Galactosemic Rat Lenses Research Articles

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components--ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)--suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, βB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, βB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.

The Unfolded Protein Response in Lens Epithelial Cells from Galactosemic Rat Lenses

Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats. Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation. The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.

Effects of Cataractogenesis on the CDP-Choline Pathway: Changes in ATP Concentration and Phosphocholine Synthesis during and after Exposure of Rat Lenses to Sugars in vitro and in vivo

We measured choline influx and phosphorylation, ATP concentration ([ATP]), choline kinase activity and lens swelling during formation and partial reversal of sugar cataracts in rat lenses incubated with xylose or galactose and in lenses of galactosemic rats. [ATP] and phosphocholine (P-Cho) synthesis decreased about 60 and 40% after 4 h in normal rat lenses incubated up to 24 h in medium containing 30 mM xylose and partially recovered when the lenses were then removed from the xylose. Incubation with the somewhat less cataractogenic sugar galactose decreased P-Cho synthesis but had little effect on [ATP]. P-Cho synthesis decreased rapidly in the lenses of rats fed a 50% galactose diet, but began recovery by the third day on this diet. [ATP] decreased for at least 10 days during the galactose diet and did not recover, even with resumption of the control diet (50% starch) after 4 or 7 days. The results of in vitro and in vivo sugar cataractogenesis differed from each other in several respects, including effects on choline influx and the degree to which the changes were reversible. The in vitro and in vivo sugar cataracts, however, could both produce swelling and opacification of the lens and decreased P-Cho synthesis and [ATP]. Neither model caused a substantial change in the choline kinase activity (as measured in cell-free assays). The data did not generally support the hypothesis that decreased [ATP] causes decreased P-Cho synthesis.

Raman Spectroscopy of Galactosemic Rat Lens Crystalline: Correlation of Microscopic Changes of Lens Proteins at Molecular Levels with Gross Cataractous Alteration

AbstractNear‐Infrared Fourier trans form (near‐IR FT) Raman spectros copy has been used to study the structural changes of lens proteins both in cortex and nucleus of galactosemic rat lenses. It was found that tyrosine doublet ratio of Raman bands, I832/I855, in creased more rapidly in the cortex than in the nucleus during a 5‐week period of galactose feeding, i.e., from 0.86 to 1.1 for the cortex and 0.88 to 0.92 for the nucleus. The ratio obtained for lens nucleus suggests that a lower ratio of less than 1 does not necessarily reflect the apparent transparent state of lens morphology. More over our results for the in crease of tyrosine doubletratio with the extent of cataract formation in galactosemic lens appear to indicate that there is more hydration in crystallins of the cortex than the nucleus since the in creased ratio of tyro sine doublet has been shown to be due to the hydrogen‐bond formation of hydroxyl groups in various tyrosines of proteins with water. The tryptophan band ratio at 880 cm−1 and 757 cm− (I880/I757) under went a precipitous de crease in the cortex and a rather gradual de crease in the nucleus, suggesting buried tryptophan residues become more exposed in the cortex than in the nucleus during galactose‐induced cataractogenesis. Based on the changes of the two ratios, I832/I855 and I880/I757, the change of lens protein environment induced by galactosemic feeding appeared to take place in the cortex first, which was consistent with the observation that the development of an opaque lens be gins in the cortex. While no sulfhydryl (‐SH) signal was detected, there was a slow in crease of disulfide (‐S‐S‐) signal in the cortex of galactose‐fed lenses as compared to control lenses with out galactose. This suggested that a loss of lens glutathione occurred early and oxidation of cysteines in crystallins started in the first week, i.e., be fore the onset of cortical cataract. In contrast, for nucleus of galactosemic lenses the signal of the‐SH group was detected and yet the ‐S‐S‐signal of crystallins could not be found. In this study, we have conclusively demonstrated using Raman vibrational band shift that galactosemic cataract be gins in the cortex, and that the lens cortex suffers more structural alteration in crystallins than the nucleus.

Functional and Structural Studies of α-Crystallin from Galactosemic Rat Lenses

Chaperone-like activity and structural changes of lens α-crystallin from rats fed with galactose at various time intervals have been studied using high-performance liquid chromatograph (HPLC), circular dichroism (CD), and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence emission. It was found that chaperone-like activity of α-crystallin from galactose-fed rats toward dithiothreitol (DTT)-induced insulin B aggregation started to decrease after 3 weeks and decreased significantly after 5 weeks. Consistent results were observed in lens morphology, and lens opacity slightly developed after 3 weeks and became obvious after 5 weeks. HPLC analysis for chaperone function showed that the formation of high molecular weight aggregates (HMWA) of α-/γ-crystallins decreases with the increase of galactose-feeding time, revealing that chaperone-like activity is concomitant with the formation of HMWA. Circular dichroism results showed the reduction of β-sheet structure and loss of microenvironment of aromatic-type amino acids for opaque lenses, indicating α-crystallin's secondary and tertiary structure changed with the development of the lens opacity. ANS binding site estimated by Klotz equation showed it is 1.5 times higher at room temperature and is 2.4 times higher at 58°C for age-matched normal α-crystallin than for 5-week galactose-fed lens α-crystallin, indicating opaque lens α-crystallin loses the ability to assemble into an appropriately placed hydrophobic regions. The overall results accordingly indicated that galactose-induced cataractous α-crystallin has disordered structure, leading to the loss of its chaperone-like activity.

