Accumulation of xylitol in the mammalian lens is related to glucuronate metabolism

Abstract

Cataract remains the major cause of blindness worldwide and a common complication of diabetes. Polyol Preparation of lens lysates accumulation in the lens is associated with cataract formation.Freshly dissected bovine or rat lenses were homgenised in 2 ml of a Here we present evidence for a novel pathway for xylitol suitable ice-chilled assay buffer, using a manual Braun Homogeniser. production in the lens involving glucuronate metabolism. Xylitol Membranes and cell debris were removed by brief centrifugation in a can be produced in rat and bovine lens from glucose, via the benchtop centrifuge (at maximum speed for 2 rain) and the cleared enzymes myo-inositol-oxygen oxidoreductase, D,glucuronate lysates were then stored on ice and assayed immediately for the enreductase, L-gulonate NAD+-3-oxidoreductase and L-iditolzyme of interest. If crude lysates failed to demonstrate significant NAD+-5-oxidoreductase, which have been found in the mammaenzyme activity, samples of lysate were separated by gel-filtration lian lens for the first time. Glucuronate reductase has been chromatography on a Pharmacia Hi Prep Sephacryl S-300 column purified and was inhibited by thiol quenching reagents. UDPand fractions in the predicted molecular weight range of the enzyme glucuronyl transferase is also present in mammalian lenses; this of interest were then assayed individually.enzyme may be an anti-toxic defense mechanism in the lens. Enzyme assaysAll substrates and buffer components, other than those specified Key words." Gulonate; myo-Inositol; Glucuronyl transferase; separately, were obtained from Sigma Biochemicals. SpectrophotoXylulose; Lens; Cataract; Aging metric analysis was carried out using a Beckman DU-70 spectrophotometer with a water-heated cuvette holder. myo-Inositol-l-phosphatesynthetase, myo-Inositol-l-phosphate synthetase was assayed by a modification of the method of Eisenberg [10]. Lens lysates contain many potentially glucose-6-phos-1. Introduction phate-utilising enzymes and addition of labeled G-6-P to a crude lens lysate would therefore yield a large number of different labeled prodXylose-fed young rats accumulate polyols, including xylitol ucts, making it difficult to identify any inositol-l-phosphate produced, myo-Inositol-phosphate synthetase has been reported in [1] and develop cataractous lesions. Kinoshita and colleagues non-lens tissues as having a molecular mass of approx. 215 kDa have proposed that 'sugar' cataract arises through osmotic [10]. HPLC gel filtration was used to isolate fractions of lens lysate damage to the lens as a direct result of polyol accumulation corresponding to that molecular mass range and those fractions were [2,3]. However, whilst xylose-fed adult rats accumulate similar then pooled and assayed for myo-inositol-l-phosphate synthetase acpolyol levels to young rats, they fail to develop cataract sugtivity. Incubations were carried out in 50 mM sodium phosphate buffer containing 1 mM NAD +, 1 mM unlabeled G-6-P and 0.1 gesting that the relationship between polyol levels...

myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience

“…MIOX, ALR1, gulonate dehydrogenase, xylulose reductase, and xylulose kinase) could be demonstrated in kidney tissues (Fig. 2D), which confirmed the existence of a G-X pathway in the kidney cortical tubules, as it has been described in the eye lens previously and is akin to the polyol pathway (25)(26)(27)(28).…”

“…Following another phenol-chloroform extraction, the RNA was reprecipitated with ethanol. About 2 g of total RNA was subjected to the first strand cDNA synthesis using Moloney murine leukemia virus-RT and oligo(dT) 25 as a primer. The cDNA was suspended in 250 l of 10 mM Tris-EDTA buffer, pH 7.4, and kept at Ϫ70°C until further use.…”

“…Later, it was found to be a glycolytic enzyme, MIOX (24), which catabolizes myo-inositol to D-glucuronate via the glucuronate-xylulose (G-X) pathway as originally described in the eye lens (25), whereas it has been proposed to be operative in the kidney as well, and its metabolites enter into the pentose pathway (26,27). Past studies indicate that the promoter region of MIOX includes osmotic, carbohydrate, and oxidant/antioxidant response elements and that its transcription is heavily regulated by organic osmolytes, high glucose ambience, and oxidant stress (20,28,29).…”

Effects of Xylose on Monkey Lenses in Organ Culture: A Model for Study of Sugar Cataracts in a Primate

“…There seems to be no reason to doubt that the xylitol detected in the lenses comes from xylose in the medium, although Hayman et al (1966) reported the presence of enzymes in rabbit lenses capable of synthesizing xylulose from glucuronate, and Goode, Lewis and Crabbe (1996) speculated that this pathway was responsible for lenticular production of xylitol from glucose (via myoinositol, glucuronate, gulonate, keto-gulonate, and xylulose, which could be converted to xylitol by SDH). However, no measurable xylitol was found in the lenses incubated without xylose, even in the presence of high concentrations of glucose or other sugars.…”

Structure/function analysis of a critical disulfide bond in the active site of l-xylulose reductase

“…The enzyme is highly expressed in liver and kidney of the human and has been shown to be localized in the brush-border membranes of proximal tubular cells of the mouse kidney, suggesting a role for the enzyme in water reabsorption and cellular osmoregulation by producing xylitol [3]. Moreover, enzymes of the glucuronic acid pathway are present in mammalian lens, and the flux of sugars and xylitol through this pathway has been suggested to be involved in the osmoregulation process of the lens and the etiology of sugar cataracts [4]. In apoptosis of human T lymphoma cells induced by 9,10-phenanthrenequinone, a major component in diesel exhaust particles, XR is upregulated and deteriorates the apoptotic signaling through the generation of reactive oxygen species [5].…”