Glucose regulates preproinsulin messenger RNA levels in a clonal cell line of simian virus 40‐transformed B cells

Abstract

In HIT-T15 cells grown in the absence of glucose, Northern blot analysis of total RNA revealed a major 0.5 kb preproinsulin (pp1) mRNA transcript which co-migrated with the mature transcript from a human insulinoma. In 4 h tissue cultures, glucose (2-20 mM) stimulated HIT cell pp1 mRNA levels in a markedly dose-dependent manner. Glucose-stimulated pp1 mRNA was (i) inhibited by actinomycin D, suggesting that regulation may be in part transcriptional, and (ii) potentiated by agents known to activate B cell protein kinases. HIT cells represent a unique model for investigating long term regulation of insulin gene expression and biosynthesis.

Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.

“…The probe hybridized with a 0.5-kb band on agarose gel fractionation oftotal HIT cell RNA consistent with HIT cell insulin mRNA (35). Under the hybridization conditions employed, the probe also labeled HIT cell 18S and 28S rRNA, and the density oflabeled 18S rRNA was proportional to total RNA over the range of total HIT cell RNA applied to gels.…”

Chronic exposure of HIT cells to high glucose concentrations paradoxically decreases insulin gene transcription and alters binding of insulin gene regulatory protein.

“…The observation that chronic exposure of HIT cells to high glucose concentration leads to decreased insulin gene expression represents an apparent time-related paradox. Acute increases in glucose concentrations have been shown to increase insulin mRNA levels (26)(27)(28)(29)(30) is transcribed (26,31,32). The chronic effect of glucose on insulin gene transcription that we describe and which can be partially prevented by chronically culturing HIT cells in lower glucose concentrations, may represent a direct glucose toxic effect on a insulin-gene specific transcription factor(s).…”

Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line, HIT-T15.

“…The membrane was prehybridized in 50% formamide, 5 ϫ SSC, 5 ϫ Denhardt's, 50 mM sodium phosphate, 0.1 mg/ml salmon sperm DNA, and 0.1% SDS at 42 Њ C overnight, then hybridized for 16 h with 32 P-labeled Syrian hamster preproinsulin cDNA probe (25) in the same solution. The membrane was then washed three times at room temperature in 2 ϫ SSC and 0.1% SDS, then twice at 60 Њ C in 0.2 ϫ SSC and 0.1% SDS, then exposed to x-ray films (model X-Omat AR; Eastman Kodak Co., Rochester, NY) for 4 to 12 h. Under the hybridization conditions employed, the probe hybridized to a single 0.5-kb band consistent with HIT cell preproinsulin mRNA (26).…”