Polyol Pathway Research Articles

Paeonol attenuates retinopathy in streptozotocin-induced diabetes in rats by regulating the oxidative stress and polyol pathway.

The current research work was planned to study the effects of paeonol in the management of diabetic retinopathy. Diabetes was induced in male Sprague Dawley rats using Streptozotocin (55 mg/kg, i.p.). After 4 weeks, the diabetic animals were treated with paeonol at a dose of 50, 100, and 200 mg/kg body weight daily for the next 4 weeks. At the end of treatment, retinal physiology was studied by recording an electroretinogram (ERG); biochemical parameters and oxidative stress were estimated. The histopathology of the retina was also carried out at the end of the study. The ERG of paeonol-treated animals showed a significant improvement in a-wave amplitude, b-wave amplitude, a-wave latency, and b-wave latency (p < 0.001) at 15 cd s/m2 when compared with the diabetic control animals. The paeonol treatment (200 mg/kg) in diabetic animals showed a significant decrease in the plasma glucose level (p < 0.001) when compared to the animals in diabetic control group. Paeonol also significantly decreased the lactate dehydrogenase, aldose reductase, and sorbitol dehydrogenase levels when compared with the diabetic control animals. The oxidative stress in the eye was significantly reduced after the paeonol treatment in the diabetic rats. The histopathology showed a significant reduction (p < 0.05) in the retinal thickness after the paeonol treatment. The results of the study indicate that paeonol can be considered an effective management option for diabetic retinopathy.

Dissecting the Activity of Catechins as Incomplete Aldose Reductase Differential Inhibitors through Kinetic and Computational Approaches.

Simple SummaryThe increased glucose levels occurring in diabetes lead to several metabolic alterations responsible for the onset of the so-called diabetic complications, which include nephropathies, neuropathies, retinopathies, and cataract. An increased flux of glucose through the polyol pathway is considered the most relevant among these alterations. For this reason, the block of the polyol pathway, through the inhibition of the enzyme aldose reductase, is considered a valuable strategy to impair the onset of diabetic complications. However, aldose reductase also exerts a beneficial effect inside cells, since it can remove toxic aldehydes. Thus, to ameliorate the outcome of the use of aldose reductase inhibitors, the use of “differential inhibitors” has been proposed. These inhibitors should block the catalytic activity depending on the substrate the enzyme is working on, thus preserving the detoxifying action of the enzyme. In this work, derivatives of catechins are analyzed to evaluate their inhibitory action on aldose reductase. The study was conducted both in vitro on the isolated enzyme and in silico through a computational approach. Results demonstrated that gallocatechin gallate and catechin gallate act as differential inhibitors and that this action may be linked to an incomplete inhibitory effect.The inhibition of aldose reductase is considered as a strategy to counteract the onset of both diabetic complications, upon the block of glucose conversion in the polyol pathway, and inflammation, upon the block of 3-glutathionyl-4-hydroxynonenal reduction. To ameliorate the outcome of aldose reductase inhibition, minimizing the interference with the detoxifying role of the enzyme when acting on toxic aldehydes, “differential inhibitors”, i.e., molecules able to inhibit the enzyme depending on the substrate the enzyme is working on, has been proposed. Here we report the characterization of different catechin derivatives as aldose reductase differential inhibitors. The study, conducted through both a kinetic and a computational approach, highlights structural constraints of catechin derivatives relevant in order to affect aldose reductase activity. Gallocatechin gallate and catechin gallate emerged as differential inhibitors of aldose reductase able to preferentially affect aldoses and 3-glutathionyl-4-hydroxynonenal reduction with respect to 4-hydroxynonenal reduction. Moreover, the results highlight how, in the case of aldose reductase, a substrate may affect not only the model of action of an inhibitor, but also the degree of incompleteness of the inhibitory action, thus contributing to differential inhibitory phenomena.

Resorcinol Derivatives as Novel Aldose Reductase Inhibitors: In Silico and In Vitro Evaluation

