Abstract
Hormone-sensitive lipase (HSL) is a key enzyme in fatty acid mobilization in many cell types. Two isoforms of HSL are known to date, namely HSL(adi) (84 kDa in rat) and HSL(tes) (130 kDa in rat). These are encoded by the same gene, with exons 1-9 encoding the parts that are common to both and an additional 5'-exon encoding the additional amino acids in HSL(tes). HSL of various tissues, among these the islet of Langerhans, is larger than HSL(adi), but not as large as HSL(tes), indicating that there may be other 5'-coding exons. Here we describe the molecular basis for a novel 89-kDa HSL isoform that is expressed in beta-cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1-9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon. The additional 5'-base pairs encode a 43-amino acid peptide, which is highly positively charged. Conglomerates of HSL molecules are in close association with the secretory granules of the beta-cell, as determined by immunoelectron microscopy with antibodies targeting two separate regions of HSL. We have also determined that the human genomic sequence upstream of exon A has promoter activity in INS-1 cells as well as glucose sensing capability, mediating an increase in expression at high glucose concentration. The minimal promoter is present within 170 bp from the transcriptional start site and maximal glucose responsiveness is conferred by sequence within 850 bp from the transcriptional start site.
Highlights
Because of the established association between type 2 diabetes and obesity, lipids have received much attention in research on the pathophysiology of type 2 diabetes
We describe the molecular basis for a novel 89-kDa hormone-sensitive lipase (HSL) isoform that is expressed in -cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1–9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon
HSL of the pancreatic -cell is slightly larger (89 versus 84 kDa) than the predominant isoform found in adipocytes, referred to as HSLadi, as evident when lysates of clonal -cell or islets from mouse and rat are subjected to immunoblot analysis [5]
Summary
RNA Extraction—Total RNA was extracted from tissues or cells according to Chomczynski and Sacchi [17]. A nested PCR was performed using the adapter primer 2 of the Marathon kit and a primer of the following sequence: 5Ј-AGGAATTCGCCCTGGCTTGAGAAGAAGGCCAT-3Ј (EcoRI site underlined). After washing in a buffer (phosphate-buffered saline supplemented with 0.1% Tween 20) for 15 min the grids were incubated during 2 h at room temperature with a secondary antibody (goat anti-rabbit IgG conjugated with 6-nm gold particles (Aurion), diluted 1:10 in the blocking buffer). Southern blotting was performed using a nylon membrane (Hybond N, Amersham Biosciences) and ExpressHyb hybridization solution (Clontech) according to the manufacturer’s instructions. Two l of this PCR was used for nested PCR using a gene-specific primer of the following sequence: 5Ј-GATGAATTCACAGGTAGGCCTCCAGTTC-3Ј (EcoRI site underlined) Both primers contain antisense sequence from exon 1 of the rat HSL gene. The nested PCR was performed using an exon A-specific reverse primer
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