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Relative roles of ATP and NADPH in anabolic and other processes


Chemistry isn't my strong point, but I'm trying to understand why NADPH is said to be the main agent necessary for anabolic reactions, and if NADPH is used for anabolic reactions what is ATP used for? Movement?

An example of what I'm wondering: When skin cells are exposed to sunlight, to synthesize melanin is the free energy of ATP hydrolysis sufficient? For example by catabolizing some protein in the cell and using the amino acids to synthesize melanin? Or is NADPH always necessary?

I have not been able to find an explanation on-line that addresses this, so suggestions of a suitable book would be appreciated.


Preamble

I don't know where you read the statement “NADPH is… the main agent necessary for anabolic reactions”, but out of context it is very misleading, and it is difficult to envisage circumstances in which it would be helpful for students of metabolism.

Put simply, NAPH is only used in anabolic pathways which involve chemical reduction.

You ask for a suitable book, but the book I'm going to quote from (Berg et al., Biochemistry) would overwhelm you (and any other student - it is grossly overweight). However an old edition is available on-line, and by citing specific sections you can read what is relevant (and spare me breaking up my answer with pirated diagrams).

ATP and NADPH: energetic similarities and differences

ATP and NADPH can both be regarded as 'energy-rich' in the sense that they can undergo reactions that have a high negative change in Gibbs Free Energy (which is what determines whether a reaction proceeds) and that this can be linked to certain other reactions with a positive free-energy change - including, but not exclusively, those in biosynthesis - to drive an overall reaction. (Incidentally both these compounds are synthesized in reactions powered by solar energy during photosynthesis.) However the nature of this linkage or coupling to other reactions differs between NADPH and ATP, and this difference determines the roles that they can play.

The oxidation of NADPH to NADP+ has a standard redox potential of +0.32V , which is an effective a negative standard free-energy change, but it can only occur in conjunction with another redox 'half-reaction' with a redox potential that will result in an overall negative free-energy change. Berg et al., section 18.2.1, presents a calculation to show how NADH (essentially similar to NADPH in this respect) can reduce pyruvate to lactate. The key point is that this overall reaction is a chemical reduction, so that the 'energy' of NADPH is only of use in reductive synthesis.

The 'energy' of ATP (more properly its group transfer potential) is in fact the negative free energy of the hydrolysis of ATP to ADP (and orthophosphate), or to AMP (and pyrophosphate). If an enzyme can couple this reaction to another with positive free energy of hydrolysis then it can drive the second reaction. This important point is dealt with in Berg et al. section 14.1.3 and it is also worth reading the preceding section. ATP is not a reducing agent so is not limited to oxido-reduction reactions. (The hydrolysis of ATP can also be used to provide e.g. mechanical or electrical or light energy, which is why Berg et al refer to it in section 14.1.2 as “… the Universal Currency of Free Energy in Biological Systems”.) In synthetic processes ATP (or other nucleotide triphosphates derived from it) is often used in reactions forming bonds (C-C, peptide, glycosidic, phophodiester). In some of these cases the hydrolysis of the ATP is used to generate an activated form of the basic component, which allows it to form the bond in a subsequent reaction.

Example of a synthetic pathway using both NADPH and ATP

In Fatty acid synthesis NADPH is used for two reduction reactions - ketone to alcohol, unsaturated C=C to saturated C-C bond - whereas ATP hydrolysis is coupled to the formation of the C-C bond of the condensing unit, malonyl CoA. This is shown in Figure 22.22 of Berg et al..

Example of synthetic pathways using ATP but not NADPH

The synthesis of the peptide bonds of proteins requires condensation of the carboxylic acid group of one amino acid with the amino group of another. This is not a reductive process and NADPH is not involved. ATP hydrolysis 'activates' each amino acid by attaching it to the ribose of a transfer-RNA in a bond that has a high enough free energy of hydrolysis to 'drive' peptide bond formation to the growing polypeptide chain at the peptidyl transferase centre of the ribosome.

Melanin synthesis?

This is an area in which I am profoundly ignorant, but from Googling it would appear that the first phase of melanin synthesis is the conversion of tyrosine to DOPA and then to indoles. These reactions are oxidative (using molecular oxygen) rather than reductive, so do not use NADPH, and the bond formation is cyclization to form the pyrole ring. This seems to proceed without ATP - presumably it is energetically favourable overall. There is a Wikipedia entry for the key tyrosinase oxidation reaction.

I suppose the anabolic phase is the polymerization of the aromatic units to form the crosslinked polymeric structure of melanin. This appears to be poorly understood, but there is certainly no indication that NADPH is involved, and it would appear to depend on chemical features of the environment of the melanosome, rather than on ATP. (There is a recent short review in this area by VJ Hearing).

You mention the role of sunlight in this process. Although sunlight in itself can provide the energy for one stage of the synthesis of Vitamin D in the skin, this is a ring-splitting step. A direct role in the polymerization of melanin doesn't seem to be suggested.


The nexus of chromatin regulation and intermediary metabolism

Living organisms and individual cells continuously adapt to changes in their environment. Those changes are particularly sensitive to fluctuations in the availability of energy substrates. The cellular transcriptional machinery and its chromatin-associated proteins integrate environmental inputs to mediate homeostatic responses through gene regulation. Numerous connections between products of intermediary metabolism and chromatin proteins have recently been identified. Chromatin modifications that occur in response to metabolic signals are dynamic or stable and might even be inherited transgenerationally. These emerging concepts have biological relevance to tissue homeostasis, disease and ageing.


Summary

Loss of mitochondrial respiratory flux is a hallmark of skeletal muscle aging, contributing to a progressive decline of muscle strength. Endurance exercise alleviates the decrease in respiratory flux, both in humans and in rodents. Here, we dissect the underlying mechanism of mitochondrial flux decline by integrated analysis of the molecular network.

Mice were given a lifelong ad libitum low-fat or high-fat sucrose diet and were further divided into sedentary and running-wheel groups. At 6, 12, 18 and 24 months, muscle weight, triglyceride content and mitochondrial respiratory flux were analysed. Subsequently, transcriptome was measured by RNA-Seq and proteome by targeted LC-MS/MS analysis with 13 C-labelled standards. In the sedentary groups, mitochondrial respiratory flux declined with age. Voluntary running protected the mitochondrial respiratory flux until 18 months of age. Beyond this time point, all groups converged. Regulation Analysis of flux, proteome and transcriptome showed that the decline of flux was equally regulated at the proteomic and at the metabolic level, while regulation at the transcriptional level was marginal. Proteomic regulation was most prominent at the beginning and at the end of the pathway, namely at the pyruvate dehydrogenase complex and at the synthesis and transport of ATP. Further proteomic regulation was scattered across the entire pathway, revealing an effective multisite regulation. Finally, reactions regulated at the protein level were highly overlapping between the four experimental groups, suggesting a common, post-transcriptional mechanism of muscle aging.


Role of Oxidative Stress in Cardiac Hypertrophy and Remodeling

From the Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md.

From the Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md.

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Cardiac adaptation in response to intrinsic or external stress involves a complex process of chamber remodeling and myocyte molecular modifications. A fundamental response to increased biomechanical stress is cardiomyocyte and chamber hypertrophy. Although this may provide initial salutary compensation to the stress, sustained hypertrophic stimulation becomes maladaptive, worsening morbidity and mortality risks because of congestive heart failure and sudden death. 1 Growing evidence highlights oxidative and nitrosative stresses as important mechanisms for this maladaptation. 2–9 Oxidative stress occurs when excess reactive oxygen species (ROS) are generated that cannot be adequately countered by intrinsic antioxidant systems. Superoxide anion (O2 − ) can further combine with NO, forming reactive compounds such as peroxynitrite, generating nitroso-redox imbalance. 4 ROS generation is a normal component of oxidative phosphorylation and plays a role in normal redox control of physiological signaling pathways. 5,8,9 However, excessive ROS generation triggers cell dysfunction, lipid peroxidation, and DNA mutagenesis and can lead to irreversible cell damage or death. 5,8,9 In this review, we discuss recent experimental evidence for the role of oxidant stress on cardiac remodeling, focusing on pressure- overload–induced hypertrophy and dilation.

ROS, Antioxidant Enzymes, and Nitroso-Redox Balance

ROS include free radicals such as superoxide (O2 − ) and hydroxyl radical and compounds such as hydrogen peroxide (H2O2) that can be converted to radicals, and they participate in both normal and pathologic biochemical reactions. 9 O2 − is formed intracellularly (Figure 1) by activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase (XO), uncoupling of NO synthase (NOS), and electron transport and “leakage” during oxidative phosphorylation in the mitochondria. 5,8,9 H2O2 can generate the highly reactive hydroxyl radical via Fenton chemistry under pathological conditions. 9

Figure 1. General schematic of generation pathways for ROS and antioxidant systems in the heart. Low levels of ROS are thought to play a role in normal cardiac signaling, growth adaptations, and matrix changes. Higher levels play a role in pathophysiologic remodeling, apoptosis, and chamber dysfunction. SOD indicates superoxide dismutase GPX, glutathione peroxidase TRX, thioredoxin.

Cells also have intrinsic antioxidant systems that counter ROS accumulation. These include enzymes such as catalase, glutathione peroxidases, and superoxide dismutase, and nonenzymatic antioxidants, such as vitamins E, C, beta carotene, ubiquinone, lipotic acid, and urate. 9,10 Superoxide dismutase converts O2 − to H2O2, which is further converted by catalase and glutathione peroxidase to water. The thioredoxin system, including thioredoxin, thioredoxin reductase, and NADPH, forms an additional integrated antioxidant defense system, which operates as a powerful protein–disulfide oxidoreductase. 10–12

NO is another important reactive molecule controlling cardiovascular homeostasis. NO stimulates the synthesis of intracellular cGMP by activating soluble guanylyl cyclase, and cGMP and its target kinase cGK-1 (protein kinase G 1), in turn, modulate myocyte function, growth, and remodeling. 13 NO also interacts with proteins via S-nitrosylation at specific cysteine residues to alter their function. 14,15 S-nitosylation is facilitated by O2 − at physiological levels however, this process is inhibited at high levels of O2 − . 9 As noted, increased O2 − interacts with NO to form peroxynitrite, a reactive species that is capable of triggering an array of cytotoxic processes, including lipid peroxidation, protein oxidation, and nitration (altering excitation–contraction coupling), 16 and activation of matrix metalloproteinases (MMPs) contributing to chamber remodeling (reviewed in References 17,18 ). NO can act as an antioxidant, inhibiting activation of XO 19,20 and NADPH oxidase 21,22 and maintaining normal O2 − /NO homeostasis. Thus, increasing peroxynitrite levels means that normal NO bioavailability and physiology would be compromised.

Mechanisms for ROS Stimulation of Cardiac Hypertrophy/Remodeling

Figure 2 summarizes the mechanisms by which ROS stimulate myocardial growth, matrix remodeling, and cellular dysfunction. ROS activate a broad variety of hypertrophy signaling kinases and transcription factors. 23 In rat neonatal cardiomyocytes, H2O2 stimulates the tyrosine kinase Src, GTP-binding protein Ras, protein kinase C, mitogen-activated protein kinases (extracellular response kinase and extracellular signal–regulated kinase), and Jun-nuclear kinase. 24–26 Phosphoinositol 3-kinase is required for H2O2-induced hypertrophy. 27 Low levels of H2O2 are associated with extracellular signal–regulated kinase activation and protein synthesis, whereas higher levels stimulate extracellular signal–regulated kinase, Jun-nuclear kinase, p38, and Akt kinases to induce apoptosis. 28 ROS also play an important role in G protein–coupled hypertrophic stimulation by angiotensin II and α-adrenergic stimulation, 29–33 the latter involving oxidative modulation of Ras thiols. 34 ROS also stimulate cellular apoptosis signaling kinase-1, a redox-sensitive kinase upstream of Jun-nuclear kinase and p38. Apoptosis signaling kinase-1 overexpression activates nuclear factor κB to stimulate hypertrophy, whereas genetic silencing of apoptosis signaling kinase-1 inhibits hypertrophy induced by angiotensin II, norepinephrine, and endothelin I. 35

Figure 2. Molecular signaling pathways linking ROS to cardiac hypertrophy and remodeling. Pathways include calcium channels (ICa), α and β adrenergic (αAR and βAR), angiotensin II (AT1) receptor agonism, modification of stress kinases, collagen and metalloproteinases, sarcomeric and excitation-contraction coupling proteins, and nuclear transcription factors. Details are provided in text. MAPK indicates mitogen-activated protein kinases PI3K, phosphoinositol 3-kinase Ask-1, apoptosis signaling kinase-1 PKC, protein kinase C NFκB, nuclear factor κB AP-1, activator protein-1.

