An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gßγ peptidic inhibitor ßARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ßARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.

Isolation of viable porcine islets by selective osmotic shock without enzymatic digestion.

Islet transplantation is a potential cure for type 1 diabetes, but clinical results have been disappointing. Currently, islet isolation is by enzymatic digestion of the pancreas which has significant pitfalls: warm ischemia exposure, collagenase-induced damage to the islet mass and viability, poor reproducibility, high cost, a relatively low number of islets obtained per whole pancreas, and selection of islets for collagenase resistance rather than for glucose responsiveness. In the present study we performed a series of experiments in a porcine model to demonstrate the feasibility of a new isolation method based on selective osmotic shock (SOS) using very high glucose solutions, doubling or tripling physiological osmotic strength. The SOS method can be carried out at room temperature or in the cold eliminating warm ischemia time which damages the islets. The SOS method does not depend on the texture of the pancreas so all pancreases can be processed identically and the process can be fully automated. The SOS method isolates all the islets of the pancreas regardless of size and shape allowing a greater number of islets to be harvested. The SOS method avoids exposure to toxins in collagenase solutions, is inexpensive and selects for islets with high concentrations of Glut 2 transporters, representing the best glucose responding islets. The SOS method showed a comparable recovery of islets from young pig pancreas and the islets showed improved viability. We conclude that the selective osmotic shock (SOS) method of separating islets from the pancreatic tissue is superior to the collagenase method.

Treatment of chronic infected hip arthroplasty wounds by radical debridement and obliteration with pedicled and free muscle flaps.

Nine patients with extensive wounds of the hip joint due to chronic infection following total hip arthroplasty or internal fixation of fractures of the femoral head and neck have been treated by serial radical debridements to remove infected bone, contaminated remnants of bone cement, and the surrounding fibrotic soft tissues. The resultant deep cavity extending down to the acetabulum has then been obliterated with either pedicled muscle flaps or free muscle flaps. Subcutaneous or transpelvic transposition of rectus abdominis muscle flaps is preferred for smaller defects, but only the free latissimus dorsi muscle flap provides sufficient volume of tissue to obliterate the more extensive hip defects. Systemic antibiotics have been continued only for a short-term course of 14 days postoperatively. There has been no recurrence of infection, with follow-up ranging between 6 months and 3 1/4 years. One patient has undergone reimplantation of a second custom hip prosthesis into the vascularized bed of a free latissimus dorsi muscle flap.

The "double barrel" free vascularized fibular bone graft.

A further modification of the free vascularized fibular bone graft is described in which a transverse osteotomy is made from the anterolateral aspect of the fibular shaft just distal to the entry of the nutrient artery. This produces two vascularized bone struts that may be folded parallel to each other but that remain connected by the periosteum and muscle cuff surrounding the peroneal artery and vein. The proximal strut is vascularized by both a periosteal and an endosteal blood supply, whereas the distal strut is vascularized by a periosteal blood supply alone. This so-called "double barrel" free vascularized fibular graft has been employed in three patients with segmental bone defects of the distal femur and in one patient with adjacent bony defects of the radius and ulna.