group with massive obesity. A low expression of IRS 1 (

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