ROS also have potent effects on the extracellular matrix, stimulating cardiac fibroblast proliferation 36 and activating MMPs, 37–39 effects central to fibrosis and matrix remodeling. MMPs are generally secreted in an inactive form and are activated posttranslationally by ROS from targeted interactions with critical cysteines in the propeptide autoinhibitory domain. 40 ROS also stimulate transcription factors nuclear factor κB, Ets, and activator protein-1 to stimulate MMP expression. 38

Cardiomyocyte apoptosis is another important contributor to hypertrophic remodeling and cell dysfunction. 41 For example, mice lacking apoptosis signaling kinase-1 display both reduced ventricular remodeling in response to pressure load or after myocardial infarction (MI) and less cellular apoptosis. 42 Apoptosis is inhibited in cells at low levels of ROS stimulation, whereas the opposite occurs at higher levels. 28 Mechanisms include DNA and mitochondrial damage and activation of proapoptotic signaling kinases.

Lastly, ROS directly influence contractile function by modifying proteins central to excitation–contraction coupling (reviewed in Reference 43 ). This includes modification of critical thiol groups (-SH) groups on the ryanodine receptor to enhance its open probability, 44 suppression of L-type calcium channel current, 45 and oxidative and nitrosative interaction with the sarcoplasmic reticular Ca 2+ ATPase to inhibit Ca 2+ uptake. 46,47

NOS3 Uncoupling: A Pathophysiologic ROS Generator

NOS3, or endothelial NOS, has not been traditionally considered a major oxidase, yet recent evidence suggests this function in cardiovascular pathologic remodeling (Figure 3). 48–51 NOS3 normally generates NO to stimulate cGMP and cGK-1, which blunt cellular cardiac hypertrophy and fibrosis via transcriptional regulation, phosphorylation, and suppression of targeted signaling, such as from Gαq stimulation. 52–58 In mice exposed to sustained pressure overload, chronic inhibition of cGMP hydrolysis by phosphodiesterase 5A induced protein kinase G activation and attenuated chamber and myocyte hypertrophy and fibrosis coupled to inhibition of multiple hypertrophic cascades. 59 ROS impede this regulation by reacting with NO to form peroxynitrite, stimulating nitrosative stress and reducing NO bioactivity, limiting soluble guanylate cyclase activity and expression. 60 In this setting, NOS3 can generate O2 − instead of NO. 61

Figure 3. Involvement of NOS3 and NOS3 uncoupling in cardiac hypertrophy and remodeling. The normal (coupled) eNOS pathway is shown to the right and is thought to provide an inhibitory influence on hypertrophy and hypertrophic signaling, MMP activation, and cardiac dysfunction. sGC indicates soluable guanylate cyclase PKG, protein kinase G Trx, thioredoxin. In the presence of oxidant stress, depletion of substrate (arginine), or cofactors (BH4), NOS can become uncoupled and generate more ROS. This enhanced ROS diverts NO (forming peroxynitirite) and stimulates pathologic cardiac remodeling.

Under normal conditions, NOS3 consumes NADPH and generates NO and l -citrulline from l -arginine and O2. In this process, electrons are passed from a reductase domain to the heme-containing oxygenase domain (catalytic core). The cofactor tetrahydrobiopterin (BH4) is essential for donating an electron and proton to versatile intermediates in this reaction cycle. Calmodulin controls the shuttling of the electrons, and a zinc-thiolate complex, as well as BH4, is required for NOS dimer formation and stability of the oxidase domain. 62,63 NOS functions normally as a homodimer, and BH4 is required to maintain its “coupled” state and, thus, to synthesize NO.

When exposed to oxidative or nitrosative stress or when deprived of BH4 or l -arginine, NOS3 becomes structurally unstable. On protein gels, it appears more as a monomer, and electrons become diverted to molecular oxygen rather than to l -arginine, resulting in O2 − formation (uncoupled state). 64–66 This change was first reported in the vasculature and has been linked to the endothelial pathophysiology in hypertension, diabetes, smoking, and atherosclerosis. 48,50,51,67–70 We reported recently that similar mechanisms also play a key role in the adverse remodeling resulting from chronic pressure overload. 49 Hearts exposed to trans-aortic constriction developed marked chamber dilation with decreased NOS3 dimer in the myocardium and elevation of oxidative stress. The latter was reduced by half by preincubating myocardial extract with the NOS inhibitor, N G -nitro- l -arginine methyl ester, suggesting that ROS were being generated by NOS itself. Similarly, animals genetically lacking NOS3 exposed to pressure overload developed more modest and compensated concentric hypertrophy, with little cavity dilation, less interstitial fibrosis, and far less oxidative stress.

A major factor that may mediate NOS3 uncoupling in pressure-overloaded hearts is a decline in BH4 levels. This is supported by both direct BH4 measurements and findings that BH4 supplementation offsets the hypertrophic/dilative phenotype. 49 Hearts with increased ROS because of uncoupled NOS3 have increased MMP activation, which, in turn, degraded extracellular matrix, facilitating left ventricular dilatation 49,71,72 and worsening cardiac function.

Given that NOS3 is expressed in vascular endothelium and myocytes, with the latter representing <20 of total myocardial NOS3, it is unclear which cell type contributes most to ROS generated by NOS3 uncoupling. Furthermore, the exact mechanisms leading to NOS3 uncoupling and a reduction in BH4 levels remain unknown. One possibility reported by Landmesser et al 51 is that initial oxidant stress (O2 − ) from NADPH oxidase enhances BH4 oxidization resulting in NOS3 uncoupling. In their study, oral supplementation with BH4 or genetic depletion of NADPH oxidase prevented uncoupling. As NADPH-dependent ROS generation increases in pressure-overload hypertrophy, 73 a similar scenario may apply. The interaction between NOS3 uncoupling and BH4 is somewhat circular NOS3 can become an O2 − generator without BH4 depletion, 48 and the consequent ROS can, in turn, oxidize BH4 to worsen the process. Evidence of the latter was shown by Bendall et al, 74 who generated stoichiometric discordance between NOS3 protein and BH4 levels by comparing endothelial-targeted overexpression of GTP cyclohydrolase 1 ([GTPCH-1] rate-limiting BH4 synthetic enzyme), NOS3, and their combination. Imbalance between BH4 and NOS3 resulted in NOS3 uncoupling. The relative role of these mechanisms may depend on the nature and/or stage of the pathology. For example, NOS3 expression increases in cardiomyopathic hamsters 75 and decreases in ischemic cardiomyopathy. 76 BH4 depletion can occur by reduced synthesis, particularly related to changes in GTPCH-1, or by the salvage pathway that uses sepiapterin as an intermediate. 77 Neopterin, a byproduct of BH4 synthesis by GTPCH-1, declines with pressure-load hypertrophy, suggesting that BH4 biosynthesis is diminished. 49 Although studies on the regulation of GTPCH-1 and its role in the myocardium have yet to be reported, recent data showing that signal transducer and activator of transcription-3 activation in endothelial cells lowers GTPCH-1 expression 78 suggest a potential mechanism, because signal transducer and activator of transcription-3 is potently activated by pressure overload in the heart. 79

NADPH Oxidases

NADPH oxidases (Figure 4A) are multimeric enzymes that consist of the membrane-bound flavocytochrome composing a catalytic Nox subunit and p22 phox subunit, and 4 cytosolic regulatory subunits, p40 phox , p47 phox , p67 phox , and the small GTP-binding protein Rac. 80 Electron transfer occurs from NADPH to molecular oxygen at the catalytic site, resulting in the formation of superoxide. There are 5 Nox isoforms (Nox1–5), expressed in a tissue-specific manner. Among these, Nox2 (known previously as gp91 phox ) and Nox4 are the main isoforms expressed in the myocardium. 5 NADPH oxidase activity increases from stimuli such as angiotensin II, 81,82 cyclic load, 83,84 α-adrenergic agonists, 85 and tumor necrosis factor-α. 86 NADPH oxidase activity and subunit expression increase during the development of pressure-overload hypertrophy in guinea pigs 73 and human heart failure. 87,88 ROS derived from NADPH oxidases can induce NOS3 uncoupling (as discussed above) and activate XO, 89 thus, the NADPH oxidases may serve as priming sources for amplification of ROS generation.

Figure 4. A, Role of NADPH oxidases in cardiac response to pressure overload, angiotensin stimulation, and myocardial infarction. Nox2 has been found to play a role in angiotensin-stimulated hypertrophy, but its genetic absence does not prevent pressure-load–induced hypertrophy. Other influences on fibrosis and contractile function, however, have been observed. Nox4 may contribute to pressure-load hypertrophy, but this has not been clarified to date. B, Role of XO system in heart failure and, potentially, pressure overload. Increased XO activity has been observed in various animal heart failure models, and its inhibition can improve contraction and energetic efficiency. C, Proposed interaction between XO and neuronal NOS (nNOS) in modifying calcium handling by the ryanodine receptor and contractile function. Genetic loss of NOS1 can result in increased XO and O2 − generation, inhibiting calcium handling and leading to contractile dysfunction.

ROS generated by NADPH oxidase seem to play a key role to angiotensin II–induced cardiac hypertrophy/remodeling. Subpressor doses of angiotensin II induce cardiac hypertrophy that is blunted in hearts lacking Nox2. 90 In addition, Rac1 null mice have reduced NADPH oxidase activity, associated with lower myocardial oxidative stress, blunted hypertrophy, and less activation of apoptosis signaling kinase-1 and nuclear factor κB in response to angiotensin II infusion. 91 Nox2 null hearts also display less remodeling after MI. 92 However, mice lacking Nox2 develop similar hypertrophic responses to pressure overload as in controls but with less fibrosis and better cardiac contraction. 93–95 Thus, alternative pathways and/or Nox isoforms (eg, Nox4) may be more relevant in this setting.

Xanthine Oxidase

Recent experimental data suggest that XO and XO-related oxidant stress also play a role in the pathogenesis of chronic heart failure (Figure 4B and 4C). Elevated XO expression and activity have been demonstrated in end-stage human heart failure 96 and in the canine rapid pacing-induced heart failure model. 97,98 Chronic treatment with allopurinol significantly reduced adverse left ventricular remodeling 99 and modestly improved survival 100 in mice after MI or rats with dilated cardiomyopathy. 101 Allopurinol also prevented myofibrillar protein oxidization and preserved cardiac function in transgenic mice harboring truncated troponin I, a model of myocardial stunning. 102

Interestingly, XO-derived superoxide seems to interfere with NO regulation of myocardial energetics. 103 Khan et al 104 reported that neuronal NOS (NOS1 or nNOS) and the superoxide generating xanthine oxidoreductase lie in physical proximity in the sarcoplasmic reticulum of cardiac myocytes. Deficiency of NOS1 increased xanthine oxidoreductase–mediated superoxide production, negatively regulating cardiac contractility, and this was reversible by allopurinol. The same group reported that NOS1 null hearts had worse remodeling and cardiac function than wild-type after myocardial infarct however, surprisingly, tissue ROS levels increased similarly in either genotype. 105 Importantly, they found different NO levels in heart tissues (increase in wild-type but not in NOS1 null hearts), suggesting that the imbalance between NOS1-mediated NO signaling and ROS, rather than the ROS level itself, was more important. Increased XO activity has been reported in late-stage pressure-overload–induced right ventricular hypertrophy. 106

Mitochondrial ROS

Excessive ROS derived from mitochondria are also likely contributors to cardiac failure (reviewed in Reference 107 ) and have also been reported in experimental models of MI and heart failure (eg, tachypacing-induced failure 108,109 ). This has been attributed to electron leakage associated with reduced activity in electron transport chain complexes and can be a source for O2 − , H2O2, and OH − . This oxidative stress plays an initial role in damaging mitochondrial DNA and organelle function and can result in membrane potential abnormalities, ROS leakage, and cellular damage. Overexpression of the mitochondrial antioxidant, peroxiredoxin-3, ameliorates mitochondrial DNA damage and inhibits adverse left ventricular remodeling after MI. 110 However, the specific role of ROS from mitochondria in pressure-overload–induced hypertrophy has not been reported to date and awaits further elucidation.

