Inhibition of Contraction-Stimulated AMP-Activated Protein Kinase Inhibits Contraction-Stimulated Increases in PAS-TBC1D1 and Glucose Transport Without Altering PAS-AS160 in Rat Skeletal Muscle

OBJECTIVE

Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays.

RESEARCH DESIGN AND METHODS

To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction.

RESULTS

Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser^21/9glycogen synthase kinase 3α/β (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser⁷⁹acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160.

CONCLUSIONS

These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt–dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt–dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.Insulin and contractile activity, the two most important physiological stimuli that increase glucose transport in skeletal muscle, can each induce the translocation of GLUT4 glucose transporters from the cell''s interior to its surface membranes (1,2). However, they regulate glucose transport via distinct signaling pathways (3). Insulin-stimulated glucose transport requires phosphatidylinositol (PI) 3-kinase activation, which leads to Akt activation without stimulating AMP-activated protein kinase (AMPK) (3–6). A great deal of evidence suggests that contraction stimulates glucose transport by a mechanism independent of PI 3-kinase/Akt (7–10) and attributable to the effects of multiple inputs, with AMPK- and calcium-mediated processes being major factors (11,12).In 3T3-L1 adipocytes, insulin stimulates phosphorylation of Akt substrate of 160 kDa (AS160; also called TBC1D4) in an Akt-dependent manner on sites identifiable by the phospho-Akt substrate (PAS) antibody (13,14). AS160 includes a Rab GTPase-activating protein domain (RabGAP) that inhibits Rab proteins involved in regulating vesicular traffic (15). The insulin-mediated increase in PAS phosphorylation of AS160 (PAS-AS160) appears to inhibit RabGAP activity, thereby allowing GLUT4 to be recruited to surface membranes and elevate glucose transport (14–17). In skeletal muscle, insulin or contraction results in elevated PAS-AS160 (18,19), and AS160 phosphorylation appears to regulate glucose transport (20).Recently, TBC1D1, a RabGAP protein paralog to AS160, was also shown to become PAS-phosphorylated (PAS-TBC1D1) in response to insulin in an Akt-dependent manner (21). However, whereas AS160 knockdown in 3T3-L1 adipocytes resulted in elevated basal cell-surface GLUT4 (17,22), TBC1D1 knockdown had no effect on basal cell-surface GLUT4 in 3T3-L1 cells (23). TBC1D1 protein is only ∼5% as abundant as AS160 protein in 3T3-L1 adipocytes, which may explain why TBC1D1 does not appear to play a major role in regulating glucose transport in these cells (23). TBC1D1 protein abundance is much greater in skeletal muscle versus adipose tissue (24), and silencing TBC1D1 in L6 myotubes resulted in increased basal cell-surface GLUT4 (25), supporting the idea that TBC1D1 inhibits GLUT4 translocation in the basal state. However, in contrast to the results for L6 cells with AS160 knockdown (which did not alter the insulin-stimulated net increase in cell-surface GLUT4), silencing TBC1D1 in L6 cells resulted in greater insulin-induced GLUT4 translocation versus control cells (25). In other words, TBC1D1 knockdown allowed insulin to induce a greater amount of GLUT4 translocation than in cells that express TBC1D1. These findings suggest that at least a portion of the inhibitory effects of TBC1D1 on GLUT4 may not be restrained by insulin. However, they do not eliminate the possibility that TBC1D1 can regulate an insulin-independent increase in glucose transport (e.g., with contraction). PAS-TBC1D1 is elevated in response to contraction in rodent skeletal muscle (19,24). Therefore, it seems possible that PAS-TBC1D1 may play a role in mediating contraction-stimulated glucose transport.Experiments using purified Akt or AMPK demonstrated that each kinase can phosphorylate both AS160 and TBC1D1 in cell-free assays (26,27). Considerable evidence indicates that the insulin-stimulated increase in PAS-AS160 is Akt dependent in skeletal muscle (18,28), and increased AS160 phosphorylation appears to be important for the full effect of insulin on glucose transport (20). However, the specific kinases responsible for contraction-stimulated PAS-AS160 need to be clarified because: 1) Wortmannin can completely inhibit the contraction-stimulated increase in PAS-AS160 in rat skeletal muscle, suggesting that Akt is responsible for the increased PAS-phosphorylation of AS160 during contraction (18), but 2) muscles from mice with genetically disrupted AMPK versus wild-type littermates had reduced contraction-stimulated increase in immunoreactivity toward PAS antibody at ∼160 kDa (PAS-160) (28,29).The primary aim of this study was to elucidate the contributions of Akt and AMPK on increases in PAS-AS160 and PAS-TBC1D1 in skeletal muscle stimulated by insulin or contraction. The PI 3-kinase inhibitor Wortmannin was used to prevent Akt activation (without altering AMPK activation), and compound C, a potent AMPK inhibitor (30), was used to prevent AMPK activation (without altering Akt activation). A secondary aim was to determine whether inhibition of insulin- or contraction-stimulated increases in PAS-AS160 or PAS-TBC1D1 was accompanied by attenuated insulin- or contraction-stimulated glucose transport. We hypothesized that in isolated rat epitrochlearis muscle: 1) Akt-dependent mechanisms are essential for the insulin-stimulated increases in glucose transport and phosphorylation of AS160 and TBC1D1; 2) Akt-dependent (but not AMPK-dependent) mechanisms are essential for contraction-stimulated increases in PAS-AS160, but not glucose transport; and 3) AMPK-dependent (but not Akt-dependent) mechanisms are essential for contraction-stimulated increases in PAS-TBC1D1 (but not PAS-AS160) and glucose transport.     

