Insulin-stimulated translocation of the fatty acid transporter CD36 to the plasma membrane is mediated by the small GTPase Rac1 in adipocytes.

Cluster of differentiation 36 (CD36) is a scavenger receptor (SR), recognizing diverse extracellular ligands in various types of mammalian cells. Long-chain fatty acids (FAs), which are important constituents of phospholipids and triglycerides, also utilize CD36 as a predominant membrane transporter, being incorporated from the circulation across the plasma membrane in several cell types, including cardiac and skeletal myocytes and adipocytes. CD36 is localized in intracellular vesicles as well as the plasma membrane, and its distribution is modulated by extracellular stimuli. Herein, we aimed to clarify the molecular basis of insulin-stimulated translocation of CD36, which leads to the enhanced uptake of long-chain FAs, in adipocytes. To this end, we developed a novel exofacial epitope-tagged reporter to specifically detect cell surface-localized CD36. By employing this reporter, we demonstrate that the small GTPase Rac1 plays a pivotal role in insulin-stimulated translocation of CD36 to the plasma membrane in 3T3-L1 adipocytes. Additionally, phosphoinositide 3-kinase and the protein kinase Akt2 are shown to be involved in the regulation of Rac1. Downstream of Rac1, another small GTPase RalA directs CD36 translocation. Collectively, these results suggest that CD36 is translocated to the plasma membrane by insulin through mechanisms similar to those for the glucose transporter GLUT4 in adipocytes.

Impaired Insulin Signaling Mediated by the Small GTPase Rac1 in Skeletal Muscle of the Leptin-Deficient Obese Mouse.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

Regulation of De Novo Lipid Synthesis by the Small GTPase Rac1 in the Adipogenic Differentiation of Progenitor Cells from Mouse White Adipose Tissue.

White adipocytes act as lipid storage, and play an important role in energy homeostasis. The small GTPase Rac1 has been implicated in the regulation of insulin-stimulated glucose uptake in white adipocytes. Adipocyte-specific rac1-knockout (adipo-rac1-KO) mice exhibit atrophy of subcutaneous and epididymal white adipose tissue (WAT); white adipocytes in these mice are significantly smaller than controls. Here, we aimed to investigate the mechanisms underlying the aberrations in the development of Rac1-deficient white adipocytes by employing in vitro differentiation systems. Cell fractions containing adipose progenitor cells were obtained from WAT and subjected to treatments that induced differentiation into adipocytes. In concordance with observations in vivo, the generation of lipid droplets was significantly attenuated in Rac1-deficient adipocytes. Notably, the induction of various enzymes responsible for de novo synthesis of fatty acids and triacylglycerol in the late stage of adipogenic differentiation was almost completely suppressed in Rac1-deficient adipocytes. Furthermore, the expression and activation of transcription factors, such as the CCAAT/enhancer-binding protein (C/EBP) ß, which is required for the induction of lipogenic enzymes, were largely inhibited in Rac1-deficient cells in both early and late stages of differentiation. Altogether, Rac1 is responsible for adipogenic differentiation, including lipogenesis, through the regulation of differentiation-related transcription.

Atrophy of White Adipose Tissue Accompanied with Decreased Insulin-Stimulated Glucose Uptake in Mice Lacking the Small GTPase Rac1 Specifically in Adipocytes.

Insulin stimulates glucose uptake in adipose tissue and skeletal muscle by inducing plasma membrane translocation of the glucose transporter GLUT4. Although the small GTPase Rac1 is a key regulator downstream of phosphoinositide 3-kinase (PI3K) and the protein kinase Akt2 in skeletal muscle, it remains unclear whether Rac1 also regulates glucose uptake in white adipocytes. Herein, we investigated the physiological role of Rac1 in white adipocytes by employing adipocyte-specific rac1 knockout (adipo-rac1-KO) mice. Subcutaneous and epididymal white adipose tissues (WATs) in adipo-rac1-KO mice showed significant reductions in size and weight. Actually, white adipocytes lacking Rac1 were smaller than controls. Insulin-stimulated glucose uptake and GLUT4 translocation were abrogated in rac1-KO white adipocytes. On the other hand, GLUT4 translocation was augmented by constitutively activated PI3K or Akt2 in control, but not in rac1-KO, white adipocytes. Similarly, to skeletal muscle, the involvement of another small GTPase RalA downstream of Rac1 was demonstrated. In addition, mRNA levels of various lipogenic enzymes were down-regulated in rac1-KO white adipocytes. Collectively, these results suggest that Rac1 is implicated in insulin-dependent glucose uptake and lipogenesis in white adipocytes, and reduced insulin responsiveness due to the deficiency of Rac1 may be a likely explanation for atrophy of WATs.