Background: The polyol pathway, an alternative way of carbohydrate metabolism, is activated by hyperglycemia. Aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, is responsible for the reduction of glucose to sorbitol. Inhibiting the aldose reductase enzyme and reducing the polyol pathway is considered an effective method to prevent and postpone the onset of diabetic complications. Objective: Therefore, in this work, we investigate the inhibition effects of certain resorcinol derivatives and the positive control compound quercetin on the AR enzyme in vitro and in silico. These phenolic compounds, whose inhibitory effects on the AR enzyme were investigated, were also compared with known drugs in terms of their drug-like characteristics. Methods: Three methods were used to determine the inhibitory effects of resorcinol derivatives on recombinant human AR enzyme. After the in vitro inhibition effects were determined spectrophotometrically, the binding energy and binding modes were determined by molecular docking method. Finally, the MM-GBSA method was used to determine the free binding energies of the inhibitors for the AR enzyme. Results: 5-pentylresorcinol compound showed the strongest inhibition effect on recombinant human AR enzyme with an IC50 value of 9.90 μM. The IC50 values of resorcinol, 5-methylresorcinol, 4- ethylresorcinol, 4-hexylresorcinol, 2-methylresorcinol, and 2,5-dimethylresorcinol compounds were determined as 49.50 μM, 43.31 μM, 19.25 μM, 17.32 μM, 28.87 μM, 57.75 μM, respectively. Conclusion: The results of this research showed that resorcinol compounds are effective AR inhibitors. These findings are supported by molecular docking, molecular mechanics, and ADME investigations undertaken to corroborate the experimental in vitro results.

GC × GC-TOFMS metabolomics analysis identifies elevated levels of plasma sugars and sugar alcohols in diabetic mellitus patients with kidney failure

Two dimensional GC (GC × GC)–time-of-flight mass spectrometry (TOFMS) has been used to improve accurate metabolite identification in the chemical industry, but this method has not been applied as readily in biomedical research. Here, we evaluated and validated the performance of high resolution GC × GC-TOFMS against that of GC-TOFMS for metabolomics analysis of two different plasma matrices, from healthy controls (CON) and diabetes mellitus (DM) patients with kidney failure (DM with KF). We found GC × GC-TOFMS outperformed traditional GC-TOFMS in terms of separation performance and metabolite coverage. Several metabolites from both the CON and DM with KF matrices, such as carbohydrates and carbohydrate-conjugate metabolites, were exclusively detected using GC × GC-TOFMS. Additionally, we applied this method to characterize significant metabolites in the DM with KF group, with focused analysis of four metabolite groups: sugars, sugar alcohols, amino acids, and free fatty acids. Our plasma metabolomics results revealed 35 significant metabolites (12 unique and 23 concentration-dependent metabolites) in the DM with KF group, as compared with those in the CON and DM groups (N = 20 for each group). Interestingly, we determined 17 of the 35 (14/17 verified with reference standards) significant metabolites identified from both the analyses were metabolites from the sugar and sugar alcohol groups, with significantly higher concentrations in the DM with KF group than in the CON and DM groups. Enrichment analysis of these 14 metabolites also revealed that alterations in galactose metabolism and the polyol pathway are related to DM with KF. Overall, our application of GC × GC-TOFMS identified key metabolites in complex plasma matrices.

Free-radical oxidation as a pathogenetic factor of metabolic syndrome

The medical and social significance of cardiovascular diseases remains high. One of the factors that determine cardiovascular risks is metabolic syndrome. As a result of excessive accumulation of lipid and carbohydrate metabolism products in metabolic syndrome, oxidative (oxidative) stress develops. The article considers both domestic and foreign scientific studies, which highlight various aspects of the influence of reactive oxygen and nitrogen species, as well as other free radicals on the formation of oxidative stress in pathological conditions that are part of the metabolic syndrome complex. This describes the mechanisms of the formation of chronic inflammation through excessive secretion of pro-inflammatory cytokines and adipokines, activation of the transcription factor NF-kB, as well as damage to the antioxidant system in obesity. Separately, a number of mechanisms of the stimulating effect of adipokines: leptin, adiponectin, chimerine, omentin 1, resistin, on the formation of oxidative stress have been noted. The ways of activating the polyol pathway, as well as diacyl-glycerol — protein kinase C — the signaling pathway of oxidative stress, the formation of mitochondrial dysfunction is described. As a result of which there is an excessive production of free radicals in insulin resistance, diabetes mellitus and macroand microvascular complications of diabetes. In addition, the influence of oxidative stress directly on the formation of cardiovascular diseases of atherosclerotic genesis, as well as arterial hypertension, has been shown.