Influence of stability on range of motion after cruciate-retaining TKA

BACKGROUND: A loosely balanced total knee arthroplasty (TKA) is reported to produce a good postoperative range of motion (ROM), but too much laxity is thought to be the cause of persistent pain and worsened functionality. METHODS: The anteroposterior and mediolateral laxity values were measured to evaluate the influence of stability after cruciate-retaining (CR) TKA on ROM and the modified Knee Society score at 4-8 years after the operation. Twenty-one knees in 15 patients with an average age of 68 years who had undergone a CR TKA for osteoarthrosis were examined. The mean preoperative and postoperative ROM was 124 degrees and 112 degrees, respectively. The mean anteroposterior and mediolateral laxity values were 9.7 mm and 10.6 degrees, respectively. RESULTS: No correlation was found between the postoperative ROM and laxity or between the modified Knee Society score and laxity. A loosely balanced TKA did not produce a good postoperative ROM. No parameters suggested that lax knees showed a higher pain score and lower functional score than stable knees.     

Basic and clinical studies of percutaneous radiofrequency ablation of osteoid osteoma using a standard electrosurgical generator

 For percutaneous radiofrequency ablation of osteoid osteoma for pain management, we used a standard electrosurgical generator instead of the radiofrequency generator system. First, we used the standard electrosurgical generator to determine the diameter of the coagulated area of normal femurs and humeruses of dogs under general anesthesia and to detect damage to normal tissue around the bone. We then used a standard electrosurgical generator to perform percutaneous radiofrequency ablation of the osteoid osteoma. All three patients were almost pain-free within the first 24 h after the procedure, and they were discharged the day after the operation, being hospitalized for only 2 days. We thus confirmed that percutaneous radiofrequency ablation using a standard electrosurgical generator produced results similar to those achieved with the radiofrequency generator system. Received: August 19, 2002 / Accepted: January 11, 2003 RID="*" ID="*" Offprint requests to: A. Takeda     

Clinicoanatomical studies on the dorsal subsegmental bile duct of the right anterior superior segment of the human liver

BACKGROUND: The dorsal subsegmental intrahepatic bile duct in the right anterior superior segment (B8c) sometimes joins the posterior sectorial duct. In such cases it can be misidentified as the right posterior superior segmental duct (B7). However, there are no published studies on the confluent pattern of B8c. MATERIALS AND METHODS: We studied B8c in the resected liver of 107 patients (65 with bile duct carcinoma and 42 with gallbladder carcinoma) who had undergone right hepatectomy or more extensive right-sided liver resection. RESULTS: B8c was identified in all cases. It joined the right posterior sectorial duct or B7 in 18 cases (16.8%). In 12 cases B8c joined independently the posterior sectorial duct or B7. In 6 cases B8c joined the posterior sectorial duct after making the common duct with the lateral subsegmental duct in the anterior superior or anterior inferior segment (B8b or B5c). CONCLUSIONS: B8c does not join the anterior sectorial bile duct in every sixth case.     

