Rac1 Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

The actin cytoskeleton–regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAK^Thr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.Insulin increases glucose uptake in skeletal muscle by stimulating translocation of GLUT4 from intracellular compartments to the plasma membrane and transverse tubuli (1–4). Skeletal muscle accounts for up to 75% of postprandial glucose disposal in humans (5), and normal insulin action in skeletal muscle is therefore crucial for maintaining glucose homeostasis.The Rho family GTPase Rac1 has been shown to regulate insulin-stimulated GLUT4 translocation and glucose transport in cultured muscle cells (6–8). Insulin activates Rac1, which leads to reorganization of the cortical actin cytoskeleton. Downregulation of Rac1 by small interfering RNA prevents this process (7,9) and also abolishes insulin-stimulated glucose uptake and GLUT4 translocation in L6 myoblasts (6,7). In addition, expression of a constitutively active Rac1 increases GLUT4 translocation to the same level seen after maximal insulin stimulation in this cell line (6).Even though cultured muscle cell lines are powerful tools to understand intracellular mechanisms, they differ from mature skeletal muscle in the expression and reliance of various proteins in the regulation of insulin-stimulated glucose uptake (10). Cultured muscle myoblasts, although able to fuse into myotubes, do not reach the same end-stage differentiation (e.g., do not have cross striations and do not develop transverse tubules) as muscles in vivo and therefore do not fully mature into a system that mimics fully developed skeletal muscles (11,12). Furthermore, the location, expression, and insulin-stimulated GLUT4 translocation are very different in cultured cells compared with mature muscle and may not require the same trafficking steps (2,3,13,14). As a consequence, it is imperative to investigate the role of Rac1 in insulin-stimulated glucose uptake in fully matured skeletal muscle in order to understand its role in glucose metabolism. Furthermore, the importance of skeletal muscle Rac1 on whole-body glucose homeostasis has not been determined.Rac1 activates p21-activated kinase (PAK) by facilitating autophosphorylation of PAK on threonine 423 (p-PAK^Thr423), and this pathway induces actin remodeling of the actin cytoskeleton (15). Accordingly, disruption of the actin cytoskeleton by actin-depolymerizing agents, such as latrunculin B, inhibits insulin-stimulated GLUT4 translocation in L6 myotubes (16,17). Dynamic rearrangement of the actin cytoskeleton is thus necessary for insulin to induce GLUT4 translocation in these cells (18).These findings also apply to mature skeletal muscle, since latrunculin B inhibits insulin-stimulated glucose uptake in rat epitrochlearis muscle (19). Furthermore, Ueda et al. (20) recently showed that Rac1 is activated by insulin in mouse skeletal muscle and that insulin-stimulated GLUT4 translocation is decreased in muscle-specific Rac1 knockout (KO) mice. PAK1 was also recently shown to be implicated in the regulation of insulin-stimulated GLUT4 translocation in mouse skeletal muscle (21). However, GLUT4 translocation does not always mimic glucose uptake, and numerous studies have reported experimental conditions where GLUT4 translocation and transport can be clearly dissociated (22–27), suggesting that GLUT4 translocation is not always an adequate measure of the functional end point, glucose uptake. Thus, the involvement of Rac1 and its downstream signaling in insulin-stimulated glucose uptake in mature skeletal muscle has not yet been investigated, and Rac1-dependent signaling has not been characterized in animal or human models of insulin resistance.A decreased ability to rearrange the cortical actin cytoskeleton in response to insulin has been proposed as a central defect in insulin-resistant muscle cells (28–30). Although exposure to insulin resistance–inducing agents decreased Rac1 activation and GLUT4 translocation (7), only small reductions in Akt signaling were observed in L6 myotubes (8). It is therefore possible that Rac1 is a major regulator of glucose uptake in mature skeletal muscle, and its dysregulation might contribute to the phenotype of muscular insulin resistance and type 2 diabetes (T2D). In the current study, we hypothesized that activation of Rac1 and its downstream target, PAK, is crucial for insulin-induced glucose uptake in mature skeletal muscle and for maintaining whole-body glucose homeostasis. We further hypothesized that Rac1-dependent signaling is downregulated in insulin-resistant states.     

Off-pump surgery for the poor ventricle?

Severely decreased ejection-fraction is an established risk-factor for worse outcome after cardiac surgery. We compare outcomes of off-pump coronary artery bypass grafting (OPCAB) and on-pump CABG (ONCABG) in patients with severely compromised EF. From 2004 to 2009, 478 patients with a decreased EF ??35% underwent myocardial-revascularization. Patients received either OPCAB (n?=?256) or ONCABG (n?=?222). Propensity score (PS), including 50 preoperative risk-factors, was used to balance characteristics between groups. PS adjusted logistic regression analysis was performed to assess mortality and major adverse cardiac and cerebrovascular events (MACCE). A composite endpoint for major non-cardiac complications such as respiratory failure, renal failure, rethoracotomy was applied. Complete revascularization (CR) was assumed when the number of distal anastomoses was larger than that of diseased vessels. There was no difference for mortality (2.3 vs. 4.1%; PS-adjusted odds ratio (PS-OR)?=?1.05; p?=?0.93) and MACCE (13.7 vs. 17.6%; PS-OR?=?1.22; p?=?0.50) including myocardial-infarction (1.4 vs. 4.9%; PS-OR?=?0.39; p?=?0.26), low cardiac output (2.3 vs. 4.7%; PS-OR?=?0.75; p?=?0.72) and stroke (2.3 vs. 2.7%; PS-OR?=?0.69; p?=?0.66). OPCAB patients presented with a trend to less frequent occurrence of the non-cardiac composite (12.1 vs. 22.1%; PS-OR?=?0.54; p?=?0.059) including renal dysfunction (PAOR?=?0.77; 95% CI 0.31?C1.9; p?=?0.57), bleeding (PAOR?=?0.42; 95% CI 0.14?C1.20; p?=?0.10) and respiratory failure (PAOR?=?0.39; 95% CI 0.05?C3.29; p?=?0.39). The rate of complete revascularization was similar (92.2 vs. 92.8%; PS-OR?=?0.75; p?=?0.50). OPCAB in patients with severely decreased EF is safe and feasible. It may even benefit these patients in regard to non-cardiac complications and does not come at cost of less complete revascularization.     

Assessing the validity of corneal power estimation using conventional keratometry for intraocular lens power calculation in eyes with Fuch’s dystrophy undergoing Descemet membrane endothelial keratoplasty

Graefe's Archive for Clinical and Experimental Ophthalmology - The present retrospective study was designed to test the hypothesis that the postoperative posterior to preoperative anterior...     

ICUD-EAU International Consultation on Bladder Cancer 2012: Non–Muscle-Invasive Urothelial Carcinoma of the Bladder

Context

Our aim was to present a summary of the Second International Consultation on Bladder Cancer recommendations on the diagnosis and treatment options for non–muscle-invasive urothelial cancer of the bladder (NMIBC) using an evidence-based approach.

Objective

To critically review the recent data on the management of NMIBC to arrive at a general consensus.

Evidence acquisition

A detailed Medline analysis was performed for original articles addressing the treatment of NMIBC with regard to diagnosis, surgery, intravesical chemotherapy, and follow-up. Proceedings from the last 5 yr of major conferences were also searched.

Evidence synthesis

The major findings are presented in an evidence-based fashion. We analyzed large retrospective and prospective studies.

Conclusions

Urothelial cancer of the bladder staged Ta, T1, and carcinoma in situ (CIS), also indicated as NMIBC, poses greatly varying but uniformly demanding challenges to urologic care. On the one hand, the high recurrence rate and low progression rate with Ta low-grade demand risk-adapted treatment and surveillance to provide thorough care while minimizing treatment-related burden. On the other hand, the propensity of Ta high-grade, T1, and CIS to progress demands intense care and timely consideration of radical cystectomy.     

Enhanced migration of human bone marrow stromal cells in modified collagen hydrogels

Purpose

Collagen I hydrogels are widely used as scaffolds for regeneration of articular cartilage defects. We hypothesised that ingrowth might be improved by removing the superficial layer of a compressed hydrogel. The control group consisted of the original unmodified product.

Methods

The migration of human bone marrow stromal cells (hBMSCs) into the hydrogel was evaluated by confocal microscopy. We quantified the DNA concentration of the hydrogel for each group and time point and evaluated the chondrogenic differentiation of cells.

Results

After one week, the detectable amount of cells at the depth of 26–50 μm was significantly higher in the modified matrix (MM) than in the non-modified matrix (NM) (p = 0.011). The maximum depth of penetration was 75 μm (NM) and 200 μm (MM). After three weeks, the maximum depth of penetration was 175 μm (NM) and 200 μm (MM). Likewise, at a depth of 0–25 μm the amount of detectable cells was significantly higher in the MM group (p = 0.003). After 14 days, the concentration of DNA was significantly higher in the samples of the MM than in the control group (p = 0.000). Staining of histological sections and labelling with collagen II antibodies showed that a chondrogenic differentiation of cells in the scaffold can occur during in vitro cultivation.

Conclusions

Removing the superficial layer is essential to ensuring proper ingrowth of cells within the compressed hydrogel. Compressed hydrogels contribute better to cartilage regeneration after surface modification.     

No need for broad-spectrum empirical antibiotic coverage after surgical drainage of orthopaedic implant infections

Purpose

Empirical broad-spectrum antibiotic treatment for orthopaedic implant infections after surgical lavage is common practice while awaiting microbiological results, but lacks evidence.

Methods

This was a single-centre cohort study from 1996 to 2010 with a follow-up of two years.

Results

We retrieved 342 implant infections and followed them up for a median of 3.5 years (61 recurred, 18 %). Infected implants were arthroplasties (n = 186), different plates, nails or other osteosyntheses. The main pathogens were S. aureus (163, 49 methicillin-resistant) and coagulase-negative staphylococci (60, 45 methicillin-resistant). Median duration of empirical antibiotic coverage after surgical drainage was three days before switching to targeted therapy. Vancomycin was the most frequent initial empirical agent (147), followed by intravenous co-amoxiclav (44). Most empirical antibiotic regimens (269, 79 %) proved sensitive to the causative pathogen, but were too broad in 111 episodes (32 %). Cephalosporins and penicillins were used only in 44 and ten cases, respectively, although they would have covered 59 % of causative pathogens identified later. Multivariate Cox regression analysis showed that neither susceptible antibiotic coverage (compared to non-susceptible; hazard ratio 0.7, 95 % confidence interval 0.4–1.2) nor broad-spectrum use (hazard ratio 1.1, 0.8–1.5) changed remission rates.

Conclusions

Provided that surgical drainage is performed, broad-spectrum antibiotic coverage does not enhance remission of orthopaedic implant infections during the first three days. If empirical agents are prescribed from the first day of infection, narrow-spectrum penicillins or cephalosporins can be considered to avoid unnecessary broad-spectrum antibiotic use.