Hydantoin based dual inhibitors of ALR2 and PARP-1: Design, synthesis, in-vitro and in-vivo evaluation

Diabetic nephropathy is one of the most dreadful diabetic complications (DCs). The polyol pathway and unified mechanism are two important pathways implicated in the progression of DCs. In this regard, targeting the key enzymes i.e., aldose reductase (ALR2) and poly (ADP-ribose) polymerase-1 (PARP-1), of these pathways can be a relevant strategy. Thus, in this study, the pharmacophoric requirements necessary for the dual inhibition of these two enzymes i.e., ALR2 and PARP-1 were identified and consequently, some hydantoin based molecules were designed. The designed molecules were subjected to structure-based molecular modelling analysis including molecular docking analysis and molecular dynamic simulations. The promising molecules were duly synthesized and examined for their ALR2 and PARP-1 dual inhibitory activities and selectivity over aldehyde reductase (ALR1) using in vitro enzymatic assays. Based on the results of in silico analysis and in vitro assays, the best three molecules were evaluated in vivo for their nephroprotective effect and antioxidant potential in the high-fat diet-streptozotocin induced diabetic rat model. The results showed that the compounds FM6B, FM7B and FM9B were having low micromolar inhibitory potential against ALR2 (IC50; 1.02, 1.14 and 1.08 μM, respectively) and PARP-1 (IC50; 0.95, 0.81 and 1.42 μM, respectively) with selectivity over ALR1 (selectivity index; 43.63, 37.03 and 45.14, respectively).

Assessing the Phytochemical Profile and Potential of Traditional Herbal Infusions against Aldose Reductase through In Silico Studies and LC-MS/MS Analysis

In the current market, there is a growing interest in traditional herbal nutraceuticals. Therefore, herbal formulations have re-emerged as products with sought-after nutraceutical and disease-preventing properties. The health-promoting effects of herbal bioactives are attributed to the active phytoconstituents of these plants. Thus, the aim of the present study was to evaluate the putative nutraceutical effectiveness of the preparations of ten herbs (chamomile, purple coneflower, lemon verbena, pennyroyal, spearmint, oregano, marjoram, headed savory, sea buckthorn, and St. John’s wort) by combining in silico techniques and LC-MS/MS analysis. The binding potential of the selected phenolic compounds, according to literature and web databases, was investigated by using molecular target prediction tools. Aldose reductase (AR), an enzyme of polyol pathway which is related to hyperglycemic-induced pathologies, emerged as the most promising molecular target. The molecular docking results showed that rosmarinic acid, caftaric acid, naringenin, and quercetin presented the highest binding affinity. In a further step, the phytochemical profile of the examined infusions, obtained by LC-MS/MS analysis, revealed that the abovementioned compounds were present, mainly in the herbs of the Lamiaceae family, designating headed savory as the herbal infusion with possible significant inhibitory activity against AR.

Iron Deficiency and Overload Modulate the Inflammatory Responses and Metabolism of Alveolar Macrophages.

Alveolar macrophages (AM) are critical to defense against respiratory pathogens. This study evaluated cellular iron imbalance to immunometabolism in endotoxin-polarized porcine AMs (PAMs). PAMs collected from five 6-week-old pigs were treated with a basal media, iron chelator, or ferric ammonium citrate to maintain iron replete or induce iron deficiency or overload, respectively. After 24 h treatment, PAMs were challenged with saline or lipopolysaccharide (LPS) for 6 h. Cells were analyzed for gene, protein, and untargeted metabolome. Cytokines were determined in culture media. Data were assessed using two-way ANOVA. Treatments successfully induced iron deficiency and overload. The mRNA of DMT1 and ZIP14 was increased up to 300-fold by LPS, but unaffected by iron. Surprisingly, both iron deprivation and overload attenuated LPS-induced inflammation, showing less TNFα production and lower mRNA of pro- and anti-inflammatory cytokines than iron-replete PAMs. Forty-eight metabolites were altered by either or both main effects. LPS enhanced the glycolysis and polyol pathways. Iron deprivation disrupted the TCA cycle. Iron overload increased intracellular cholesterol. Interestingly, iron deprivation augmented, whereas iron overload diminished, LPS-induced itaconic acid production, which has anti-microbial and anti-inflammatory properties. Therefore, iron-deficient PAMs may be more resistant to intracellular pathogens which use PAMs as a conduit for infection.

Aldose reductase promotes diet-induced obesity via induction of senescence in subcutaneous adipose tissue.

Aldose reductase (AKR1B1 in humans; Akr1b3 in mice), a key enzyme of the polyol pathway, mediates lipid accumulation in the murine heart and liver. The study objective was to explore potential roles for AKR1B1/Akr1b3 in the pathogenesis of obesity and its complications. The study employed mice treated with an inhibitor of aldose reductase or mice devoid of Akr1b3were used to determine their response to a high-fat diet. The study used subcutaneous adipose tissue-derived adipocytes to investigate mechanisms by which AKR1B1/Akr1b3 promotes diet-induced obesity. Increased expression of aldose reductase and senescence in the adipose tissue of humans and mice with obesity were demonstrated. Genetic deletion of Akr1b3 or pharmacological blockade of AKRIB3 with zopolrestat reduced high-fat-diet-induced obesity, attenuated markers of adipose tissue senescence, and increased lipolysis. AKR1B1/Akr1b3 modulation of senescence in subcutaneous adipose tissue contributes to aberrant metabolic responses to high-fat feeding. These data unveil new opportunities to target these pathways to combat obesity.