Hepatectomy with portal vein resection for hilar cholangiocarcinoma: audit of 52 consecutive cases

OBJECTIVE: To better determine the role of portal vein resection and its effect on survival, as well as to appreciate the impact of portal vein invasion on prognosis in hilar cholangiocarcinoma. SUMMARY BACKGROUND DATA: Hepatectomy with portal vein resection is sometimes performed for locally advanced hilar cholangiocarcinoma. However, the significance of microscopic invasion of the portal vein has not been determined. METHODS: Medical records of 160 patients with hilar cholangiocarcinoma who underwent macroscopically curative hepatectomy with (n = 52) or without portal vein resection (n = 108) were reviewed. Invasion of the portal vein was assessed histologically on the surgical specimen, and results were correlated with clinicopathologic features and survival. RESULTS: Surgical mortality, including all hospital deaths, was similar in patients who did and did not undergo portal vein resection (9.6% vs. 9.3%), but the primary tumor was more advanced in patients who underwent portal vein resection. Histologically, no invasion was found in 16 (30.8%) of resected portal veins. However, dense fibrosis adjacent to the portal vein was common, and the mean distance between the leading edge of cancer cells and the adventitia of the portal vein was 437 +/- 431 mum. The prognosis was worse in patients with than without portal vein resection (5-year survival, 9.9% vs. 36.8%; P < 0.0001). The presence or absence of microscopic invasion of the resected portal vein did not influence survival (16.6 months in patients with microscopic invasion vs. 19.4 months in those without; P = 0.1506). Multivariate analysis identified histologic differentiation, lymph node metastasis, and macroscopic portal vein invasion as independent prognostic factors. CONCLUSIONS: Microscopic invasion of the portal vein may be misdiagnosed clinically in patients with hilar cholangiocarcinoma. However, the distance between tumor and adventitia is so narrow that curative resection without portal vein resection is unlikely to be possible. Gross portal vein invasion has a negative impact on survival, and hepatectomy with portal vein resection can offer long-term survival in some patients with advanced hilar cholangiocarcinoma.     

Combined aortic valve replacement and coronary artery bypass grafting with in situ arterial grafts for porcelain aorta.

Patients with porcelain aorta carry a high risk of cerebral as well as systemic embolism during cardiac surgery. Here we describe a case of severe aortic stenosis and coronary artery disease combined with the circumferentially calcified aorta. The patient was a 77-year-old man who successfully received four coronary artery bypass grafts with in situ arterial grafts without clamping the aorta and aortic valve replacement. Aortic valve replacement and two distal coronary artery anastomoses to the left circumflex artery and obtuse marginal branch were performed under cardiac arrest during hypothermic perfusion with endoaortic balloon occlusion, followed by partial endarterectomy and closure of the aorta buttressed with bovine pericardium under deep hypothermic circulatory arrest. While rewarming, the other two distal coronary anastomoses to the left anterior descending artery and diagonal branch were done on the beating heart in order to minimize cardiac arrest time. On-pump beating heart coronary artery bypass grafting (CABG) can be useful especially for combined complex cardiac surgery.     

Changes of D-dimer after total hip arthroplasty in patients with and without intraoperative heparin

Introduction Marked activation of thrombosis is common in patients undergoing total hip arthroplasty, especially during reaming of the femur and after insertion of the femoral prosthesis. This suggests that management designed to minimize deep vein thrombosis and fatal pulmonary embolism after total hip arthroplasty should be focused on the period during insertion of the femoral component. In some previous studies, a low dose of heparin administered intraoperatively was shown to suppress the formation of fibrin. Objective The present study was performed to evaluate the influence of intraoperative heparin administration on the D-dimer level and on the prevention of pulmonary embolism after total hip arthroplasty. Material/methods A total of 22 and 26 consecutive patients respectively underwent total hip arthroplasty with and without intraoperative administration of unfractionated heparin. Postoperatively, all patients wore knee-high elastic stockings and were fitted with calf-to-thigh intermittent pneumatic compression devices. Active ankle flexion and extension exercises were commenced as soon as motor function recovered. None of the 48 patients received prophylactic anticoagulants postoperatively. Results There was a significant difference of the mean D-dimer level on the 1st day between the patients with and without intraoperative administration of heparin (8.9 ± 6.6 vs. 15.7 ± 12.7, P < 0.05). Although there were no patients with symptomatic deep venous thrombosis and pulmonary embolism, asymptomatic pulmonary embolism was detected by pulmonary perfusion scintigraphy in three patients who did not receive intraoperative heparin. The operative blood loss and postoperative drainage were similar in both groups and no bleeding complications were observed. In conclusion, we recommend a safe and inexpensive regimen comprising 1,000 U of intravenous unfractionated heparin intraoperatively, postoperative pneumatic compression, and early active mobilization for prevention of thoromboembolic complications after total hip arthroplasty.     

