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Insulin biosynthesis in HIT cells. Effects of glucose, forskolin, IBMX, and dexamethasone
G Gold1, R L Qian, G M Grodsky
1Metabolic Research Unit, University of California, San Francisco 94143.
Abstract:
Glucose, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone were tested as regulators of proinsulin biosynthesis in HIT T-15 cells, which are glucose-responsive simian virus 40-transformed hamster beta-cells. Rate of [3H]leucine incorporation into proinsulin was increased as glucose concentrations were raised from 0 to 20 mM. Biosynthetic rate increases were significant after 48 but not at 4 or 24 h of glucose and were greater for proinsulin than for total extractable proteins. After 48 h, glucose-stimulated proinsulin biosynthesis was unaffected by 10(-6) M forskolin and/or 3 x 10(-5) M IBMX but was specifically and significantly inhibited by 10(-6) M dexamethasone. Four hours of exposure to dexamethasone had no effect. When cells were incubated for 24 h and then continuously labeled for an additional 24 h, cellular conversion of labeled proinsulin to insulin was increased by glucose, and this increase was reversed or inhibited by 10(-6) M dexamethasone. Therefore, proinsulin biosynthesis in transformed HIT T-15 cells is regulated in several ways by metabolites and hormones in a manner that compares with biosynthetic regulation in normal beta-cells.
Insights
Glucose stimulates proinsulin biosynthesis in HIT T-15 cells, with significant increases observed after 48 hours. Dexamethasone inhibits this glucose-stimulated process and the conversion of proinsulin to insulin.
Area of Science:
- Endocrinology
- Cell Biology
- Metabolic Research
Background:
- HIT T-15 cells are glucose-responsive hamster beta-cells used to study insulin regulation.
- Understanding proinsulin biosynthesis is crucial for diabetes research.
Purpose of the Study:
- To investigate the regulatory effects of glucose, forskolin, IBMX, and dexamethasone on proinsulin biosynthesis.
- To compare regulatory mechanisms in transformed beta-cells with normal beta-cells.
Main Methods:
- HIT T-15 cells were treated with varying glucose concentrations.
- Incorporation of [3H]leucine was measured to assess biosynthetic rates.
- Effects of forskolin, IBMX, and dexamethasone on proinsulin biosynthesis and conversion were evaluated.
Main Results:
- Increased glucose concentrations significantly enhanced proinsulin biosynthesis after 48 hours.
- Dexamethasone (10(-6) M) specifically inhibited glucose-stimulated proinsulin biosynthesis and proinsulin-to-insulin conversion.
- Forskolin and IBMX did not affect glucose-stimulated proinsulin biosynthesis.
Conclusions:
- Proinsulin biosynthesis in HIT T-15 cells is regulated by glucose in a time-dependent manner.
- Dexamethasone plays a significant inhibitory role in proinsulin biosynthesis and maturation.
- These findings highlight similarities between transformed and normal beta-cell regulation.