Ascorbate deficiency increases the rate of galactose-induced cataract formation in guinea pig: A study of the possible mechanism: Yokoyama, T., Sasaki, H., Giblin, F.J. and Reddy, V.N. Eye Research Institute, Oakland University, Rochester, MI 48309-4401, U.S.A.

An early decrease in 86Rb uptake was found in lenses of galactose-fed rats. Dissections in normal rat lenses showed that 86Rb acoumulation occurred in areas closer to the lens epithelium. The decreased uptake in lenses of galactosemic rats occurred in all lens areas; however, these lenses recovered their ability to accumulate 86Rb following galactose withdrawal from the diet.High dulcitol (galactitol) levels were found in lenses of galactose-fed rats. Dulcitol accumulations in the lens suggested a possible inhibitory role of this metabolite on the concentrating mechanisms of cations and amino acids in the lens during the development of galactose cataract in rats.

Abnormal Redox Status Without Increased Lipid Peroxidation in Sugar Cataract

In conflict with a previous report, we find that phenolic inhibitors of lipid peroxidation (butylated hydroxytoluene [BHT] and butylated hydroxyanisole [BHA]) do not have significant inhibitory effect on galactosemic cataract formation. This is consistent with the lack of enhancement of stable products of lipid peroxidation (measured by the thiobarbituric acid assay) in the lenses of galactosemic rats. This does not imply that oxidative stress plays no role in galactosemic cataract formation (indeed, we find that galactosemic lens homogenates contain increased amounts of an Fe2+ oxidant, possibly a peroxide), but rather that BHT- and BHA-inhibitable lipid peroxidation specifically has no role to play. In instances where drugs appear to inhibit galactosemic cataract formation, other effects caused by the drugs, e.g., inhibition of feeding or induction of general detoxification pathways, must be considered.

Distribution of total and non-freezable water contents of galactosemic rat lenses.

Galactosemic cataracts were induced in Sprague-Dawley rats by keeping them on a 50% galactose diet for 36 days. The galactosemic rat lenses as well as normal rat lenses were sliced into 0.50 mm sections and differential scanning calorimetric (DSC) and thermogravimetric (TGA) measurements were performed. The TGA measurements gave the total water content. This was much higher in cataractous lenses than in normal lenses. The total water content also showed different gradients in normal and cataractous lenses. In normal lenses the percentage of water decreases as one proceeds from cortex to nucleus. In galactosemic cataractous lenses the trend is the opposite. The non-freezable (bound) water content was obtained as the difference between the total and freezable (DSC measurements) water content. In both cataractous and normal lenses the non-freezable water content increases when one proceeds from cortex to nucleus. It seems that most of the water influx generated by the accumulation of galactitol ends up as free water in lakes and pools rather than water of hydration.

A temporal and spatial study of the synthesis and degradation of water-soluble and insoluble proteins in galactosemic rat lenses

Events of synthesis and degradation of water-soluble and -insoluble rat lens proteins were examined during a 21-day period of galactose-induced cataractogenesis. Epithelial cells continued to synthesize both fractions of proteins throughout the duration of the experiment. There were few qualitative changes in the pattern of proteins expressed and the rate of synthesis did not decrease. In contrast, synthesis of both water-soluble and -insoluble proteins by the cortical cells decreased and then became totally inhibited indicating no selective or differential inhibition of synthesis of one protein fraction, but that both water-soluble and -insoluble proteins in the cortical cells are equally affected by the metabolic changes associated with galactose-induced cataractogenesis. The cells within the nuclear region of control lenses were capable of incorporating [35S]-methionine into protein. This synthesis was completely abolished in 7- and 14-day nuclear cells. Labeled proteins were found to be present in the 21-day nuclear preparation to a level almost three times greater than that observed in the control nucleus. Degradation was minimal in both fractions of epithelial proteins, whereas selective degradation of proteins from both fractions was apparent in 14- and 21-day cortical cells. At day 14, the water-soluble protein profile from nuclear cells became less complex with the loss of several major bands, but these bands as well as some unique bands appeared at day 21. The profile of water-insoluble nuclear proteins remained unchanged over 21 days.