Antioxidant Systems and Pressure Overload

Several intrinsic antioxidants have been shown to ameliorate the evolution of heart failure or hypertrophy in experimental models. For example, chronic treatment with the nonspecific antioxidant vitamin E improved cardiac function and blunted heart failure in a guinea pig pressure-overload model, 111 although an antihypertrophic effect was not observed. 2N-merocaptoproninyl glycine or N-acetyl cysteine blunted cardiac hypertrophy in mice with pressure overload. 93,112 The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin also can act as an antioxidant and has been shown to prevent in vitro cardiomyocyte and in vivo pressure-overload–induced hypertrophy linked to inhibition of Rac1 and, thus, reduction of NADPH oxidase activity. 113

Enzyme antioxidant pathways have been genetically manipulated to reveal a prominent role in hypertrophic remodeling. Thioredoxin is a ubiquitous thiol oxidoreductase composed of thioredoxin, thioredoxin reductase, and NADPH and limits oxidative stress by direct ROS scavenging and by interaction with other signaling kinases. 12 Inhibition of endogenous thioredoxin-1 results in enhanced cardiac hypertrophy with increased myocardial oxidative stress to pressure overload, whereas overexpression of the protein reduces hypertrophy and oxidative stress. 11 Interestingly, thioredoxin is upregulated by cGMP/protein kinase G in human neuroblastoma cells, protecting cells from oxidative stress–induced apoptosis. 114 A similar mechanism might play a role in NO/cGMP/protein kinase G–mediated amelioration of cardiac hypertrophy/remodeling (Figure 3). Another intrinsic antioxidant enzyme, glutathione peroxidase, important for removing H2O2 and detoxifying lipid hydroperoxides, has also been overexpressed in mouse heart, and this ameliorated post-MI remodeling. 115

Targeting Oxidative/Nitrosative Stress: A Clinical Strategy

Although recent basic experimental studies strongly support a key role of oxidative/nitrosative stress in the pathophysiology of cardiac hypertrophic remodeling and dysfunction, clinical data testing these findings remain scant. Although small, often uncontrolled clinical studies have been supportive, larger prospective and randomized, controlled trials have failed to show clinical benefit from antioxidants such as vitamin C and vitamin E. 116–118 However, these studies have not examined hypertrophic heart disease or heart failure, per se, and the bulk of the data, which are from cancer trials, may not predict cardiac disease responses. Recent clinical trials of allopurinol and oxypurinol to treat class II to III congestive heart failure found reduced plasma urate yet no impact on clinical outcome. 119 However, angiotensin-converting enzyme inhibitors, the β-blocker carvedilol, and angiotensin receptor blockers can block NADPH oxidase, whereas statins inhibit Rac1 and, thus, NADPH oxidase, and this may contribute to part of their efficacy. More specific targeting of the source of oxidative stress, such as recoupling of NOS or enhancing intrinsic antioxidants, 120 may ultimately provide more effective approaches to reversing cardiac remodeling. BH4 and its precursor sepiapterin are well tolerated and are currently used to treat some forms of the genetic disease phenylketonuria in humans. 121 The experimental data seem compelling, and it seems premature to abandon this tactic for clinical treatment. Further studies using more potent and better-targeted agents will hopefully establish their use in the future.


Physiological barriers

Although Hb readily inactivates · NO through reaction 3, irreversible · NO consumption is limited by a factor of

1,000 because of Hb’s packaging into RBCs (20, 55, 77, 108, 167). Substrate availability is thus the rate-limiting factor in the dioxygenation reaction, which otherwise possesses an extremely high rate constant (k =

9 × 10 7 M −1 s −1 ) (71, 108). · NO produced by eNOS at the vascular endothelium encounters four successive barriers before reaching Hb in RBCs: 1) the endothelial cell membrane, 2) an RBC-free zone at the vascular edge of the migrating RBC column created by blood flow velocity gradients (104, 167, 168), 3) an unstirred layer of blood around moving RBCs (7), and 4) the RBC membrane and cytoskeleton.

The relative importance of these physiological barriers is the subject of continuing investigation. It is generally accepted that cell membranes do not represent important obstacles for · NO diffusion because · NO is equally soluble in aqueous and lipid compartments and, therefore, freely permeates membranes. However, chemical and physical modifications of the RBC membrane result in significant changes in the rate of RBC · NO uptake (66, 77). These changes could be due to changes in essential Hb-protein interactions that modulate · NO uptake by RBCs rather than changes in the nature of the membrane. By contrast, the cell-free zone at the vascular wall, with a width of

5 μm, accounts for a necessary · NO transit time of

7.5 ms, during which its derivatives can bind to albumin or glutathione for instance (18). The rate-limited diffusion of · NO in the static plasma layer around RBCs is essentially similar to the rate-limiting diffusion at the blood-endothelium interface. There, rapid · NO uptake by the RBC creates inhomogeneous · NO concentrations (96). Interestingly, · NO itself may influence the rate of RBC uptake of further · NO, as demonstrated by Liao and coworkers (67, 77).

Although physiological barriers certainly limit · NO uptake by RBCs and thereby prevent chronic systemic hypertension, they do not preclude the interactions of · NO with Hb. Alternative, stable NO stores formed in and ferried by the RBC could ensure that NO bioactivity reaches the tissues when needed (see below).


Mechanisms of amino acid sensing

The findings described above identify the molecular platform necessary for mTORC1 activation at the lysosomal surface and have helped in the understanding of the amino acid-dependent Rag-mediated regulation. However, increasing evidence suggests other pathways to positively regulate mTORC1 in response to amino acids. Indeed, it is now clear that mTORC1 senses both, intralysosomal and cytosolic amino acids through distinct mechanisms (Figure 2). All these mechanisms cooperate to provide a sensory readout from more than one subcellular compartment with varying sensitivity to amino acids, thus leading to the tight control of the metabolic state.


MATERIALS AND METHODS

Strains

We used the wild-type strain 972 h - (Leupold, 1970) and two isogenic mutant strains sty1Δ(sty1::ura4 + ura4-D18 h - ) and atf1Δ(atf1::ura4 + ura4-D18 h - ). sty1Δ and atf1Δwere derived from auxotrophic strains (Millar et al., 1995Takeda et al., 1995, respectively) by crossing out markers.

Stress Experiments, Cell Collection, and RNA Isolation

The three strains were cultured in yeast extract (YE) medium (http://www.bio.uva.nl/pombe/handbook/) at 30°C, shaken in flasks at 170 rpm until reaching OD600 = 0.2 (∼4 × 10 6 cells/ml). Cells were harvested immediately before as well as 15 and 60 min after stress treatment from the same culture. Stress conditions were as described below. Oxidative stress: hydrogen peroxide (H2O2 H1009 Sigma, St. Louis, MO) was added to a final concentration of 0.5 mM. Heavy metal stress: cadmium sulfate (CdSO4 C2919 Sigma) was added to a final concentration of 0.5 mM. Heat stress: cells were quickly transferred from 30°C to a large prewarmed flask in a 39°C water bath, reaching temperature equilibrium after 2 min. Osmotic stress: cells were grown to OD600 = 0.4, and an equal volume of prewarmed YE + 2 M sorbitol was added to a final concentration of 1 M sorbitol. Alkylating agent: methylmethane sulfonate (MMS 64294 Fluka, Buchs, Switzerland) was added to a final concentration of 0.02% (w/v). Cells were collected by gentle centrifugation (2000 rpm for 2 min), and pellets were frozen immediately in liquid nitrogen. We isolated total RNA using a hot-phenol protocol (for details, see our website:http://www.sanger.ac.uk/PostGenomics/S_pombe/).

Target Labeling, Microarray Hybridization, and Data Acquisition

Twenty micrograms of total RNA was labeled by directly incorporating Cy3- and Cy5-dCTP through reverse transcription and the resulting cDNA was hybridized onto DNA microarrays containing probes for 99.3% (H2O2 and cadmium experiments) or 99.9% (heat, sorbitol, and MMS experiments) of all known and predicted fission yeast genes printed in duplicate onto glass slides (for details on protocols and microarrays, see our website). Microarrays were scanned using a GenePix 4000B laser scanner (Axon Instruments, Foster City, CA) and analyzed with GenePix Pro software. Unreliable signals were filtered out, and data were normalized using a customized Perl script (local adjustment of median of ratios to 1 within running windows of 1000 spots G. Burns, R. Lyne, J. Mata, G. Rustici, D. Chen, D. Vetrie, and J. Bähler, manuscript submitted).

Experimental Design

The five stress time course experiments with the wild-type andsty1Δ strains were performed as two independent biological repeats (except the H2O2and cadmium experiments in sty1Δ, which were done once), and the experiments with the atf1Δ cells were done once each. Labeled samples from each stress time point of the wild-type and mutant experiments were hybridized with a labeled reference pool, containing an equal amount of all the RNA samples from the wild-type time points of the corresponding stress. For duplicate experiments, the Cy dyes were swapped for the experimental and reference samples. After data acquisition and within-array normalization, the ratios of each gene (time point/reference pool) were divided by the corresponding ratios of untreated wild-type cells (0 h wild type/reference pool). Thus, the reported ratios represent the expression levels at each time point relative to the expression levels of the untreated wild-type cells from the same stress experiment. Because of the relative importance of the measurements for untreated wild-type cells, we performed two technical repeats of these arrays (with swapping of fluorochromes) and used the averaged data to “zero-transform” the data of all stress time points from wild-type and mutant cells. Expression ratios of biological repeat experiments (wild-type andsty1Δ strains) were averaged. In total, 67 microarrays were used in this study. The complete processed data set is available from our website, and all raw data will be available from the ArrayExpress repository: www.ebi.ac.uk/arrayexpress.

Data Evaluation, Hierarchical Clustering, and Gene Classification

We used SAM (Tusher et al., 2001) and GeneSpring (Silicon Genetics, Redwood City, CA) to discard genes that did not behave reproducibly between biological duplicate experiments. Hierarchical clustering was performed with preselected log-transformed gene sets using Cluster and TreeView software (Eisen et al., 1998), with uncentered Pearson correlations and average linkage clustering. Genes with 50% of data points missing were not used. The criteria used to select various groups of stress genes (using GeneSpring) are given below. Gene annotations were taken from GeneDB at the Sanger Institute:http://www.genedb.org/genedb/pombe/index.jsp.

Identification of CESR and SESR Genes.

Genes induced at least twofold at either 15 or 60 min were identified. Among those genes, we selected induced CESR genes as those that were up-regulated in at least four of the five stress conditions. Repressed CESR genes were selected as those being down-regulated twofold or greater in at least three of the five stress conditions. We subtracted these induced CESR genes from the genes that were up-regulated twofold or greater in at least one stress to identify induced SESR genes.

Identification of “Stress-Specific” and Super-Induced Genes.

Among the genes that were induced at least twofold at either 15 or 60 min in a given wild-type stress experiment, we selected “stress-specific” genes as those that were induced at least twice as highly in the stress of interest than in any of the other four stresses. This procedure also identified CESR genes that were more highly induced in the given stress. These “super-induced” genes were subtracted from the “stress-specific” genes and listed separately (lists available from our website).

Identification of Sty1p- and Atfp1-Dependent Genes.

We selected genes that required Sty1p and/or Atf1p for induction among the genes that were induced at least twofold in a given wild-type stress experiment and were also induced at least twice as highly in the wild-type cells than in sty1Δ or atf1Δ cells in the same stress. Among those genes, we selected genes that were Sty1p- or Atf1p-dependent in at least three of the five stress conditions. Various groups of Sty1p- and/or Atf1p-dependent genes were also selected based on cluster analysis (Figure 5A).

We selected genes that required Sty1p and/or Atf1p for repression among the genes that changed <2-fold in a given wild-type stress experiment and were also induced at least twice as highly insty1Δ or atf1Δ cells than in wild-type cells in the same stress. We then selected Sty1p- or Atf1p-repressed genes as those that were repressed in at least three of the five stress conditions. Comparison of the Sty1p- and Atf1p-repressed genes allowed us to identify genes that were repressed by both Sty1p and Atf1p and genes that were repressed by Sty1p but not by Atf1p.

Comparisons with Budding Yeast Data

Gene lists of S. cerevisiae CER/ESR genes were downloaded from the accompanying websites of Gasch et al.(2000) and Causton et al. (2001). Genes with a prospectiveS. pombe ortholog were determined using a table of curated orthologs created by mutual highest gene hits using FASTA aided by manual inspection of pairwise alignments and domain organization (Val Wood, personal communication available from the Sanger Institute FTP site: http://www.sanger.ac.uk/Projects/S_pombe/ftp.shtml) and imported into GeneSpring. The total number of orthologs available at the time of analysis was 2842. CESR genes that were induced (314 genes) or repressed (424 genes) were selected based on cluster analysis (Figure 1A lists available from our website). These genes were translated into S. cerevisiaehomologs using the ortholog table and list comparisons were performed with GeneSpring.

Fig. 1. Changes in gene expression in response to five environmental stresses. (A) Approximately seventeen hundred genes whose transcript levels changed significantly by at least twofold in one or more of the stress conditions were hierarchically clustered based on their expression patterns in the five time course experiments (Eisenet al., 1998 see “Materials and Methods”). Horizontal strips represent genes, and columns represent experimental time points. The fold changes in expression, relative to the untreated wild-type sample (time point 0), are color-coded as shown in the bar. The labels on the right indicate CESR induced genes (red Table 1) and repressed genes (green) that were chosen based on conservative criteria as described in “Materials and Methods.” (B) Average expression patterns of the CESR-induced (red) and -repressed (green) genes as labeled in A in the five stress conditions.