Tibial condylar valgus osteotomy (TCVO): Surgical technique and clinical results for knee osteoarthritis with varus deformity.

Tibial condylar valgus osteotomy (TCVO) is an intra-articular proximal tibial osteotomy developed in 1989 and has since been used for the treatment of knee osteoarthritis (OA) associated with genu varum. This article describes the surgical technique and clinical results of TCVO. TCVO can be used for all grades of varus knee OA in patients of any age. he preoperative range of movement should be at least 90°. Preoperative screening showed varus-valgus instability due to an intra-articular deformity of the proximal tibia. Using intraoperative image intensification, a sagittally oriented "L"-shaped osteotomy is made from the medial to the tibial tuberosity to the center of the tibial plateau between the medial and lateral tibial spines. The separation of the osteotomy using the lamina spreader is gradually increased using an image intensifier guidance until the articular surface of the lateral tibial plateau comes in contact with the articular surface of the lateral femoral condyle. Adequate correction is indicated by parallelism of the lateral tibial plateau and a line tangential to the distal convexity of the lateral femoral condyle on an anteroposterior (AP) image and the elimination of the valgus instability with the knee in extended position. A "T"-plate (locking or non-locking plate or circular external fixator) is used to fix the osteotomy in the corrected position. Synthetic or autologous bone grafts can be used. We used the Japanese Orthopaedic Association score to evaluate the patient's function and also measured the %MAD, medial plateau opening angle, medial plateau angle, and lateral plateau opening angle on an AP view of the long length roentgenogram of the lower limb (standing position). The JOA score, radiologically measured values, and instability of the knee joint remarkably improved.

Distal tibial oblique osteotomy for reconstruction of ankle joint congruity and stability.

Teramoto distal tibial oblique osteotomy (DTOO) is a joint-preserving surgery for ankle osteoarthritis (AOA). However, there are few articles on the radiological assessment of DTOO. The purpose of this study was to report the clinical outcomes and radiological evaluations of weight-bearing radiographs before and after DTOO. We retrospectively reviewed 52 patients who underwent DTOO between 2007 and 2018. We recorded the Tanaka-Takakura classification, fixation methods, Japanese Society for Surgery of the Foot Ankle/Hindfoot Scale (JSSF scale), and complications. The tibial articular surface angle (TAS), medial malleolar angle (MMA), tibial lateral surface angle (TLS), talar tilt angle (TTA), and tibiotalar surface angle (TTS) were evaluated using weight-bearing ankle radiographs. The median patient age was 66 years, and the mean follow-up duration was 46 ± 23 months. Two stage 2, 9 stage 3a, 30 stage 3b, and 11 stage 4 according to the Tanaka-Takakura classification were performed using DTOO. The JSSF scale improved significantly from 39.9 ± 13.8 before surgery to 87.2 ± 7.5 after surgery. Seven cases were fixed using a locking plate, and 45 cases were fixed using a circular external fixator. The TAS, MMA, TLS, TTA, and TTS significantly changed before and after DTOO. Radiological evaluation indicated that DTOO influences talar behavior during weight-bearing, and improves the clinical outcomes of AOA.

Radiological assessments and clinical results of intra-articular osteotomy for traumatic osteoarthritis of the ankle.

