Waka Omata

A Single Intraportal Administration of Follistatin Accelerates Liver Regeneration in Partially Hepatectomized Rats

BACKGROUND/AIMS Activin A is an autocrine negative regulator of DNA synthesis in rat hepatocytes and is expressed in remnant liver after partial hepatectomy. To determine the role of activin A in liver regeneration, the effects of exogenous follistatin, which blocks the action of activin A, were examined. METHODS Human recombinant follistatin was infused into the portal vein immediately after 70% hepatectomy. Changes in body weight, remnant liver weight, liver regeneration rate, and nuclear bromodeoxyuridine labeling were measured. RESULTS In control rats, nuclear labeling was observed at 24 hours and peaked at 36 hours after the hepatectomy. In follistatin-treated rats, nuclear labeling was first observed after 18 hours and was significantly (P < 0.05) greater than that in control rats at 24 hours. In follistatin-treated rats, both remnant liver weight and liver regeneration rate were significantly greater at 120 hours. Serum concentrations of albumin and glucose remained reduced for up to 120 hours in control rats but recovered in follistatin-treated rats. CONCLUSIONS A single administration of follistatin accelerates the initial round of DNA synthesis after partial hepatectomy. Activin A produced in remnant liver may exert tonic inhibitory effect on liver regeneration. Follistatin may be useful as a potential therapeutic agent to promote liver regeneration.

The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes

The small ubiquitin-related modifier (SUMO) conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in L6 myoblast cells, although the mechanism of action has remained undefined. Here we investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector–mediated overexpression and by small interfering RNA (siRNA)-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in an inhibition of GLUT4 degradation and promoted its targeting to the unique insulin-responsive GLUT4 storage compartment (GSC), leading to an increase in GLUT4 amount and insulin-responsive glucose transport in 3T3-L1 adipocytes. Overexpression of Ubc9 also antagonized GLUT4 downregulation and its selective loss in GSC induced by long-term insulin stimulation. By contrast, siRNA-mediated depletion of Ubc9 accelerated GLUT4 degradation and decreased the amount of the transporter, concurrent with its selective loss in GSC, which resulted in attenuated insulin-responsive glucose transport. Intriguingly, overexpression of the catalytically inactive mutant Ubc9-C93A produced effects indistinguishable from those with wild-type Ubc9, suggesting that Ubc9 regulates GLUT4 turnover and targeting to GSC by a mechanism independent of its catalytic activity. Thus, Ubc9 is a pivotal regulator of the insulin sensitivity of glucose transport in adipocytes.

Direct interaction of Rab4 with syntaxin 4.

In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5′-O-(3-thiotriphosphate) (GTPγS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1–273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPγS treatment of the cells significantly increased, but guanosine 5′-O-(2-thiodiphosphate) (GDPβS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1–273) from the cell lysates; 2) GTPγS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPβS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4Q67L), but not a GTP-binding-defective mutant (Rab4S22N), was bound to GST-syntaxin 4-(1–273). Although insulin stimulated [γ-32P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4Q67L to GST-syntaxin 4-(1–273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.

Dictyostelium differentiation‐inducing factor‐1 induces glucose transporter 1 translocation and promotes glucose uptake in mammalian cells

The differentiation‐inducing factor‐1 (DIF‐1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF‐1 and its analogs have been shown to possess antiproliferative activity in vitro in mammalian tumor cells. In the present study, we investigated the effects of DIF‐1 and its analogs on normal (nontransformed) mammalian cells. Without affecting the cell morphology and cell number, DIF‐1 at micromolar levels dose‐dependently promoted the glucose uptake in confluent 3T3‐L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3‐kinase). DIF‐1 affected neither the expression level of glucose transporter 1 nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6‐phosphate kinase, pyruvate kinase, and glucose 6‐phosphate dehydrogenase. Most importantly, stimulation with DIF‐1 was found to induce the translocation of glucose transporter 1 from intracellular vesicles to the plasma membranes in the cells. In differentiated 3T3‐L1 adipocytes, DIF‐1 induced the translocation of glucose trasporter 1 (but not of glucose transporter 4) and promoted glucose uptake, which was not inhibited with wortmannin. These results indicate that DIF‐1 induces glucose transporter 1 translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3‐kinase/Akt‐independent pathway in mammalian cells. Furthermore, analogs of DIF‐1 that possess stronger antitumor activity than DIF‐1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF‐1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth.