Rapid reduction and return of surface insulin receptors after exposure to brief pulses of insulin in perifused rat hepatocytes

Cellular respiratory rates are normally determined by metabolic activity, but become rate limited by O2 availability if the cell O2 tension (PO2) falls below a critical value (typically 1–10 Torr). An ability to reduce metabolic activity and energy demand in response to a falling O2 availability might confer an increased resistance to a diminished O2 supply. Isolated rat hepatocytes were studied in primary culture under controlled O2 tensions. Cells were obtained by collagenase digestion and seeded into nutritive media in control and experimental spinner flasks at identical cell densities. Cells subjected to rapid reduction in PO2 (100⇢0 Torr over < 40 min) exhibited undiminished O2 uptake until PO2 fell below 10 Torr. By contrast, when cell PO2 was reduced over several hours, significant decreases in O2 uptake became evident at O2 tensions as high as 70 Torr. These decreases were associated with a reduction in ATP concentration and an increase in NAD(P)H, compared with rapidly deoxygenated cells at the same PO2. No loss in cell viability was detected after 24 h at reduced PO2. The decrease in respiratory rate was associated with an increased rate of lactic acid production relative to normoxic controls. Restoration of normoxia was associated with an immediate return of O2 uptake to control levels. These results demonstrate that hepatocytes are capable of reversibly decreasing metabolic activity and O2 demand during sustained moderate reductions in PO2, via a mechanism that appears to involve an inhibition of mitochondrial function other than O2 supply limitation. This response may alter cellular susceptibility to physiological stresses including hypoxia.

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Internalized insulin receptors are recycled to the cell surface in rat hepatocytes.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.79.19.5921 ◽

1982 ◽

Vol 79 (19) ◽

pp. 5921-5925 ◽

Cited By ~ 86

Author(s):

M. Fehlmann ◽

J. L. Carpentier ◽

E. Van Obberghen ◽

P. Freychet ◽

P. Thamm ◽

...

Keyword(s):

Cell Surface ◽

Insulin Receptors ◽

Rat Hepatocytes

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Insulin stimulates tyrosine phosphorylation of its receptor β-subunit in intact rat hepatocytes

Biochemical Journal ◽

10.1042/bj2410099 ◽

1987 ◽

Vol 241 (1) ◽

pp. 99-104 ◽

Cited By ~ 16

Author(s):

R Ballotti ◽

A Kowalski ◽

M F White ◽

Y Le Marchand-Brustel ◽

E Van Obberghen

Keyword(s):

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Biological Effects ◽

Insulin Receptors ◽

Rat Hepatocytes ◽

Enzymic Activity ◽

Beta Subunit ◽

Isolated Rat Hepatocytes ◽

Receptor Phosphorylation ◽

Insulin Receptor Kinase

We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin.

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