In vitro incubation paralleling changes occurring during mouse cataract formation

Certain biochemical and morphological changes involved in the formation of osmotic cataracts in the Nakano mouse and galactosemic rat lenses also occur in vitro. Incubation of young, normal, albino mouse lenses in glucose-free medium for 48 hr resulted in a gain in wet weight due to dramatic hydration. The glucose deprived lenses showed a marked decrease in the soluble protein content as is noted in cataract formation in vivo. Chromatography of the soluble protein demonstrated a substantial increase in heavy molecular weight material in the lenses incubated without glucose in comparison to the fresh lenses. A decrease in β H-crystallin was also striking and might be correlated with the disappearance of a 31 000 mol. wt polypeptide band seen by SDS-PAGE gel. Interestingly, the membrane polypeptides in the in vitro incubated lenses mimicked changes observed during cataract formation in vivo. A 23 000 mol. wt polypeptide band became prominent in the membrane fraction of these lenses and there was a concomitant decrease in the 26 000 mol. wt component. Messenger RNAs were present in the glucose deprived lenses as shown by cell-free translation in vitro, although no [ 35S]methionine incorporation into crystallin was noted. Both the hexokinase and glyceraldehyde phosphate dehydrogenase activities in the glucose deprived lenses fell rapidly and were virtually absent at the end of 48-hr incubation period. The data in this report suggest that the glucose deprived incubated mouse lens may serve as an in vitro model to the study of some biochemical and morphological changes occurring in the development of osmotic cataract in vivo.

Crystallin synthesis and crystallin mRNAs in galactosemic rat lenses

Changes in crystallin synthesis and crystallin mRNA content were followed in the epithelial and fiber cells of rat lenses during the development and reversal of galactose cataracts. Crystallin synthesis was not lowered in the lens epithelial cells throughout the 18 days of galactose feeding. By contrast, crystallin synthesis appeared partially reduced in the fiber cells of cultured lenses after the rats had been on a diet of 50% galactose for 6 days, and was essentially arrested in the lens fiber cells after the rats were fed galactose chow for 12 or 18 days. In vitro translation tests in a rabbit reticulocyte lysate demonstrated that control lenses contained α-, β- and γ-crystallin mRNAs in the cortical and nuclear fiber cells. The lens fiber cells of the 6-day galactosemic rats still possessed a full complement of crystallin mRNAs but those of the 15-day galactosemic rats had no detectable crystallin mRNAs. After 12 days the galactosemic lenses had lost approximately half of the crystallin mRNAs from the cortical fiber cells and all of the mRNAs from the nuclear fiber cells. Both crystallin synthesis and crystallin mRNA content recovered in the cortical fiber cells, but not in the nuclear fiber cells, during reversal of the galactose cataract. These results indicate that the reduction in crystallin synthesis is confined to the fiber cells and occurs initially by a decreased efficiency of utilization of crystallin mRNAs and subsequently by the degradation of the mRNAs. Previous experiments provide evidence that the impaired utilization of the crystallin mRNAs is due to the alterations in electrolytes in the cataractous lenses.

Role of nonenzymatic glycosylation in experimental cataract formation

The relevance of nonenzymatic glycosylation of lens proteins to cataract formation was studied in rats on a normal and high galactose diet, treated with and without sorbinil, an aldose reductase inhibitor. All galactosemic rats not receiving sorbinil had cataracts; none receiving sorbinil had cataracts. Lens homogenate was treated with a 200 fold molar excess of [ 3H]-borohydride and the extent of glycosylation was estimated from radioactivity incorporation and quantitation of hexitol-lysine adduct after extensive dialysis. We found no differences in the radioactivity uptake nor the amounts of hexitol-lysine in the lenses of galactosemic rats treated with and without sorbinil. Thus, nonenzymatic glycosylation was not responsible for the sugar-induced cataracts.

The absence of cataracts in mice with congenital hyperglycemia

Investigations were conducted to determine the biochemical basis of the absence of cataracts in genetically diabetic mice. The resistance of these mice to cataracts appears to be due to the low activity of aldose reductase in the lens. Consequently only low amounts of polyols accumulate even after persistent hyperglycemia. The finding thus lends further support to the polyol theory of sugar cataractogenesis. The accumulation of sugar alcohols in galactosemic mice lenses was also much lower as compared to that in the lenses of galactosemic rats. A study was made of the enzymes that influence the polyol level in the lenses of various mouse strains. The activities of aldose reductase, polyol dehydrogenase, hexokinase and glucose-6-phosphate and 6-phosphogluconate dehydrogenases in the mouse and rat lenses were determined. The activities of all the enzymes except that of aldose reductase were similar in the lenses of both species. The aldose reductase activity in the mouse lens was only one tenth of that present in the rat lens.

Rubidium-86 accumulation and dulcitol distributions in lenses of galactose-fed rats

An early decrease in 86Rb uptake was found in lenses of galactose-fed rats. Dissections in normal rat lenses showed that 86Rb acoumulation occurred in areas closer to the lens epithelium. The decreased uptake in lenses of galactosemic rats occurred in all lens areas; however, these lenses recovered their ability to accumulate 86Rb following galactose withdrawal from the diet. High dulcitol (galactitol) levels were found in lenses of galactose-fed rats. Dulcitol accumulations in the lens suggested a possible inhibitory role of this metabolite on the concentrating mechanisms of cations and amino acids in the lens during the development of galactose cataract in rats.