Discovery of Statistically Significant Sequence Motifs

We searched upstream intergenic regions of limited length (up to 1000 base pairs) for sequence motifs that were statistically overrepresented for our sets of coexpressed stress-response genes. The sequences were extracted from S. pombe chromosome release 22.03.2002 on the Sanger Institute FTP site in EMBL format. The search was carried out by the SPEXS tool available online fromhttp://ep.ebi.ac.uk (Brazma et al., 1998 Vilo, 1998). Given a set of upstream sequences, this tool searches exhaustively for all possible sequence patterns that are common to a minimum number of sequences in the set. For each of these motifs, SPEXS calculates the statistical significance of its occurrence with respect to a control set of sequences, which in our case was the total set of intergenic sequences.

Generally, we limited the query motif to substrings of arbitrary length that contained up to one “wild-card” (N), but in some cases, we used more general patterns (i.e., with one or two group character symbols). The statistical significance was calculated according to the binomial distribution (Vilo et al., 2000). To assess the significance thresholds for each set, we repeated this process on sets containing the same number of intergenic sequences selected at random, repeating the randomization three times independently. We reported only the patterns clearly above the significance threshold (with the binomial probabilities at least 10 times smaller than the lowest probabilities in any of the randomized sets) and with the selected group of upstream sequences enriched for the motif at least twofold compared with all intergenic regions.


Ketogenic Diet - Powerful Dietary Strategy for Certain Conditions

True. Fatty acids seem to be a major source of energy for many cancers. It seems that cancer cells will burn whatever is available.

When you consider Gerson therapy (which supposedly had great results), it limited most fats and animal products from the diet and focused primarily on nutrients and juices. So many factors appear to be at play.

Here's a paper I read some months ago on fatty acid oxidation in cancer cells:

Warburg suggested that the alterations in metabolism that he observed in cancer cells were due to the malfunction of mitochondria. In the past decade, we have revisited this idea and reached a better understanding of the ‘metabolic switch’ in cancer cells, including the intimate and causal relationship between cancer genes and metabolic alterations, and their potential to be targeted for cancer treatment. However, the vast majority of the research into cancer metabolism has been limited to a handful of metabolic pathways, while other pathways have remained in the dark. This Progress article brings to light the important contribution of fatty acid oxidation to cancer cell function.

The process of cellular transformation and cancer progression involves genetic mutations and epigenetic alterations, as well as the rewiring of cellular signalling and the reprogramming of metabolic pathways1. We now perceive these processes as intimately interconnected and interdependent2. Emanating from the initial hypothesis of Warburg3 (now known as Warburg's hypothesis), the latest research has revealed that metabolic reprogramming occurs as a consequence of mutations in cancer genes and alterations in cellular signalling. Much of the hype in cancer metabolism comes from the genuine observation that most cancer cells are programmed to increase glucose uptake, but to reduce the proportion of glucose oxidized in the Krebs cycle. Rather than oxidizing glucose for ATP production, glucose in cancer cells tends to be used for anabolic processes, such as ribose production, protein glycosylation and serine synthesis4–7. Cancer cells use additional nutritional inputs for anabolism besides glucose. From its metabolism to pyruvate, glutamine is key for providing reduced NADPH, which is needed for lipid synthesis, and to refill the Krebs cycle (anaplerosis)8,9. The control of this pathway by key oncogenes, such as MYC and mutant RAS, has further enforced the importance of this route in cancer. This view of cancer metabolism takes the focus away from ATP as the key product of glucose and glutamine catabolism. The fact is that in most biological contexts (but not all, as we discuss below), ATP production is sufficient for cancer cell function.

In addition to glucose and glutamine, fatty acids are an extremely relevant energy source. They can be incorporated from the extracellular media, or can be potentially obtained from hydrolysed triglycerides (in cells accumulating lipid droplets) by neutral (N) hydrolases in the cytoplasm or acid (A) hydrolases through a novel autophagic pathway: lipophagy10. De novo synthesis of fatty acids is required for membrane synthesis and therefore for cell growth and proliferation. Fatty acid synthesis is an anabolic process that starts from the conversion of acetyl CoA to malonyl CoA by acetyl CoA carboxylase. Malonyl CoA is then committed to fatty acid synthesis (FAS) and is involved in the elongation of fatty acids through fatty acid synthase (FASN). Additional modifications of fatty acids can be carried out by elongases and desaturases. Fatty acids are catabolized by the fatty acid oxidation (FAO also known as β-oxidation) pathway.

With most cancer researchers focusing on glycolysis, glutaminolysis and fatty acid synthesis, the relevance of FAO for cancer cell function has not been carefully examined, and its relevance has remained obscure. However, studies in the past 4 years have started to bring to light a relevant role for this metabolic pathway in cancer, and this is accompanied by new and exciting therapeutic implications. The focus of this Progress article is to enumerate, highlight and integrate these recent findings into our current understanding of metabolic reprogramming in cancer cells.

Extra ATP when needed
Relative to their dry mass, fatty acids provide twice as much ATP as carbohydrates (six times more when comparing stored fatty acids to stored glycogen), and in turn they are the preferred nutrient for storage (in the form of triglycerides in adipose tissue) under conditions of nutrient abundance. FAO is composed of a cyclical series of reactions that result in the shortening of fatty acids (two carbons per cycle) and that generate in each round NADH, FADH2 and acetyl CoA, until the last cycle when two acetyl CoA molecules are originated from the catabolism of a four-carbon fatty acid (FIG. 1). NADH and FADH2 that are generated by FAO enter the electron transport chain (ETC) to produce ATP (FIG. 2). FAO is carried out in energy-demanding tissues (such as the heart and skeletal muscle) and in the liver as a central organ for nutrient supply and conversion.

We have summarized the metabolic switch as a programme in which the utilization of metabolic intermediates for anabolism prevails beyond ATP production. But there are situations in which cancer cells seem to require increased ATP production. This is exemplified by loss of attachment (LOA) to the extracellular matrix. Cells derived from solid tumours that undergo LOA display inhibition of glucose uptake and catabolism, which results in the loss of ATP, NADPH (as a result of decreased flux through the pentose phosphate pathway (PPP)) and increased production of reactive oxygen species (ROS) (FIG. 3). Schafer and co-workers11 showed that in these settings ROS inhibit FAO, and that antioxidants counteract ROS accumulation and can reactivate FAO, increase ATP levels and prevent LOA-induced anoikis, although the exact mechanism by which an increase in ATP rescues anoikis remains unclear.

[..]
Cancer metabolism can be perceived as a network of pathways with plasticity, feedback loops and crosstalk that ensure the fitness of tumour cells. Plasticity is key, and FAO might provide some of this plasticity by enabling the production of ATP and NADPH when required, eliminating potentially toxic lipids, inhibiting pro-apoptotic pathways and providing metabolic intermediates for cell growth. However, FAO cannot be perceived as a metabolic pathway that is active independently of the microenvironment of the cancer cell. Indeed, in ovarian cancers, which have a predilection to metastasize to the omentum (an adipocyte-rich tissue), the interaction with adipocytes is necessary for the transfer of lipids to the cancer cell, the activation of FAO and the establishment of metastasis38.

A big challenge is to unify the idea of FAO as an essential pathway in cancer cells with the fact that cancer cells also require active FAS in order to grow and divide. Dogma states that FAO and FAS are incompatible. In principle, ACC determines which pathway is active, on the basis of acetyl CoA and malonyl CoA levels. Therefore, as ACC is a ‘one-way street’, both metabolic activities cannot coexist. However, we might need to rethink such a rigid regulatory framework. The group of Nissim Hay32 showed that genetic manipulation of ACC1 or ACC2 in cancer cells yielded different outcomes in terms of FAS and FAO. In addition, FAO metabolism can contribute to the accumulation of acetyl CoA in the cytoplasm that is needed to initiate FAS, so that FAS and FAO can support each other23. On the basis of this idea, we can speculate that, rather than a total pool of acetyl CoA and malonyl CoA, there might be ACC1 and ACC2 localization-dependent compartmentalization39 of these metabolites that allows both metabolic pathways to be active simultaneously and independently from each other.

The data suggesting a greater requirement of FAO in undifferentiated cells also raise an interesting possibility. It is plausible that in quiescent and undifferentiated cells the competition between FAS and FAO may be less prominent (as these cells display a lower membrane synthesis rate), thus indicating that these cells might derive a full survival benefit from FAO activation and its biological output. In turn, their dependence on FAO could make them vulnerable, providing a unique therapeutic opportunity from the pharmacological manipulation of this metabolic pathway.

For all the reasons stated above, there is an exciting therapeutic potential for the pharmacological blockade of FAO in cancer. Two key enzymes in the FAO pathway are particularly interesting as potential targets for pharmacological intervention. CPT1 is considered the rate-limiting enzyme in FAO and can be pharmacologically targeted. Drugs that target 3-ketoacylthiolase (3-KAT), which catalyses the final step in FAO, are also available (TABLE 1).


8. Acknowledgment

This work was funded in part by Touro University California. The authors are grateful to Dr. Ricardo Hermo for critical reading of the manuscript.

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Relative roles of ATP and NADPH in anabolic and other processes - Biology

Fig. 1. Diagrammatic section through the eye.

Fig. 2. A schematic 3-D computer-assisted drawing showing tracks of laser beams transmitted through a lens. The beams are brought to a sharp focus in a single plane. (Kuszak JR, Sivak JG, Herbert KL et al: The relationship between rabbit lens optical quality and sutural anatomy after vitrectomy. Exp Eye Res 71:267, 2000)

For the eye to focus the image of a near object on the retina, there must be an increase in ocular refractive power. The mechanism that achieves this change is accommodation, and the eye at rest is said to be unaccommodated. The mechanism of accommodation varies among species. 2 , 3 Not all animals accommodate. Some fish retract their lenses to focus on distant objects, whereas in snakes and frogs and many mammals, the position of the lens moves forward for near vision. In certain other fish and horses, the retina is tilted so that the lens-retina distance alters generally in a dorsal-ventral plane. In humans, the ability of the eye to alter focus is facilitated by a change in the shape of the lens there is no change in the curvature of the cornea or the length of the eyeball. In the human, the accommodative mechanism involves the ciliary muscle, the zonules, and the lens itself. However, the role of the lens is passive in the sense that its shape change takes place as the result of contraction or relaxation of the ciliary muscle.

When the ciliary muscle is relaxed, the zonules are under tension, exerting a centrifugal force on the lens equator that causes the lens to take on a flattened form. Contraction of the ciliary muscle makes it shorten and move forward and toward the equator of the lens. As a result, the zonules become less taut their pull on the lens capsule is reduced and the lens changes to its relaxed, more spherical, and, thus, accommodated, shape. This results in an increase in the dioptric power of the lens, allowing a near object to come into focus on the retina. To shift the focus back from a near to a distant object, the ciliary muscle relaxes and the elasticity of Bruch's membrane causes it to move posteriorly, widening the ciliary ring so that the zonules become tense once again and flatten the lens. The principle of this currently accepted theory for the mechanism of accommodation was first suggested by Helmholtz 4 in 1855, and the important role of the lens capsule was emphasized by Fincham 5 in 1937.

During accommodation, the principal change in the lens shape is seen at the anterior surface. The anterior surface of the unaccommodated lens has a spherical radius of curvature of about 12 mm. When the eye accommodates, the anterior surface tends to bulge centrally, attaining a radius of curvature of about 3 mm. The more peripheral anterior surface shows relatively little or no increase in curvature. Minimal changes occur in the curvature of the posterior lens surface Fincham 5 detected changes from 5.18 to 5.05 mm in one case and from 5.74 to 4.87 mm in another case for an accommodative change amounting to 8 diopters. The axial thickness of the lens increases during accommodation and the diameter decreases. The increase in lens axial thickness is paralleled by a shallowing of the anterior chamber of the eye.