INTRODUCTION: Traumatic osteoarthritis of the ankle joint caused after malleolar fractures of the ankle and tibial plafond fractures are frequently observed in comparatively young and highly active patients. Since the ankle movement in these patients is in general, comparatively favorable, orthopedists may sometimes have difficulty in deciding on a treatment policy. In our department, when treating traumatic osteoarthritis patients having a movable range within their ankle joints, we proactively applied distal tibial oblique osteotomy (DTOO) developed by Dr. Teramoto in 1994 or intra-articular osteotomy developed based on DTOO concepts such as distal tibial intra-articular osteotomy (DTIO) and distal fibular oblique osteotomy (DFOO).The objectives of the current study are to radiologically assess the ankle joint after intra-articular osteotomy for traumatic ankle osteoarthritis and evaluate the change in configuration of the ankle joint. This study summarizes the clinical results of intra-articular osteotomy obtained through the above-mentioned study. PATIENTS AND METHODS: The subjects of this study were 20 patients diagnosed with traumatic osteoarthritis who were surgically treated for a total of 20 ankles. All patients underwent treatment with intra-articular osteotomy and were evaluated retrospectively for the following parameters: surgical procedure, fixation devices, clinical results based on the Japanese Society for Surgery of the Foot ankle/hindfoot scale (hereafter, JSSF scale) and post-operative adverse events. They were also assessed radiologically with pre- and post-operative anterior-posterior (AP) and lateral weight-bearing ankle radiographs. RESULTS: The 20 patients consisted of 12 males and 8 females. The median age at surgery was 49 years old (range 14 - 87 years old) and the average follow-up period was 42 months (range 19 to 121 months). DTOO was applied to 10 cases, DFOO to 2 cases, DTOO and DFOO to 2 cases, medial-distal tibial intra-articular osteotomy (M-DTIO) and DFOO to 1 case, lateral-distal tibial intra-articular osteotomy (L-DTIO) and DFOO to 3 cases, M-DTIO followed by DTOO and DFOO to 1 case, and DTOO followed by low tibial osteotomy (LTO) to 1 case. Fixation devices utilized included circular external fixator for 15 cases, locking compression plate (LCP) to 3 cases, LCP and Kirschner-wire (K-wire) to 1 case, and screw and K-wire to 1 case. Radiological assessment revealed significant changes in the following parameters after surgery: tibial ankle surface angle (TAS, P= 0.0203), tibiotalar surface angle (TTS, P= 0.0021), medial malleolar angle (MMA, P= 0.0217), empirical axis (EA, P= 0.0019), fibular angle (FA, P= 0.0002), talar tilt angle (TTA, P= 0.0374), and tibial lateral surface angle (TLS, P= 0.0279). The JSSF scale also improved significantly after surgery (pre-operative JSSF scale: 51.1±11.0, post-operative JSSF scale: 89.2±8.2), p=0.0001. CONCLUSION: Intra-articular osteotomy may change the radiological configuration of the ankle in a weight-bearing state. The present study showed very good short-term clinical results. Intra-articular osteotomy can prove a viable surgical option applicable for treatment of patients with traumatic ankle osteoarthritis having a reasonable range of motion within their ankle joints.

Correction of equinus deformity by means of a new unconstrained ilizarov frame system.

PURPOSE: This study aimed to clarify the effectiveness of a novel technique utilizing the new unconstrained Ilizarov frame system by evaluating the clinical outcomes of equinus deformity correction. METHODS: From January 1998 to December 2012, a total of 9 consecutive patients (median age: 33 years) with equinus deformity were treated by using a simple, unconstrained, hinge-less Ilizarov frame, which was developed to correct talar subluxation using an unconstrained frame system. All patients had equinus deformity >30°, although preoperative radiographs showed a congruous ankle joint with no fixed bony deformity. Preoperative equinus deformity was evaluated as well as dorsal flexion (DF) in Ilizarov at 3 months after removal and at final follow-up. Furthermore, the presence or absence of talar subluxation at the time removal of the Ilizarov apparatus, and whether or not ankle arthrodesis was finally indicated, was evaluated. RESULTS: Median follow-up period was 76 months. Median preoperative equinus deformity was -40° None of the patients showed anterior or posterior subluxation of the talus at the time of removal. Three months after removal of the Ilizarov apparatus, the median DF angle was -5° However, 4 patients showed less than -15° of DF and underwent ankle arthrodesis with the ankle joint in the 5° DF position. At the final follow-up, median DF angle was 5°. CONCLUSION: This technique allows for safe, gradual correction of equinus deformity without talar subluxation, although additional procedures, such as ankle arthrodesis, may be needed in some cases.

The guanine nucleotide exchange factor FLJ00068 activates Rac1 in adipocyte insulin signaling.

Insulin stimulates glucose uptake via the translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes. Several lines of evidence suggest that the small GTPase Rac1 plays an important role in insulin-stimulated glucose uptake in skeletal muscle and adipocytes. The purpose of this study is to investigate the mechanisms whereby Rac1 is regulated in adipocyte insulin signaling. Here, we show that knockdown of the guanine nucleotide exchange factor FLJ00068 inhibits Rac1 activation and GLUT4 translocation by insulin and a constitutively activated form of the protein kinase Akt2. Furthermore, constitutively activated FLJ00068 induced Rac1 activation and Rac1-dependent GLUT4 translocation. Collectively, these results suggest the involvement of FLJ00068 downstream of Akt2 in insulin-stimulated glucose uptake signaling in adipocytes.

Comparison of histopathology and preoperative 18F-FDG-PET/CT of osteomyelitis aiming for image guided surgery: A preliminary trial.

OBJECTIVE: There are currently no robust methods for accurately localizing the infection focus of osteomyelitis. Accumulation of fluorodeoxyglucose (FDG) is nonspecific, and it is well-known that it can indicate inflammatory cells and sites of inflammation, and its effectiveness in detecting osteomyelitis has been reported recently. However, the optimal cut-off value for the Standardized Uptake Value (SUV) in detecting the focus of osteomyelitis through 18F-FDG-PET/CT is not known. We investigated the optimal SUV cut-off values using 18F-FDG positron emission tomography (PET)computed tomography (CT) to visualize the infection focus of osteomyelitis accurately. PATIENTS AND METHODS: Initially, we investigated a case where osteomyelitis was bacteriologically detected after orthopedic surgery on lower limb. Based on the surgical pathology, we explored the optimal SUV cut-off value of the 18F-FDG PET/CT image taken before surgery. The SUV cut-off value was varied, using the GE Rainbow Color Scale on a dedicated workstation. We searched for the most accurate visualization of the extent of the infectious lesion. Subsequently, using the SUV cut-off value decided on the basis of the first case studied, we investigated the accuracy for diagnosing osteomyelitis. A total of sixteen patients underwent 18F-FDG PET/CT for suspected osteomyelitis (one case involved the upper extremity and 15 cases the lower one). All patients underwent surgery. The final diagnosis was made by means of bacteriologic culture of surgical specimens and histopathologic analysis. We compared surgical pathology and preoperative 18F-FDG PET/CT. RESULTS: In the first case studied, the infection was most accurately localized with a SUV with a lower level of 2.00 and an upper of 8.00. Upon comparing the pathological findings and the 18F-FDG PET/CT, we set a SUV with a lower level of 2.00 and an upper level of 8.00. In thirteen cases, infection was detected with positive pathological findings. Preoperative 18F-FDG PET/CT showed high accumulation in these cases. In the remaining three cases, no infection was detected on either pathological findings nor 18F-FDG PET/CT findings. CONCLUSIONS: The infection focus of osteomyelitis was accurately visualized by setting the SUV cut-off lower level to 2.00 and upper level to 8.00. We believe that this 18F-FDG PET/CT technique is helpful for image guided surgery of osteomyelitis.

A Crucial Role for the Small GTPase Rac1 Downstream of the Protein Kinase Akt2 in Insulin Signaling that Regulates Glucose Uptake in Mouse Adipocytes.

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.