Predictive value of blood flow in the gastric tube in anastomotic insufficiency after thoracic esophagectomy

Anastomotic insufficiency is considered to be one of the most serious complications associated with esophageal reconstruction. The purposes of this study were to identify (1) the relationship between anastomotic insufficiency and tissue blood flow (TBF) in the gastric tube in the perioperative period, and (2) the effects of intravenous prostaglandin E1 (PGE1) on TBF in the gastric tube. The study group consisted of 44 patients who were to undergo esophagectomy for esophageal cancer. Intraoperative and postoperative TBF on the serosal side of the gastric tube were measured by laser-Doppler tissue blood flowmetry. The TBF of the Leakage(+) group (n = 5) was poorer than that of the Leakage(?) group (n = 39) during the intraoperative and postoperative periods. There was a significant difference in TBF between the two groups at postoperative day (POD) 3. There was a tendency in the PGE1(+) group (n = 18) to exhibit richer blood flow through the anastomosis than the PGE1(?) group (n = 26), intraoperatively, but the difference was not significant. Two of five Leakage(+) cases were also in the PGE1(+) group. There was no relationship between intraoperative medication with PGE1 and incidence of leakage. The TBF of three-field lymph node dissection and reconstruction of the retrosternal route group (n = 21) was poorer than that of the two-field lymph-node dissection and reconstruction of the posterior mediastinal route group (n = 23). The TBF in the gastric tube after esophagectomy may be a predictor of anastomotic insufficiency. However, PGE1 treatment in the intraoperative period alone is not effective in preventing anastomotic insufficiency.     

Hemangiopericytoma of the Greater Omentum

A 41-year-old Chinese woman was admitted to our hospital with epigastric pain. Computed tomography detected a heterogeneous enhancement tumor fed by the left gastroepiploic artery in the left lower quadrant and cholelithiasis. Excision of the tumor in the greater omentum and cholecystectomy were performed laparoscopically. Histological findings confirmed a diagnosis of hemangiopericytoma with low-grade malignancy. To our knowledge, hemangiopericytoma of the greater omentum is very rare, and only 12 cases were reported in English literature. We report a case of hemangiopericytoma arising in the greater omentum and review the literature.     

Laparoscopic Intragastric Full-thickness Excision (LIFE) of Posterior Gastric Lesions under Flexible Endoscopic Control—A Feasibility Study

Background We have developed a new technique for treatment of intramucosal carcinoma which exceeds the standard indication for endoscopic mucosal resection and carcinoma invading the submucosa without lymph node metastasis that are located in the posterior wall of the stomach, which we refer to as laparoscopic intragastric full-thickness excision (LIFE) under flexible endoscopic control. Surgical Technique Three pigs were used for the study. Three trocars were used. The first trocar (trocar # 1) was placed in the subumbilical region to introduce the videoscope, whereas the second and third trocars (trocar # 2 and trocar # 3) were punctured percutaneously into the abdominal cavity. A straight needle with 3-0 silk suture was attached to a T-bar on the wire side and inserted into the abdominal cavity. An area adjacent to the lesion in the posterior wall of the stomach was pierced by the straight needle, which was then pulled into the stomach using the forceps of the endoscope. The T-bar, after being passed through the abdominal wall, was fixed outside the gastric wall, and trocar # 3 was repositioned in the stomach by the percutaneous transgastric route. The posterior wall of the stomach was pulled inward by the T-bar, and the lesion was removed by several excisions with laparoscopic stapling devices inserted through trocar # 3; extraction of the specimen was achieved through trocar # 3. The gastrotomy site was suture-closed using instruments positioned through trocar # 2 and trocar # 3 under laparoscopy. Conclusions Based on a feasibility study in pigs, the LIFE procedure can be performed for lesions of the posterior wall of the stomach.