The amount that an eye can alter its refractive power between focus on a far object and a near object is called the amplitude of accommodation. The amplitude of accommodation decreases progressively with age, a condition known as presbyopia 6 (Fig. 3). The decline in accommodative amplitude means that the near point becomes more distant from the eye and, thus, small objects must be held farther away to be seen clearly. It is doubtful that there is a single cause of presbyopia. Theories proposed to explain the development of presbyopia include changes in the elastic properties of the capsule, a change in the deformability of the lens cell mass, loss of elasticity in Bruch's membrane, and weakening of the ciliary muscle. 7𠄹 There is little evidence to support the concept of a weakened ciliary muscle, although age-related morphologic changes are known to occur in this tissue. 10 In presbyopic eyes, the ciliary muscle is still able to contract vigorously under the influence of pilocarpine. 11 Presbyopia does not appear to be simply the result of changes in the rate of lens thickening or anterior chamber shallowing with age. 12 Changes in the aging capsule have been studied by Fisher, 13 who concluded that reduction in capsular elasticity in the senile lens is a contributing factor in presbyopia. The principal cause of presbyopia was once considered to be “lenticular sclerosis,” a progressive hardening of the lens with age so that it becomes less deformable. However, in his study of lens mechanics, Fisher 14 determined that age-related changes are not entirely consistent with the timetable for the onset of presbyopia.

Fig. 3. Decrease in the amplitude of accommodation with age in the human. Open symbols, female solid symbols, males. A. Left eye, based on subjectively reported clarity of target viewed through trial lenses of progressively decreasing plus or increasing minus power. B. Phenylephrine-dilated right eye, based on Hartinger coincidence refractometry while subject accommodated to view target with left eye, as in A. (Koretz JF, Kaufman PL, Neider MW et al: Accommodation and presbyopia in the human eye𠅊ging of the anterior segment. Vis Res 29:1685, 1989)

Fig. 4. Scheme to show the development of the lens. A. Lens thickening. B. Lens pit. C. Lens pit closing. D. Lens vesicle. E. Elongation of cells of the posterior wall of the lens vesicle. F. Obliteration of the cavity of the lens vesicle by cells of the posterior wall. G. Formation of lens sutures by the meeting of fibers developed in the equatorial region. (Mann I: The Development of the Human Eye. New York, Grune & Stratton, 1950)

The cells of the lens vesicle are positioned so that their apical surfaces face inward and their basal surfaces are in contact with the lens capsule. The cells at the posterior of the lens vesicle elongate and by approximately the 16-mm stage (7-weeks gestation) they have grown to form primary lens fibers that fill the cavity of the lens vesicle. The primary lens fibers eventually lose their nuclei and organelles but what remains of each cell is retained throughout life as the 𠇎mbryonic nucleus” of the lens.

Although the cells at the posterior portion of the embryonic lens grow to become primary lens fibers, the cells at the equator have a different fate. They divide and give rise to new cells. In most cases the newly formed cells differentiate and elongate to form secondary lens fibers. A few of the newly formed cells probably remain as undifferentiated epithelial cells, populating the anterior epithelial monolayer as the lens grows. The equatorial or germinative zone of the anterior lens epithelium continues to divide and produce secondary lens fibers throughout life. It is the only region of the adult lens where the cells remain capable of mitosis. Neither lens fibers nor the central anterior lens epithelium undergo cell division.

As the cells in the equatorial zone of the epithelium elongate, the portion of each cell that is anterior to the nucleus grows forward under the undifferentiated epithelium, lying just beneath the anterior capsule the posterior portion of each growing lens fiber simultaneously elongates backward beneath the posterior capsule. When the ends of similarly elongated cells meet each other close to either the anterior or posterior pole of the lens, both cells stop growing. New fiber cells are formed continually throughout life and older cells are displaced toward the center of the lens. Like tree rings, the youngest cells are at the lens periphery, the oldest at the center. Understandably, the lens center becomes denser. It is referred to as the lens nucleus. The more superficial layers of younger fiber cells are called lens cortex (see diagram, Fig. 5). An interesting feature of the differentiation of lens epithelium into lens fibers is that as the fibers are displaced to the interior of the lens, they reach a stage at which they lose their nuclei and other cytoplasmic organelles.

Fig. 5. Diagrammatic cross section through the lens.

The zonular fibers, which are suspensory ligaments that support the lens, develop from the neuroepithelium at approximately the 65-mm stage (3-months gestation), when the ciliary body and iris begin to develop from the optic vesicle. At the 110-mm stage (4-months gestation) the zonules are well developed, running from the inner surface of the ciliary body to the lens capsule. Still later, at the 240-mm stage (7-months gestation), the vascular hyaloid system within the vitreous regresses it is absent by 8.5-months gestation, leaving the lens without a blood supply for the rest of its life.

The regulatory factors involved in lens development are complex and have been nicely reviewed by McAvoy and colleagues 15 and van Doorenmaalen. 16 Lens induction depends on the interaction between presumptive lens ectoderm and other tissues, particularly the retina. Transcription factors Pax-6, as well as Eya, Six, Sox, and Prox play an important role. 15 The communication between developing tissues is a two-way street signals emanating from the lens have a significant influence the development of other parts of the eye 17 (Fig. 6). The involvement of specific lens-inducing substances in lens differentiation seems certain. 15 , 18 , 19 Chamberlain and McAvoy 19 have suggested that fibroblast growth factor promotes lens cell differentiation, and Beebe and colleagues 18 demonstrate a role for insulin-like growth factors.

Fig. 6. Eye development following lens transplantation in Astyanax mexacanus, a fish with an eyeless cave-dwelling form and an eyed surface-dwelling form. The lens of the surface fish stimulates development of the eye when it is transplanted into the optic cup of the blind cave fish. The figure shows head regions of (A to D) cave fish or (E to H) surface fish hosts shown after 2 days (A and E) and 2 months (B to D and F to H) of development. Panels A and E are ventral views, panels B and F are dorsal views, and panels C and G are lateral views. The transplant side is to the right in panels A, B, E, and F (indicated by an arrow in panel A and panel E). Panels D and H show sections through restored and degenerate eyes in cave fish and surface fish hosts, respectively. (Yamamoto Y, Jeffery WR: Central role for the lens in cave fish eye degeneration: Science 289:631, 2000)

Cell division and, thus, growth of the lens continue throughout life as new lens cells are formed, the older cells are displaced toward the interior of the lens. As a result, the lens grows in size and weight as humans age 20 (Figs. 7 and 8). It has been estimated that the human lens thickness increases at about 0.02 mm per year. 21 , 22 The anterior-posterior dimension of the lens in the newborn is 3.5 to 4.0 mm, reaching 4.75 to 5.0 mm at 90 years of age. The equatorial diameter in infants is about 6.5 mm, increasing to 9.0 with age. The radius of curvature of the anterior surface of the lens is 8 to 14 mm, and at the posterior surface it is 4.5 to 7.5 mm. There is, however, variation in curvature with age and accommodation.

Fig. 7. The increase in weight of the human lens from birth to 90 years of age. The graph constructed using results presented originally by Scammon and Hesdorffer. 20

Fig. 8. 3-D drawings illustrating changes in the lens equatorial and pole-pole dimensions with age. At the time of birth (panel A) the lens is an asymmetric ellipse with an equatorial diameter approximately 1.5 times the anterior-posterior dimension. In the young adult (panel B), the equatorial diameter is close to twice the length of the anterior-posterior dimension, illustrating the unequal growth rate in the two lens axes. Panel C and D also show that throughout adult life, the equatorial dimension of the lens increases faster than the polar dimension. (Al-Ghoul KJ, Nordgren RK, Kuszak AJ et al: Structural evidence of human nuclear fiber compaction as a function of aging and cataractogenesis. Exp Eye Res 72:199, 2001)

Fig. 9. A. Light micrograph of the zonular fibers (a), capsule (b), lens epithelium (c), and cortex (d) approximately 0.7 mm anterior to the equator of the lens. The zonules insert into the most superficial portion of the capsule. The capsule contains scattered linear densities (arrows) because of the presence of coarse fibrils. The epithelial cells are tall and columnar their base faces the capsule, and their apex faces the lens cortex. The round nuclei are either central in the cytoplasm or displaced slightly toward the apex. Elaborate basal infoldings of the cells membranes are apparent. The Golgi zone appears as a dense accumulation in the apical cytoplasm (e).The rectangular or hexagonal cells of the lens cortex are seen in cross section. They have a fairly homogeneous cytoplasm. Interlocking processes occur along both the short and the long sides of the hexagon in the superficial layers of the cortical cells. In deeper cortical cells the interlocking processes are found chiefly along the long sides of the hexagons (× 1300). B. 3-D drawing of the lens indicating the interrelation of the capsule and underlying lens cells. The capsule (a) shows inclusions of fine filamentous material. The anterior lens epithelium (b) shows interdigitation of its basal surface with adjacent cells. The superficial cells of the cortex show the hexagonal shape and interdigitations at their hexagonal ends, as well as along their edges (c1, arrows). The deeper cortical cells (c2) also show a tongue-and-groove type of interdigitation along their long sides (arrows), but the interlocking is absent at the short ends. (Hogan MJ, Alvarado JA, Weddell JE: Histology of the Human Eye. Philadelphia, WB Saunders, 1971)

The lens capsule is analogous to a basement membrane. It is composed mainly of type IV collagen combined with about 10% glycosaminoglycan. 28� As with other basement membrane material, the lens capsule stains positive with periodic acid-Schiff reagent in histologic section. The capsule is also easily digested by collagenase. Autoradiographic studies by Young and Ocumpaugh 31 demonstrated that the capsule is synthesized by the lens epithelium and superficial posterior lens fibers synthesis of capsular material appears to persist throughout life at the anterior of the lens but not at the posterior surface. 26 The zonules, or suspensory ligaments, are anchored in the lens capsule. The zonules hold the lens in place and influence the shape of the lens by the degree of tension they exert. The zonules are structurally and biochemically similar to elastic microfibrils in other tissues. 32� The capsule and zonules play an important yet passive role in the process of accommodation. Their degree of elasticity is important. As described previously, the shape of the lens changes according to the degree of tension applied to the zonules by the ciliary muscle.

The plasma membrane domains at the lateral and apical aspect of the epithelium have desmosomes for cell adhesion. The epithelial cell plasma membranes have also been shown to have gap junctions, 36 , 37 which facilitate intercellular communication between the adjacent epithelial cells and less often with the underlying lens fiber cells. The role of gap junctions in the physiology of the lens is discussed later. All lens epithelial cells possess a nucleus together with granular cytoplasm, mitochondria, Golgi apparatus, ribosomes, rough endoplasmic reticulum (ER), and numerous small filaments. 35 , 38 Such intracellular organelles are consistent with the competent metabolic function of the epithelial cells. In addition to cell division, the epithelium is responsible for much of the active solute transport activity in the lens and also for secretion of capsular material. Cytochemical studies of lens epithelium membranes have demonstrated the presence of sodium-potassium adenosine triphosphatase (Na, K-ATPase the sodium pump enzyme) at the lateral and apical surface membranes. 39 , 40 The epithelium contains a high specific activity of many other enzymes, including acid phosphatase and aldose reductase. 41 , 42 In simple terms, lens epithelial cells have the characteristics of “normal” cells. The same is not true for lens fibers. Although they are differentiated epithelial cells, fibers are highly specialized and have several uncommon features.

Fig. 10. Scanning electron micrographs illustrating the tightly packed mass of fiber cells in the human lens. Panel A shows a low-magnification view of a lens from a 45-year-old donor. The lens has been split in half. Panel B shows a higher magnification view of a lens from a 38-year-old donor. (Al-Ghoul KJ, Nordgren RK, Kuszak AJ et al: Structural evidence of human nuclear fiber compaction as a function of aging and cataractogenesis. Exp Eye Res 72:199, 2001)

The newly formed elongating lens cells have a hexagonal cross section that they retain throughout life (Fig. 11). Fibers are enormously long but thin. Within the cortical region of the lens, the fiber cells are 8 to 12 mm long, 7 μm wide, and 4.5 μm thick. 35 The lens fibers are very densely packed, forming a highly structured, honeycomb-like array with an intercellular distance of only about 20 nm. The intercellular dimension is small compared with the wavelength of visible light, and this, together with the high degree of order, minimizes light scatter and favors transparency. A recently formed lens fiber has its center approximately at the equator, whereas its two ends project forward and backward to the anterior and posterior poles of the lens. If the system were perfectly symmetric, all the fibers would end at the same location at either pole of the lens this does not happen, perhaps because it would detract from the optical properties of the optical axis of the lens. Instead, the fiber ends meet in complex patterns called lens sutures. The human embryonic lens has Y sutures (λ anteriorly Y posteriorly), but in the adult lens there is a more complicated four-pointed star arrangement. 25

Fig. 11. Replica of obliquely fractured chicken lens at low magnification demonstrating the homogeneous cytoplasm of the hexagonally shaped and packed lens fibers. Because of the narrow extracellular space, the fracture plane jumps between the p and e fracture faces. The narrow extracellular space does not widen at the fiber threefold axes (arrows), revealing a narrow and tortuous route for diffusion of molecules through extracellular spaces. (Goodenough DA: Lens gap junctions: A structural hypothesis for nonregulated low-resistance intercellular pathways. Invest Ophthalmol Vis Sci 18:1107, 1979)

Superficial lens fiber cells show some moderate interdigitations between adjacent cells, but deeper lens fibers have distinct interlocking processes resembling ball-and-socket joints. 25 , 45 Moving farther into the nuclear region, the lens fiber cells retain their hexagonal shape, but the interlocking processes are replaced by ridges. The purpose of these interlocking mechanisms between lens fiber cells may be stabilization of the packing arrangement to prevent slippage of cells against each other when the lens changes shape during the process of accommodation.

