The sentrin-conjugating enzyme mUbc9 interacts with GLUT4 and GLUT1 glucose transporters and regulates transporter levels in skeletal muscle cells.

Glucose transport in insulin-regulated tissues is mediated by the GLUT4 and GLUT1 transporters. Using the yeast two-hybrid system, we have cloned the sentrin-conjugating enzyme mUbc9 as a protein that interacts with the GLUT4 COOH-terminal intracellular domain. The mUbc9 enzyme was found to bind directly to GLUT4 and GLUT1 through an 11-aa sequence common to the two transporters and to modify both transporters covalently by conjugation with the mUbc9 substrate, sentrin. Overexpression of mUbc9 in L6 skeletal muscle cells decreased GLUT1 transporter abundance 65%, resulting in decreased basal glucose transport. By contrast, mUbc9 overexpression increased GLUT4 abundance 8-fold, leading to enhanced transport stimulation by insulin. A dominant-negative mUbc9 mutant lacking catalytic activity had effects opposite to those of wild-type mUbc9. The regulation of GLUT4 and GLUT1 was specific, as evidenced by an absence of mUbc9 interaction with or regulation of the GLUT3 transporter isoform in L6 skeletal muscle cells. The mUbc9 sentrin-conjugating enzyme represents a novel regulator of GLUT1 and GLUT4 protein levels with potential importance as a determinant of basal and insulin-stimulated glucose uptake in normal and pathophysiological states.

In vitro secretion of interleukin-1 beta and interferon-gamma by peripheral blood lymphomononuclear cells in diabetic patients.

There is evidence that cytokines, in particular interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) might mediate beta cell destruction in type 1 diabetes. Therefore the secretion of these cytokines by peripheral blood lymphomononuclear cells (PBMNC) was investigated in basal conditions and 48 h after stimulation with T-cell mitogen phytohaemagglutinin (PHA) in 33 diabetic patients and in 10 normal controls. The patients were divided in 4 groups (Group 1, 10 controls; Group 2, 13 newly diagnosed type 1 diabetics, the onset had occurred from 5 days to 3 months before the study; Group 3, 10 Long Standing (LS) type 1 diabetics with duration of the disease between 2 years and 10 years; and Group 4, 10 type 2 diabetics). No difference was found among the 4 groups considered in IL-1 beta secretion by unstimulated cultures, although the percentage of TAC+ cells was significantly higher in type 1 newly diagnosed diabetic patients with respect to the LS, the type 2 diabetics and the controls. After PHA stimulation a significant increase of IL-1 beta was found in newly diagnosed type 1 diabetic patients in comparison with the control subjects, the LS and type 2 diabetic patients (P < 0.001). The supernatants of newly diagnosed type 1 diabetics also showed a significant reduction in IFN-gamma production both in basal (P < 0.01) and in stimulated conditions (P < 0.001) in comparison with the controls, the LS (P < 0.002 in basal, and P < 0.001 in stimulated conditions) and the type 2 diabetic patients (P < 0.001 both in basal and stimulated conditions).(ABSTRACT TRUNCATED AT 250 WORDS)