Reduction of postprandial hyperglycemia in patients with type 2 diabetes reduces NF-kappaB activation in PBMCs.

AIMS/HYPOTHESIS: Short-lasting hyperglycemia results in activation of the transcription factor NF-kappaB in peripheral blood mononuclear cells. We therefore studied whether the postprandial increase in glucose is sufficient to induce mononuclear NF-kappaB activation and whether blunting postprandial hyperglycemia with the alpha-glucosidase inhibitor acarbose reduces NF-kappaB activation. METHODS: 20 patients with type 2 diabetes were included in a double-blind randomized trial receiving 100 mg acarbose or placebo three times a day over a period of eight weeks. Peripheral blood mononuclear cells were isolated before and 120 minutes after a standardized breakfast. NF-kappaB binding activity was estimated by electrophoretic mobility shift assay and NF-kappaB-p65; translocation was determined by Western blot. RESULTS: Eight weeks of treatment with acarbose significantly reduced postprandial hyperglycemia (p = 0.004 when compared to placebo), postprandial mononuclear NF-kappaB-binding activity (p = 0.045) and nuclear translocation of NF-kappaB-p65 (p = 0.02). CONCLUSION: Reduction of postprandial glucose peak levels by acarbose reduces postprandial mononuclear NF-kappaB activation.

Impaired membrane traffic in defective ether lipid biosynthesis.

The first steps of ether lipid biosynthesis are exclusively localized to peroxisomes and hence some peroxisomal disorders are characterized by a severe deficiency of plasmalogens, the main ether lipids in humans. Here we report on gene defects of plasmalogen biosynthesis, chromosomal localization of the corresponding genes and, as a consequence of plasmalogen deficiency, on structural alterations of caveolae, clathrin-coated pits, endoplasmic reticulum and Golgi cisternae, as well as on the reduced rate of transferrin receptor cycling. The data suggest that plasmalogens, analogous to cholesterol, are essential for correct membrane functioning and their deficiency results in impaired membrane trafficking.

ARF- and coatomer-mediated peroxisomal vesiculation.

The authors characterized on a molecular level the clofibrate-inducible 26-kDa integral peroxisomal membrane protein (Pmp26p, Pex11-1p) of rat liver. By screening cDNA databases with the obtained Pex11-1p-cDNA, a second homologous cDNA was identified that codes for a polypeptide with slightly larger molecular mass than Pex11-1p. The authors call this polypeptide Pex11-2p. Studies on the topology of Pex11-1p revealed two transmembrane domains with the N- and C-terminus facing the cytoplasm. The C-terminal tail of Pex11-1p ends in a consensus dilysine motif of the type -KXKXX-COOH, which is known to be involved in the ADP-ribosylation factor (ARF)1-coat protein (COP) I coat (ARF)1-dependent membrane recruitment to Golgi membranes. Studies with isolated peroxisomes incubated in the presence of cytosol, adenosine triphosphate and GTP gamma S, indeed, provided evidence for specific binding of ARF and coatomer to peroxisomes. Expression of Pex11-1p in Chinese hamster ovary (CHO) wild-type cells led to a twofold increase in the number of peroxisomes, but expression in a temperature-sensitive CHO mutant, defective in coatomer, induced elongation and tubulation of peroxisomal structures, rather than numerical proliferation. The obtained results for the first time offer a mechanism explaining Pex11-1p-, as well as ARF- and coatomer-mediated peroxisomal vesiculation. Two models are presented that may explain how these observations fit in with peroxisome biogenesis.

Synthesis of plasmalogens in eye lens epithelial cells.

The present paper describes cloning and sequencing of the mouse cDNA encoding dihydroxyacetonephosphate acyltransferase (DAPAT), the peroxisomal key enzyme of plasmalogen (PM) biosynthesis. Using monospecific antibodies, we localized DAPAT and alkyl dihydroxyacetonephosphate synthase to peroxisomes of mouse lens epithelial cells (LECs) and determined their enzymatic activity. By electrospray ionization mass spectrometry of mouse lens lipid extracts, we identified phosphatidyl ethanolamine including plasmenyl ethanolamine species as major constituents. Our data demonstrate the capacity of LECs to synthesize PMs and the high coincidence between deficiency of PM and early manifestation of cataract in patients with peroxisomal disorders suggests that ether-bonded lipids may play an important role in maintaining lens transparency.

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.