Fig. 12. Freeze fracture replica of a rat lens cortical fiber cell. The image encompasses the width of an entire fiber. The large number of fiber cell junctions is apparent. Although this field is deceptively rich in gap junction, it illustrates the unusual numbers of such junctions that can be found between cortical fiber cells. (× 11,500.) (Fitzgerald PG, Bok D, Horwitz J: The distribution of the main intrinsic membrane polypeptide in the ocular lens. Curr Eye Res 4:1204, 1985)

Fig. 13. Results from a study in which a relatively small fluorescent solute, carboxyfluorescein (CF) and a large, 40 kDa solute, tetramethylrhodamine dextran (TD) were injected into a lens fiber cell. After 120 minutes, CF was observed to diffuse away from the injected fiber into neighboring cells (A), whereas the larger solute, TD, was retained (B). When a similar injected is made into a fiber cell deep in the lens core, both CF and TD diffuse from the injected cell this is seen in panel C in which TD is clearly visible 3 hours after injection into a peripheral fiber (arrow) but has mostly diffused away from the injection site in a core fiber (arrowhead). The results demonstrate the ability of solutes to pass from fiber to fiber via gap junctions and, in the lens core, via regions of cell-cell fusion. (Shestopalov VI, Bassnett S: Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core. J Cell Sci 113:1913, 2000)

The distribution of gap junctions does not appear to be spatially uniform across the fiber cell mass, and the extent of functional cell-cell coupling is high at the equator and low at the anterior and posterior poles. 47 Fiber cells located within 400 μm of the lens surface are able to reversibly uncouple in response to stimuli such as a change of pH the fiber cell connexons can apparently regulate their conductance. More deeply located, older fibers are not capable of uncoupling, and this may stem from posttranslational modification of the connexin proteins or the absence of essential cofactors. There could, however, be other pathways that couple deeply located fibers. It has been found that cell-to-cell fusion occurs in the lens core 54 (Fig. 14).

Fig. 14. A 3-D representation showing the structure of two neighboring fiber cells in the lens core illustrating that although the fibers are present as discrete, separate cells throughout most of their length, they are fused in zone b. Such fusion of adjacent fiber cells is likely to permit cell-to-cell movement of fairly large molecules as illustrated in Figure 13. (Shestopalov VI, Bassnett S: Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core. J Cell Sci 113:1913, 2000)

Water66% of wet wt
Protein33% of wet wt
Lipids28 mg/g wet wt
Na + 17 mM*
Cl - 26 mM
K + 125 mM
Ca 2+ 0.3 mM
Glucose0.6 mM
Lactic acid14.0 mM
Glutathione12.0 mM
Ascorbic acid1.0 mM
Inositol5.9 mM

*Solute concentrations are shown as mmol/kg lens H2O.

The low water content of the lens partly reflects the unique protein composition of fiber cell cytoplasm. It also reflects a lack of extracellular space. Although the adult human lens capsule is about 80% water, 55 the cortex contains 68.6% water and the nucleus 63.4% water. 56 The normal human lens shows no significant alteration in hydration with age. 56 , 57 Not all the water in the lens is freely diffusible. A fraction is involved in protein hydration and is, thus, considered bound. Earlier evidence for different fractions of lens water was obtained using flux studies with radioactively labeled water. Nuclear magnetic resonance (NMR) studies suggest that about half of the lens water is physically associated with lens protein. 58 Because the fibers are packed tightly, very little of the lens water is located in the spaces between the cells. The extracellular space of the lens has been measured by determining the distribution of tracer substances (e.g., inulin) that do not penetrate the lens cell membranes. In the rabbit lens, the inulin space has been reported to be about 5%. 55 , 59 Nearly a fourth of the measured extracellular space is located in the capsule. 55

Lens hydration can be significantly higher in certain forms of cataract. Precise regulation of lens hydration is critical for the maintenance of lens transparency. Cell swelling, cell shrinkage, or enlargement of the extracellular spaces result in disruption of the highly ordered arrangement of lens fibers, causing light scatter. Fischbarg and his coworkers 60 suggest that the lens epithelium is able to transport water inward to the fiber mass. Water may be shifted out of the lens at the equator. 47 The expression of specialized water channel proteins, aquaporins, might enable the lens to perform these functions.

Lens proteins can be broadly separated on the basis of their solubility in water. The water-soluble component accounts for 80% to 90% of mammalian lens protein and is composed mainly of the lens crystallins. In humans, the major proteins are α-, β-, and γ-crystallin in reptilian, avian, and invertebrate species there are other crystallins (Table 2). The water-insoluble fraction of lens proteins can be further separated on the basis of its solubility in urea. The urea-soluble fraction contains cytoskeletal proteins and modified crystallins. The urea-insoluble fraction contains mainly membrane protein. The original separation of lens proteins into soluble and insoluble fraction was accomplished in 1894 by Morner. 61

Lens Crystallin*Functional AbilitySpecies
αSmall heat shock protein/chaperonesAll vertebrates
βγMembers of microbial stress protein superfamilyAll vertebrates
εLactate dehydrogenase BDucks, crocodiles
δArgininosuccinate lyaseBirds, reptiles
τα-EnolaseTurtles, ducks, other selected vertebrates
ζNovel quinone oxidoreductaseGuinea pigs, camels, degus, llamas, rock cavies
μRelative of bacterial ornithine cyclodeaminaseAustralian marsupials
ηRetinaldehyde dehydrogenaseElephant screws
ρRelative of aldo-keto reductaseFrogs
λRelative of hydroxyl CoA dehydrogenaseRabbits, hares
πGlyceraldehyde-3-phosphate dehydrogenaseGeckos

*As described by Piatigorsky (1998) some lens crystallins are functional proteins expressed at low levels in other tissues but expressed in high abundance to serve as refractive proteins in lens cells. In different species, the different proteins have been recruited as lens crystallins.

α-Crystallin accounts for as much as 40% of total lens protein. α-Crystallin is not a single protein but a complex or aggregate of 30 to 40 copies of αA-crystallin and αB-crystallin in roughly a 3:1 molar ratio. The αA-and αB-crystallin polypeptides have a molecular mass of approximately 20 kilodaltons (kDa). At the time of synthesis, the αcrystallin complex has a molecular weight of about 7 × 10 5 daltons (Da). With aging, larger aggregates of α-crystallin are formed with molecular weights as high as 50 × 10 6 Da these aggregates of αcrystallin become water insoluble and have a tendency to bind to the plasma membrane of fiber cells. 62 , 63 This might contribute to a decrease in transparency.

From a three-dimensional standpoint, about half of the α-crystallin polypeptide chains are in a β-sheet configuration there is also some β-turn and helical configuration. 64 , 65 The amino acid sequence indicates approximately 55% homology between αA and αB. 66 The αA-crystallin gene is an evolutionary sibling of the αB-crystallin gene. 67 Although there is much similarity between αA- and αB-crystallin, there are marked differences in their expression. αA-crystallin is almost exclusively found in lens fibers, whereas αB-crystallin is expressed in numerous tissues including heart, skeletal muscle, brain, lungs, and kidney, as well as lens. 68 Studies on knockout mice indicate differences in the role of αA- and αB-crystallin. Deletion of αB-crystallin has little apparent effect on lens morphology, whereas αA-crystallin deletion causes cataract. 69

Similarity of amino acid sequences suggest that β- and γ-crystallins are products of the same gene family, which is distinct from α-crystallin. 65 The β-crystallins are the most abundant of all lens proteins, representing about 54% of the total water-soluble protein. The molecular weight of the β-crystallins ranges from 4 × 10 4 to 2.5 × 10 5 Da. The β-crystallins are heterogeneous and can be divided into a number of subgroups. The first four subgroups of aggregates to be identified have molecular weights estimated to be 250,000, 130,000, 60,000, and 37,000 Da 66 of these, the two larger species are called β-high and the smaller species are called β-low. Each of the β-crystallins contains two subunits the major subunit, βBp, has a molecular weight of approximately 24 × 10 3 Da. The βBp chain is a characteristic β-crystallin polypeptide found in a great number of mammalian species. The three-dimensional structure of β-crystallin is principally a β-sheet configuration. 64 , 70 Although β-crystallins are abundant, the γ-crystallins represent only 1% to 2% of the total lens protein. γ-Crystallins are found in the monomeric form with a molecular weight in the range of 20,000 to 27,000 Da. Like α- and β-crystallin, γ-crystallin has relatively little α-helical structure its configuration is mainly β-sheet and some β-turn. γ-Crystallin has been implicated in the so-called cold cataract if the temperature is reduced, the γ-crystallin can undergo a conformational change, resulting in opacification of the lens with warming, the cold cataract reverses. 70 , 71

It has been known for a long time that crystallins comprise the bulk of lens proteins and that their expression gives the lens unique optical properties. However, the original notion that these proteins are lens specific turned out to be incorrect. Analysis of crystallin amino acid sequences revealed that many of the proteins are either closely related or, in some cases, identical to enzymes and stress (or heat-shock) proteins found in nontransparent tissues 67 (see Table 2).Consistent with its identity as a stress protein, α-crystallin is able to suppress the heat-induced nonspecific aggregation of a range of soluble proteins including insulin, α-lactalbumin, and alcohol dehydrogenase. In this respect, α-crystallin is a molecular chaperone. In a variety of tissues as well as in the lens, α-crystallin likely plays a role in ensuring the correct folding of proteins. Evolution has led to gene sharing, 67 which is an apparent recruitment of functional gene products that are normally produced in modest amounts elsewhere but are expressed in high abundance in the lens as refractive proteins.

The protein associated with lens cell membranes represents 20% to 30% of the water-insoluble fraction of the lens proteins 72 the membrane proteins are insoluble in urea. Membrane proteins are classified as either intrinsic or extrinsic. The intrinsic membrane proteins are an integral part of the lens cell membrane, whereas extrinsic membrane proteins are associated only with the membrane surface. The principal intrinsic membrane protein is a 26,000-Da polypeptide, MIP-26, found in fibers but not epithelium. 72� MIP-26 is abundant it accounts for more than 60% of fiber membrane protein. Initially, MIP-26 was thought to be the unique protein component responsible for fiber-fiber coupling by gap junctions. However, subsequent studies have implicated connexin proteins in the formation of lens gap junctions. Connexin expression has been described previously. It appears MIP-26 may be a water channel. 76 MIP-26 is, in fact, a member of the aquaporin family, AQP-O. Lens epithelium expresses a different, higher conductance, aquaporin, AQP-1. 77 In the older lens there are increasing amounts of a 22,000-Da species of membrane protein (MIP-22). 78 It has been suggested that MIP-22 is an age-dependent degradation product of MIP-26. 78

Extrinsic membrane proteins have not been studied extensively in the lens. The presence of the glycoprotein fibronectin (MP 220�) has been demonstrated. Found in many tissues, fibronectin is generally thought to be involved with the interaction between the cell surface and the extracellular matrix. A 43,000-Da (MP 43) extrinsic membrane protein is of some interest because its relationship with the lens cell membrane surface appears to be calcium dependent this 43,000-Da polypeptide is thought to link aggregated crystallins to the cell membrane. The extrinsic membrane proteins of the lens are fully discussed by Alcala and Maisel. 72

Many enzymes are associated with the cell membrane, including the transport enzymes Na, K-ATPase and Ca-ATPase, as well as alkaline phosphatase and adenyl cyclase. 66 , 72 Enzymes can be either intrinsic or extrinsic membrane proteins. Glyceraldehyde-3-phosphate dehydrogenase is an extrinsic membrane protein (MP 37), whereas Na, K-ATPase (MP 90�) is an intrinsic membrane protein. There are a number of membrane proteins that are associated with carbohydrates, predominantly galactose. 79 , 80 Many of these membrane glycoproteins are intrinsic proteins associated with membrane receptors and surface recognition.