Ankle joint reconstruction by circular frame external fixator for a severely comminuted and contaminated open tibial pilon fracture (AO 43-C3.3). Case report.

A severely comminuted and contaminated open tibial pilon fracture is one of the most challenging fractures we face. Although nowadays conducting multiple operations over various stages is a common treatment option taking into account the possibility of soft tissue damage, a gold standard protocol for severe pilon fractures has not yet been established. This case concerns a 56-year-old gentleman who suffered a severely comminuted and contaminated Gustilo 3b open tibial pilon fracture (AO 43C3.3) that was successfully treated using a circular frame external fixator without flap. Two years six months after the injury, there were no indications of any skin conditions at the site of the open wound, the range of ankle motion had been maintained and independent walking was possible. The score under the JSSF (Japanese Society of Surgery of the foot) ankle/hind foot scale was 81. This indicates that use of a circular frame external fixator is a useful treatment method in the event of a severe open pilon fracture where there is a large osteochondral bone defect.

Involvement of the protein kinase Akt2 in insulin-stimulated Rac1 activation leading to glucose uptake in mouse skeletal muscle.

Translocation of the glucose transporter GLUT4 to the sarcolemma accounts for glucose uptake in skeletal muscle following insulin administration. The protein kinase Akt2 and the small GTPase Rac1 have been implicated as essential regulators of insulin-stimulated GLUT4 translocation. Several lines of evidence suggest that Rac1 is modulated downstream of Akt2, and indeed the guanine nucleotide exchange factor FLJ00068 has been identified as an activator of Rac1. On the other hand, the mechanisms whereby Akt2 and Rac1 are regulated in parallel downstream of phosphoinositide 3-kinase are also proposed. Herein, we aimed to provide additional evidence that support a critical role for Akt2 in insulin regulation of Rac1 in mouse skeletal muscle. Knockdown of Akt2 by RNA interference abolished Rac1 activation following intravenous administration of insulin or ectopic expression of a constitutively activated phosphoinositide 3-kinase mutant. The activation of another small GTPase RalA and GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated form of phosphoinositide 3-kinase, but not Rac1, were also diminished by downregulation of Akt2 expression. Collectively, these results strongly support the notion that Rac1 acts downstream of Akt2 leading to the activation of RalA and GLUT4 translocation to the sarcolemma in skeletal muscle.

The Teramoto distal tibial oblique osteotomy (DTOO): surgical technique and applicability for ankle osteoarthritis with varus deformity.

We have devised a medial peri-articular osteotomy, the distal tibial oblique osteotomy (DTOO), and have used this technique since 1994 for ankle osteoarthritis of advanced and late stages associated with varus inclination. This report describes the surgical technique and its applicability. DTOO can be used for cases of varus ankle osteoarthritis with a range of the ankle joint movement of at least 10° or more. The osteotomy is obliquely directed cut across the distal tibia from proximal-medial to distal lateral and is of an opening-wedge type with the centre of rotation coincident with the centre of the tibiofibular joint. A laminar spreader instrument is inserted in the osteotomy to open the wedge until the lateral surface of the talar body is seen on X-ray to be in contact and congruent with medial articular surface of the lateral malleolus. Common obstacles which may prevent this contact and congruency are bony spurs present on the anterior side of fibula or on the lateral side of the tibia; these require removal. The opening-wedge osteotomy is held in position by an Ilizarov external fixator or internally fixed with a plate. Bone graft is taken from the iliac crest and inserted into the open wedge. If, after completion of the osteotomy, the dorsiflexion angle of the ankle joint does not exceed 0°, a Z-lengthening is performed of the Achilles tendon. In the DTOO for ankle osteoarthritis, the contact area of the ankle joint increases and decreases the load pressure per unit area. Furthermore, as the width of the ankle mortice is restored through the realignment of the body of the talus, instability at the ankle joint decreases. There is additional improvement with restoration of the inclination of the distal tibial articular surface as this directs the hindfoot valgus and corrects the alignment of the foot, with consequent improvement of ankle pain.

In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action.

Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

Role for RalA downstream of Rac1 in skeletal muscle insulin signalling.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. The small GTPase Rac1 has been implicated in this insulin signalling, but the mechanism whereby Rac1 stimulates GLUT4 translocation remains obscure. In the present study, we examined the role of the small GTPase RalA downstream of Rac1 in skeletal muscle fibres isolated from genetically modified mice. A dominant-negative mutant of RalA, when ectopically overexpressed, significantly reduced GLUT4 translocation in response to insulin or either one of constitutively activated mutants of Rac1 and its upstream regulators, including the guanine-nucleotide-exchange factor FLJ00068, the protein kinase Akt2 and phosphoinositide 3-kinase. Constitutively activated Rac1 also failed to induce GLUT4 translocation in mouse skeletal muscle fibres in which the expression of RalA was abrogated by specific siRNA molecules. Furthermore, we applied a novel approach to detect the activated form of RalA in situ by immunofluorescence microscopy of mouse skeletal muscle fibres, demonstrating that constitutively activated mutants of Rac1 and its upstream regulators as well as insulin indeed cause the activation of RalA. Notably, this RalA activation was remarkably impaired in rac1-deficient skeletal muscle fibres. Taken together, these results provide evidence that RalA is indeed activated and involved in the regulation of GLUT4 translocation in response to insulin downstream of Rac1 in mouse skeletal muscle.

Immunofluorescent detection of the activation of the small GTPase Rac1 in mouse skeletal muscle fibers.

The small GTPase Rac1 acts as a molecular switch of intracellular signaling in mammals. For understanding the regulatory mechanism, it is important to identify subcellular locations in which Rac1 is activated following multiple extracellular stimuli. However, it is difficult to detect Rac1 activation in situ in animal tissues, and thus a novel method is highly desirable. Here, we report a simple method to visualize the activation of endogenous Rac1 in mouse skeletal muscle fibers. In this assay, specific interaction between activated Rac1 and a binding polypeptide is detected by immunofluorescent microscopy. This approach is readily applicable to other small GTPases.

Phospholipase cε, an effector of ras and rap small GTPases, is required for airway inflammatory response in a mouse model of bronchial asthma.

BACKGROUND: Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma. METHODS: We prepared a mouse model of allergic asthma using PLCε+/+ mice and PLCεΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells. RESULTS: After OVA challenge, the OVA-immunized PLCεΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCεΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCεΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency. CONCLUSIONS: PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.

Role of the guanine nucleotide exchange factor in Akt2-mediated plasma membrane translocation of GLUT4 in insulin-stimulated skeletal muscle.

The small GTPase Rac1 plays a key role in insulin-promoted glucose uptake mediated by the GLUT4 glucose transporter in skeletal muscle. Our recent studies have demonstrated that the serine/threonine protein kinase Akt2 is critically involved in insulin-dependent Rac1 activation. The purpose of this study is to clarify the role of the guanine nucleotide exchange factor FLJ00068 in Akt2-mediated Rac1 activation and GLUT4 translocation in mouse skeletal muscle and cultured myocytes. Constitutively activated FLJ00068 induced GLUT4 translocation in a Rac1-dependent and Akt2-independent manner in L6 myocytes. On the other hand, knockdown of FLJ00068 significantly reduced constitutively activated Akt2-triggered GLUT4 translocation. Furthermore, Rac1 activation and GLUT4 translocation induced by constitutively activated phosphoinositide 3-kinase were inhibited by knockdown of FLJ00068. In mouse gastrocnemius muscle, constitutively activated FLJ00068 actually induced GLUT4 translocation to the sarcolemma. GLUT4 translocation by constitutively activated FLJ00068 was totally abolished in rac1 knockout mouse gastrocnemius muscle. Additionally, we were successful in detecting the activation of Rac1 following the expression of constitutively activated FLJ00068 in gastrocnemius muscle by immunofluorescence microscopy using an activation-specific probe. Collectively, these results strongly support the notion that FLJ00068 regulates Rac1 downstream of Akt2, leading to the stimulation of glucose uptake in skeletal muscle.