This component of the lens water-insoluble protein fraction is soluble in urea. The microfilaments of the cytoskeleton are composed of actin found in two filamentous forms (β and γ) and in the globular form. The intermediate filaments are composed of a 57,000-Da molecular-weight polypeptide called vimentin. Beaded chain filaments, unique to the lens, consist of globular proteins attached to a backbone chain. Beaded filament composition may involve the expression of lens fiber-specific proteins CP49 and filensin. The microtubules are a complex structure containing two subunits of tubulin, each with a molecular weight of about 50,000 Da. The composition of the lens cytoskeleton has been reviewed by Quinlan and colleagues. 81

The relative proportions of the various lens proteins change with aging. 82 The amount of soluble protein fraction remains fairly constant with aging, but the insoluble protein fraction continues to increase. In the young lens the insoluble protein fraction might contribute only 1% of the total dry weight, whereas in a 70-year-old lens it can contribute more than 50%. This increase in the insoluble fraction is related primarily to a conversion of soluble protein into insoluble protein as a result of the formation of aggregates. The formation of large protein aggregates most probably contributes to a deterioration of lens transparency. With aging there is also an accumulation of low-molecular-weight polypeptides in the lens. These appear to be degradation products of crystallins as a result of proteolysis. 83

Because the lens is more or less a functional syncytium of tightly packed cells, the electrolyte composition of lens as a whole resembles that of a single cell. Relative to the surrounding environment of aqueous humor and vitreous, the lens has a high potassium content and a low sodium content. 84 The potassium level is about 125 mmol/kg of lens water, and sodium is 14 to 26 mmol/kg of lens water. Lens chloride, which closely parallels lens sodium, is about 26 mmol/ kg of lens water. 85 Cataractous lenses with any cortical involvement always manifest deranged electrolyte and water balance. 86 This is not the case for nuclear cataracts.

The divalent cation calcium is found at very low levels in the normal clear lens. Studies with calcium-sensitive fluorescent dyes suggest that most of the intracellular calcium is not freely diffusible the free calcium concentration in lens cytoplasm is only about 100 nM. 87 Calcium levels rise dramatically in most forms of cataract with cortical involvement. 88 , 89 Normally, calcium is important in the maintenance of lens cell membrane permeability. 90 , 91 Abnormally high concentrations of intracellular calcium are cytotoxic they are likely to promote apoptosis (programmed cell death), to cause changes in protein aggregation, and almost certainly to contribute to the mechanism of cataractogenesis. 92

Cytoplasmic pH in lens cells has been reported in the range 6.9 to 7.2, which is slightly more acidic than aqueous humor. 93 , 94 The concentration of free amino acids in the mammalian lens is higher than that in the aqueous humor, vitreous, or plasma. 95 , 96 There is good evidence that amino acids are transported into the lens by means of an energy-dependent carrier system. Little is known about the precise distribution of amino acids within the lens, but it is safe to assume that they are present at high levels in the epithelium and outer most fibers, the major site of protein synthesis.

Table 3. Phospholipid Composition of Lens Fiber Membranes

 SPHPCPEPSPI
Human47�2𠄵9�6�1𠄴
Rabbit14�23�28�11�2
Monkey4672091

The values indicate the amount of each phospholipid as a percent of the total lens phospholipid. SPH, sphingomyelin PC, phosphatidylcholine PE, phosphatidylethanolamine PS, phosphatidylserine PI, phosphatidylinositol. The table is based on an original study by Zelenka (1984).

Fig. 15. The structure of dihydrosphingomyelin, a phospholipid that is abundant in human lens but not in other tissues. The arrow indicates the location of the saturated double bond that makes this phospholipid so unusual. (Courtesy D. Borchman and M.C. Yappert, University of Louisville.)

Substantial changes in lens lipid composition and distribution occur with aging. There is a doubling of lens cholesterol from about age 25 to 75 years and a concomitant increase in sphingomyelin on the other hand, phosphatidylethanolamine and phosphatidylcholine levels decrease with aging. These changes, which reflect alteration in cell membrane structure, probably influence lens cell membrane function as the lens ages and may contribute to changes of ion permeability that occur with age. 89

The lens, like all cellular tissues, must generate a continual supply of ATP. The ATP is used to provide energy for processes such as active solute transport, synthesis of protein and lipid, and cell division. About 10% of the ATP generated is used for protein synthesis. The greatest need for energy in the lens appears to be within the epithelium. In the lens, as in most other tissues, a large fraction of the available ATP is used by Na, K-ATPase.

Anaerobic glucose metabolism plays an important role in the lens (unlike most other tissues). Anaerobic glycolysis produces about two thirds of the ATP in the lens. Anaerobic glycolysis generates 2 moles of ATP for each mole of glucose. Although the enzymes necessary for glycolysis are found throughout the lens (see Cheng and Chylack 116 for review), the first enzyme of the glycolytic pathway, hexokinase, which catalyzes the conversion of glucose to glucose-6-phosphate, is present at very low levels. 117 The low level of hexokinase appears to be the rate-limiting factor for lens glucose metabolism and, therefore, restricts the generation of glucose-6-phosphate for both glycolysis and the hexose monophosphate shunt pathway 118 (see later discussion). Anaerobic glycolysis, although not as efficient as the aerobic glucose metabolism process, obviates the problem of oxygen starvation in a tissue totally dependent on the aqueous humor, which has an unusually low oxygen tension. Indeed, lenses can survive under incubation conditions in the complete absence of oxygen as long as an adequate supply of glucose is available. However, when the lens is deprived of glucose, it rapidly uses up endogenous resources and begins to deteriorate, which results in loss of transparency.

About 80% of the glucose entering the lens is converted to lactic acid by means of anaerobic glycolysis. Some of this lactic acid is metabolized further by the Krebs cycle, but most of it simply diffuses out into the aqueous humor to be eliminated from the eye. This explains why the aqueous leaving the eye has a much higher lactate concentration than newly formed aqueous humor. 119

The aerobic metabolism of glucose is considerably more efficient than glycolysis, producing 38 moles of ATP from each mole of glucose. In the lens, however, ATP production by means of the Krebs cycle is mostly limited to the epithelium and perhaps some of the newly formed fiber cells at the lens equator. The epithelium possesses the necessary enzymes and probably has an adequate oxygen supply from the newly formed aqueous humor that flows across the anterior surface of the lens. It has been estimated that only about 3% of the total glucose is metabolized by means of the Krebs cycle, but because of the efficiency of the pathway this could generate up to 20% of the total ATP in the lens. 120

Two other significant pathways of glucose metabolism operate in the lens: the hexose monophosphate shunt and the sorbitol pathway. 121 Although these pathways do not generate a very large amount of ATP, they have received a lot of attention because of the relationship of nicotinamide adenine dinucleotide phosphate reduced form/nicotinamide adenine dinucleotide phosphate (NADPH/NADP) to glutathione metabolism and the link of the sorbitol pathway to sugar cataract. In the rabbit lens, about 14% of the glucose is metabolized by the hexose monophosphate shunt. This pathway uses glucose-6-phosphate as its initial substrate and generates pentoses, which are used in nucleic acid synthesis, and NADPH, which is an essential cofactor in many biochemical reactions (Fig. 16). NADPH is required for the maintenance of reduced glutathione by glutathione reductase and is also a necessary cofactor in the sorbitol pathway. Some of the pentoses are recycled to reenter the glycolytic pathway. Carbon dioxide produced by the hexose monophosphate shunt diffuses into the aqueous humor.

Fig. 16. A simplified scheme of the major pathways of glucose metabolism in the lens.

The sorbitol pathway of the lens converts glucose to sorbitol and then to fructose using the enzymes aldose reductase and polyol dehydrogenase, respectively (see Fig. 16). Under normal conditions, no more than about 5% of the glucose used by the lens is metabolized by the sorbitol pathway. Although it was first believed to be an ancillary ATP-generating mechanism, the sorbitol pathway has also been proposed as a means of protecting the lens from osmotic stress in hyperglycemia (see Cheng and Chylack 116 for review). The important link between the sorbitol pathway and sugar cataract has caused researchers to focus on the possible role of aldose reductase in the cataractogenic mechanism. 122 The availability of inhibitors for aldose reductase produced the encouraging finding that galactose cataracts in laboratory animals can be prevented by aldose reductase inhibition. However, the advances in the development of aldose reductase inhibitors for the treatment of human diabetic cataract have been complicated by the apparent differences between the human lens sorbitol pathway and sorbitol metabolism in animal lenses. 116 , 122

Protein synthesis takes place mainly in the lens epithelium and superficial cortex. 123 The process occurs slowly. Merriam and Kinsey 124 estimated that glycine and serine turnover was no more than 5% per day. This makes sense when one considers that, once made, most lens proteins, at least in mature fibers cells, are retained for life. Specific crystallins are synthesized at different times during the development of the lens. The pattern of protein synthesis in the lens epithelium is different from that in lens fibers. α-Crystallin is expressed early in lens morphogenesis and characteristically appears in all lens cells. β- and γ-crystallins appear later in lens morphogenesis and are only synthesized by lens fibers. 125 In the rat, epithelium and fibers express different isoforms of Na, K-ATPase. 126 The expression of aquaporins and connexons is also different in fibers and epithelium. 47 , 50 Using immunofluorescence techniques to study lens development, McAvoy and colleagues 15 determined that β- and γ-crystallin synthesis can be detected when the epithelial cells differentiate and elongate to become lens fibers. Shestopalov and Bassnett 54 demonstrated that fibers that retain their nucleus remain competent in their ability to synthesize proteins but this ability is probably lost once the fiber cell ages to the point when nucleus and other organelles are degraded.

Because the proteins in the mature fiber cell remain in the lens for life, attention has been paid to the molecular modifications of the primary gene products that take place following their synthesis. There has been considerable study of the aggregation of lens proteins, 127 , 128 because high-molecular-weight aggregates could cause light scattering and threaten the transparency of the lens. There have been a number of studies suggesting that, during the aging process, lens proteins may be susceptible to changes through oxidative mechanisms and other biochemical reactions such as phosphorylation and nonenzymatic glycation. 129�

The breakdown of obsolete or damaged proteins in the lens is catalyzed by peptidases and proteases. The lens contains a number of different endopeptidases, including neutral proteinases, and also the calcium-activated calpains. 132 Proteins can also be broken down by a multicatalytic proteinase complex (proteasome). 133 The turnover of proteins in the epithelium is consistent with the need for proteases. Their presence in lens fibers may be connected with fiber maturation, as well as with the degradation of proteins that are damaged during aging.

The finding of a large amount of glutathione in the lens, particularly in the epithelium and superficial cortex, together with the fact that the glutathione level is markedly reduced in cataractous lenses, has attracted a great deal of interest. 107 One of the principal duties of glutathione is to maintain lens protein sulfhydryl groups (-SH) in the reduced state. Oxidation of lens protein sulfhydryl groups is unwanted because it leads to cross linkage of proteins, resulting in aggregation and loss of lens transparency. 134 When the system works correctly, it is glutathione (GSH) that becomes oxidized, not the lens protein. Two molecules of oxidized glutathione link as G-S-S-G. The recycling of oxidized glutathione back to the reduced form is accomplished by a mechanism in which glutathione reductase couples the reduction of glutathione to the change of NADPH to NADP (see reviews in Spector 129 and Augusteyn 135 ). Glutathione may play a role in a number of other oxidation-reduction systems. 135 , 136 For example, Giblin and coworkers 137 have demonstrated a clear link between glutathione, glutathione peroxidase, the hexose monophosphate shunt, and hydrogen peroxide breakdown. In addition, glutathione metabolism is also linked to the removal of xenobiotics from the lens. 108

In 1951, Harris and Gehrsitz presented some of the first evidence illustrating that the lens sodium and potassium levels are controlled by metabolically dependent transport processes. 138 Refrigeration of rabbit lenses led to a fall in potassium content and an increase in sodium content the cation redistribution could be reversed by warming the lens back to 37°C in a suitable culture medium. This recovery phenomenon can be reduced or abolished by metabolic inhibitors. 84 The principal site of active cation transport in the lens is the epithelium, although sodium and calcium transport mechanisms are also present in the cortex. 139� Curiously, the abundance of Na, K-ATPase protein in lens epithelium is similar to the abundance in fiber cells even though Na, K-ATPase activity in the fibers is much lower. 142 Na, K-ATPase mRNA 126 is detectable in the epithelium but not in the fibers.

The dominant active ion transport mechanism is Na, K-ATPase, the sodium pump, which actively extrudes sodium and accumulates potassium. The ion gradients established by Na, K-ATPase provide the driving force for other processes such as sodium-calcium exchange, sodium-bicarbonate cotransport, 143 and amino acid transport. 144 The mechanistic details of the sodium pump are well documented. 145 The combination of passive diffusion and anteriorly located energy-dependent transporters has been described as a “pump-leak” system 146 (Fig. 17). Inhibition of Na, K-ATPase causes the lens to lose potassium and gain sodium. When this takes place, the osmotic equilibrium of the lens is disturbed and it swells as water enters the cytoplasm from the surrounding medium. Swelling and the consequent rupture of cells in the lens periphery obviously impair transparency. A number of investigators have explored whether the lens sodium pump has failed to operate in the human cortical cataract in which deranged lens electrolyte and water levels are a common finding. 86 Although some investigators have detected a lower Na, K-ATPase activity in human cataractous lenses, 147� others have reported no such difference. 150 , 151 It has been suggested that lens epithelial cells are capable of synthesizing additional Na, K-ATPase protein to boost sodium pump activity in response to an increase of membrane permeability. 152

Fig. 17. A diagram of the pump-leak system in the lens. Energy-coupled transport mechanisms, located anteriorly, are shown as broad arrows. Passive exchange, at both anterior and posterior surfaces, is shown as broken lines. The model was devised originally by Kinsey and Reddy, 1965.

Because high calcium levels are cytotoxic, 92 and because the lens calcium level rises dramatically during the formation of cortical cataract, attention has been given to the mechanism by which a normal low concentration of calcium is maintained by the lens. It has been shown that the lens expresses calcium-stimulated ATPase, Ca-ATPase, both in the epithelium and cortical fibers. 140 , 153 The plasma membrane-localized Ca-ATPase is capable of transporting calcium outward from the cytoplasm and plays a role in extruding calcium from the lens. 139 Calcium is also exported via Na-Ca exchange, which couples the inward flow of sodium to the efflux of calcium. In addition, calcium is removed from the cytoplasm by a different Ca-ATPase, localized on the surface of ER. 154 In the epithelium, calcium that has been shifted into the ER can be released as part of a signaling mechanism that is triggered when the lens is exposed to certain agonists. 87 , 155

The specific permeability characteristics of lens membranes, which are clearly vital to the pump-leak mechanism of cytoplasmic ion regulation, hinge on the function of ion channels. Patch clamp studies have identified potassium conductance pathways near the lens surface. At least three different potassium channels contribute to potassium conductance inward rectifiers, delayed rectifiers, and large-conductance calcium-activated channels. 47 Chloride channels have also been detected, and it has been proposed that their function is essential to volume regulation of lens cells. 156

Amino acids are accumulated by the lens, principally by the epithelium, and are therefore included in the pump-leak concept 157 (see Fig. 17). There are a number of selective uptake mechanisms including one showing preference for alanine, another for leucine, and a third for glycine and other small amino acids. 144 The sodium gradient between the lens cytoplasm and the surrounding fluid drives the amino acid accumulation mechanism Marcantonio and Duncan 158 have reported that the high sodium content of human cataractous lenses markedly reduces amino acid uptake. The uptake of glucose and other sugars has been discussed by a number of authors, including Kern. 144 Unlike the active transfer of cations and amino acids, the transfer of glucose appears to occur across both the anterior and posterior surfaces of the lens the mechanism appears to be insulin independent. Facilitative glucose transporters GLUT-1 and GLUT-3 are expressed in lens cells. 159 Once a solute such as glucose enters the lens, a considerable time would be required for simple diffusion from the surface cells to the nucleus in the human lens it has been calculated that 4 to 8 days would be necessary! 47 However, it is thought that an inward movement of fluid occurs along the intercellular clefts at the anterior and posterior poles of the lens, and this would speed the penetration of glucose and other solutes into the lens cell mass.

Fig. 18. A diagrammatic representation of the flow of electric current outward at the lens equator and inward at the anterior and posterior poles. The currents are thought to drive water flow along the same path. As described in the text, currents arise because of unequal spatial distribution of potassium channels, gap junctions, and Na, K-ATPase in the lens cell mass. For example, potassium channels are more abundant in the surface zones (S). Also, there are important differences between differentiating fibers (DF) and mature fibers (MF) in the way neighboring cells are coupled. (Baldo GJ, Gong X, et al: Gap junctional coupling in lenses from alpha (8) connexin knockout mice. J Gen Physiol 118:447, 2001)

In addition to the state of lens crystallins, the tight packing of the lens cells and the regulation of ion and water balance also play significant roles in maintaining the transparency of the normal lens. Consequently, the development of protein aggregates, cell membrane degeneration, the appearance of vacuoles, and the distortion of lens structure can all produce light scatter and the clinical observation of cataract. 162 It has been proposed that lens calcium and possibly pH levels may have an impact on the opacification process. 92 , 170 , 171

Although the lens is transparent, the refractive index throughout the organ is not uniform. As a result of the more dense nature of the nucleus, it has a higher refractive index (1.41) than the cortex (1.38). The difference in refractive index between the nucleus and cortex results in the total refractive power of the lens being greater than if the refractive index were uniform throughout. Although from an anatomic standpoint, the cortex blends into the nucleus, distinct zones of optical discontinuity may be seen by slit lamp biomicroscopy. The zones are believed to correspond with various periods during the development and continual growth of the lens.

The incidence of cataract is high. In the Framingham Eye Study, which was restricted to a small sector of the population, it was found that detectable lens changes were observed in over 70% of all persons older than 65 years and that cataract was diagnosed in about 18% of that same age-group. In the age range 75 to 85 years the incidence of lens changes was above 90%, with senile cataract in about 46% of those studied. 174 Within the United States it has been estimated that about 40,000 persons are legally blind from age-related cataract, representing 35% of existing visual impairment. 175 More than 400,000 persons develop cataract each year. Cataract is a massive health problem worldwide it is the greatest cause of global blindness. The World Health Organization estimated that 45 million persons throughout the world are blind, half of them as the result of cataract. 176

Several studies have attempted to identify risk factors for cataract development. 177� These include exposure to ultraviolet light, ionizing radiation, microwave radiation, toxic chemicals including prescription drugs, diabetes, elevated blood pressure, family history of high cataract prevalence, and a variety of inborn errors of metabolism. Several arguments have also been made to demonstrate a relationship between cataract and a variety of nutritional factors, including antioxidant intake. Recently the role of genetics in the development of cataract has come under scrutiny. 180 Data from the Twin Eye Study suggest the relative influence of genetics and environmental factors on the development of human nuclear cataract is 48% and 14%, respectively. 181 For cortical cataract, findings from the Twin Eye Study and the Beaver Dam Eye Study estimate that genetic factors could account for as much as 58% of cataract occurrence. 182 , 183

The development of age-related cataract is obviously superimposed on the normal changes that take place in the aging lens. It is sometimes difficult to distinguish the alterations that normally occur with age from those lens changes that are precataractous. However, it is evident that the development of nuclear cataract and cortical cataract is not the same. Nuclear cataract is associated with coloration of the lens substance, but the lens electrolyte composition is unchanged 134 (Fig. 19). In contrast, cortical cataract is almost always associated with deranged electrolyte and water balance. 184 Lens sodium and calcium levels rise dramatically while there is a precipitous fall in lens potassium this electrolyte imbalance causes an increase in cellular hydration and, thus, swelling. As might be expected, the development of cataract is accompanied by loss of the organized lens structure. Studies by Harding 185 have demonstrated swollen and degenerate lens fibers, aberrant epithelial morphology and cell differentiation, and altered cell surfaces.

Fig. 19. A model illustrating one of the current theories on nuclear cataract formation. In this model, the human lens is considered to be a two-compartment system. The outer cortex region of the lens may remain unchanged even though an advance nuclear cataract develops in the center. In the core region, the entry of antioxidants from the cortex becomes insufficient to offset oxidative stress, which occurs because of factors such as the auto-oxidation of ascorbic acid and 3-hydroxykynurenine. The reduced flux of antioxidants from the outer cortex is due to the development of a diffusion barrier. The putative diffusion barrier also slows the exit of small molecules from the lens nucleus. Importantly, this could lead to a buildup of oxidized glutathione (GSSG) and a reduction in the ability of the glutathione system to afford antioxidant protection. (Truscott RJW: Age-related nuclear cataract: A lens transport problem. Ophthalmic Res 32:185, 2000)

There has been extensive evaluation of changes in lens proteins associated with cataract. 129 , 186 , 187 One certain fact is that with cataract development there is an abnormal increase in the fraction of water-insoluble protein in the lens and a decrease in total water-soluble proteins. The principal source of the increasing water-insoluble protein is the aggregation of soluble proteins to insoluble high-molecular-weight proteins. Aggregation of proteins also seems to be related to an increase in lenticular calcium. Some of the high-molecular-weight aggregates formed have been shown to be large enough to scatter light. However, it is unlikely that the formation of insoluble and high-molecular-weight proteins is alone sufficient to result in extensive lens opacification. It is known that some of the protein aggregates found in cataract are formed because of covalent disulfide bonds arising from the oxidation of thiol groups. In addition to altered membrane physiology and protein biochemistry, cataract development is accompanied by changes in lipid biochemistry, 188 decreased enzyme activity, 189 reduced glutathione levels, 108 and diminished ATP production. 190 Although the extensive alterations contribute to the demise of the cataractous lens, it is uncertain whether any single event initiates cataract development.

There is an accumulation of evidence that suggests that oxidative damage might be one of the primary biochemical events leading to cataract development. 191 In normal lenses there is only relatively minor oxidation, and this is principally associated with cell membranes. In cataract, however, there is considerable oxidation both at the level of the lens proteins and the cell membranes. If oxidation of lens constituents is indeed an initiating factor in cataract development, the potentially harmful species of oxygen responsible for the damage are superoxide, hydrogen peroxide, and the hydroxide radical. 192 There is some evidence that the concentration of hydrogen peroxide is elevated in the aqueous humor of some patients with cataract. 193 The source of hydrogen peroxide in the eye is uncertain, but ascorbic acid, present at normally high concentrations in the aqueous humor, can be a source of hydrogen peroxide in the presence of light and a metal ion. However, the lens normally has a number of mechanisms to protect it from oxidative damage, including catalase and glutathione peroxidase. 108 , 135 Therefore, these mechanisms must be impaired or overcome if the lens is to become more susceptible to oxidative damage.

Calcium might also play a significant role in eliciting molecular changes that would contribute to the loss of lens transparency. Elevation of calcium levels in cortical cataract was noted by Burge 194 in 1909. A detailed discussion of calcium and cataract is given by Duncan and Jacob. 88 Increased intracellular calcium levels can result in depressed glucose metabolism, inhibition of protein synthesis, induction of high-molecular-weight protein aggregates, and the activation of proteases, such as calpain 92 and apoptosis. Calcium might directly inhibit the lens sodium pump, 195 , 196 and intracellular calcium has been shown to modulate lens gap junction structure and coupling. 197� Finally, elevation of cellular calcium may also be linked directly, from a physiochemical standpoint, to loss of transparency. 199�

In diabetes mellitus the blood glucose level is elevated, resulting in an increase in aqueous humor glucose levels to nearly 90% of blood levels. As a result of the increased level of glucose in the aqueous humor, more glucose enters the lens by facilitated diffusion. In the normal lens virtually all the glucose entering is metabolized by way of hexokinase to enter the glycolytic pathway. However, at higher concentrations of glucose the enzyme aldose reductase is activated this enzyme converts glucose to sorbitol, which accumulates within the fibers. Similarly, in galactosemia, a condition caused by deficiency of galactose-1-phosphate-uridyl transferase or galactokinase, galactose enters the lens and is metabolized through aldose reductase to dulcitol (galactitol). Dulcitol also accumulates within the lens fibers, causing an influx of water and swelling of the lens cells and eventually opacification (Fig. 20). Galactose cataract forms more rapidly than diabetic cataract because of the higher affinity of aldose reductase for galactose. The situation is worsened because the lens is unable to metabolize dulcitol. In contrast, sorbitol, the sugar alcohol of glucose, can be oxidized to fructose by the enzyme polyol dehydrogenase.

Fig. 20. Changes that occur during the development of a galactose cataract. (Kinoshita J: Cataracts in galactosemia. Invest Ophthalmol 4:786, 1965)

Sugar cataract can be induced in experimental animals by feeding galactose or by inducing diabetes. In studies with rats fed a diet of 50% galactose, the earliest visible change is the appearance of swollen peripheral lens fibers that eventually rupture, leading to water- and protein-filled clefts in the cortex. 207 Later the lens epithelium proliferates into multiple layers. As the cortex becomes more liquefied, the lens temporarily attains a relatively clear appearance, but further changes result in a densely opaque nuclear zone, then total opacification. The accumulation of cellular water following polyol synthesis results in a number of further metabolic disturbances, including changes in membrane permeability, depletion of NADPH, reduction in glutathione levels, diminished ATP, and deranged electrolyte and balance. 208 It has also been suggested that high levels of sugar may cause some direct modification of lens protein by a mechanism called nonenzymatic glycosylation. 209 Glycosylation involves the attachment of glucose to amino acid residues in lens proteins, resulting in the formation of carboxyl groups that are able to cross link and, thus, cause protein aggregation.

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