Development, Molecular Docking, and In Silico ADME Evaluation of Selective ALR2 Inhibitors for the Treatment of Diabetic Complications via Suppression of the Polyol Pathway.

Diabetic complications are associated with overexpression of aldose reductase, an enzyme that catalyzes the first step of the polyol pathway. Osmotic stress in the hyperglycemic state is linked with the intracellular accumulation of sorbitol along with the depletion of NADPH and eventually leads to oxidative stress via formation of reactive oxygen species and advanced glycation end products (AGEs). These kinds of mechanisms cause the development of various diabetic complications including neuropathy, nephropathy, retinopathy, and atherosclerotic plaque formation. Various aldose reductase inhibitors have been developed to date for the treatment of diabetic complications, but all have failed in different stages of clinical trials due to toxicity and poor pharmacokinetic profiles. This toxicity is rooted in a nonselective inhibition of both ALR2 and ALR1, homologous enzymes involved in the metabolism of toxic aldehydes such as methylglyoxal and 3-oxyglucosazone. In the present study, we developed a series of thiosemicarbazone derivatives as selective inhibitors of ALR2 with both antioxidant and antiglycation potential. Among the synthesized compounds, 3c exhibited strong and selective inhibition of ALR2 (IC50 1.42 μM) along with good antioxidant and antiglycative properties. The binding mode of 3c was assessed through molecular docking and cluster analysis via MD simulations, while in silico ADME evaluation studies predicted the compounds’ druglike properties. Therefore, we report 3c as a drug candidate with promising antioxidant and antiglycative properties that may be useful for the treatment of diabetic complications through selective inhibition of ALR2.

Genome-wide identification and comparative evolutionary analysis of sorbitol metabolism pathway genes in four Rosaceae species and three model plants

In contrast to most land plant species, sorbitol, instead of sucrose, is the major photosynthetic product in many Rosaceae species. It has been well illustrated that three key functional genes encoding sorbitol-6-phosphate dehydrogenase (S6PDH), sorbitol dehydrogenase (SDH), and sorbitol transporter (SOT), are mainly responsible for the synthesis, degradation and transportation of sorbitol. In this study, the genome-wide identification of S6PDH, SDH and SOT genes was conducted in four Rosaceae species, peach, mei, apple and pear, and showed the sorbitol bio-pathway to be dominant (named sorbitol present group, SPG); another three related species, including tomato, poplar and Arabidopsis, showed a non-sorbitol bio-pathway (named sorbitol absent group, SAG). To understand the evolutionary differences of the three important gene families between SAG and SPG, their corresponding gene duplication, evolutionary rate, codon bias and positive selection patterns have been analyzed and compared. The sorbitol pathway genes in SPG were found to be expanded through dispersed and tandem gene duplications. Branch-specific model analyses revealed SDH and S6PDH clade A were under stronger purifying selection in SPG. A higher frequency of optimal codons was found in S6PDH and SDH than that of SOT in SPG, confirming the purifying selection effect on them. In addition, branch-site model analyses revealed SOT genes were under positive selection in SPG. Expression analyses showed diverse expression patterns of sorbitol-related genes. Overall, these findings provide new insights in the evolutionary characteristics for the three key sorbitol metabolism-related gene families in Rosaceae and other non-sorbitol dominant pathway species.

Novel Hydroxychalcone-Based Dual Inhibitors of Aldose Reductase and α-Glucosidase as Potential Therapeutic Agents against Diabetes Mellitus and Its Complications

We designed a novel series of bifunctional inhibitors of α-glucosidase and aldose reductase (ALR2) based on the structure of hydroxychalcone. The two enzymes relate to blood glucose level and anomalously elevated polyol pathway of glucose metabolism under hyperglycemia, respectively. Most compounds in the series exhibited a potent inhibitory activity for both enzymes, and a significant antioxidant property was shown. Further in vivo studies of 11j and 14d using streptozotocin (STZ)-induced diabetic rats as a model found that 11j achieved not only good antihyperglycemic and glucose tolerance effect in a dose-dependent manner (p < 0.01) but also showed effective inhibition of polyol pathway. 14d significantly suppressed the maltose-induced postprandial glucose elevation. Additionally, they effectively improved lipid metabolisms and restored an antioxidant ability. Therefore, the two compounds may be promising agents for the prevention and treatment of diabetic complications.

Characterization of carbohydrate metabolism in in vivo- and in vitro-grown and matured mouse antral follicles†.

Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid [TCA] cycle, pentose phosphate pathway [PPP], polyol pathway, and hexosamine biosynthetic pathway), as well as the antioxidant capacity, were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate, and increases TCA cycle and small molecules antioxidant capacity activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfill their role in providing proper support for acquiring oocyte competence.

The polyol pathway is an evolutionarily conserved system for sensing glucose uptake.

Cells must adjust the expression levels of metabolic enzymes in response to fluctuating nutrient supply. For glucose, such metabolic remodeling is highly dependent on a master transcription factor ChREBP/MondoA. However, it remains elusive how glucose fluctuations are sensed by ChREBP/MondoA despite the stability of major glycolytic pathways. Here, we show that in both flies and mice, ChREBP/MondoA activation in response to glucose ingestion involves an evolutionarily conserved glucose-metabolizing pathway: the polyol pathway. The polyol pathway converts glucose to fructose via sorbitol. It has been believed that this pathway is almost silent, and its activation in hyperglycemic conditions has deleterious effects on human health. We show that the polyol pathway regulates the glucose-responsive nuclear translocation of Mondo, a Drosophila homologue of ChREBP/MondoA, which directs gene expression for organismal growth and metabolism. Likewise, inhibition of the polyol pathway in mice impairs ChREBP's nuclear localization and reduces glucose tolerance. We propose that the polyol pathway is an evolutionarily conserved sensing system for glucose uptake that allows metabolic remodeling.

Ocular Formulation Development of a Standardized Herbal Extract for the Prevention and Treatment of Diabetic Cataract

Diabetes-induced Cataract is a major concern regarding the cause of blindness in developed and developing countries. Recent basic research studies have emphasized the role of the polyol pathway in the initiation of the disease process. Diabetes induced cataract is also referred to as “Diabetic cataract”. Demographic studies have certainly enhanced our knowledge concerning an association between diabetes and cataract formation and have defined the risk factors for the development of cataracts. Diabetic patients also have a higher risk of complications after phacoemulsification cataract surgery as compared to non-diabetic people. Aldose-reductase inhibitors and antioxidants have been proven beneficial in the prevention or treatment of this sight-threatening condition in in vitro experimental studies. Aldose reductase inhibitors have a vital importance in the treatment and prevention of diabetic complications. Nowadays, use of natural, herbal, and naturopathic medications has increased, as they have more therapeutic potential and lesser side effects as compared to the allopathic and ayurvedic medications. This research work provides an insight into herbal standardized extract and its ocular formulation development, Eyedrop, as an easy, sterile, safe, and alternative to phacoemulsification. Development of eyedrop as an ocular formulation and its standardization with an in vitro evaluation of the herbal extract for the prevention as well as treatment of Diabetic cataract.

Systematic Investigations on the Metabolic and Transcriptomic Regulation of Lactate in the Human Colon Epithelial Cells.

Lactate, primarily produced by the gut microbiota, performs as a necessary “information transmission carrier” between the gut and the microbiota. To investigate the role of lactate in the gut epithelium cell–microbiota interactions as a metabolic signal, we performed a combinatory, global, and unbiased analysis of metabolomic and transcriptional profiling in human colon epithelial cells (Caco-2), using a lactate treatment at the physiological concentration (8 mM). The data demonstrated that most of the genes in oxidative phosphorylation were significantly downregulated in the Caco-2 cells due to lactate treatment. Consistently, the levels of fumarate, adenosine triphosphate (ATP), and creatine significantly decreased, and these are the metabolic markers of OXPHOS inhibition by mitochondria dysfunction. The one-carbon metabolism was affected and the polyol pathway was activated at the levels of gene expression and metabolic alternation. In addition, lactate significantly upregulated the expressions of genes related to self-protection against apoptosis. In conclusion, lactate participates in gut–gut microbiota communications by remodeling the metabolomic and transcriptional signatures, especially for the regulation of mitochondrial function. This work contributes comprehensive information to disclose the molecular mechanisms of lactate-mediated functions in human colon epithelial cells that can help us understand how the microbiota communicates with the intestines through the signaling molecule, lactate.

Mechanistic approach towards diabetic neuropathy screening techniques and future challenges: A review.

Diabetic neuropathy, also called peripheral diabetic neuropathy (PDN), is among the most significant diabetes health consequences, alongside diabetic nephropathy, diabetic cardiomyopathy and diabetic retinopathy. Diabetic neuropathy is the existence of signs and indications of peripheral nerve damage in patients with diabetes after other causes have been governed out. Diabetic neuropathy is a painful and severe complication of diabetes that affects roughly 20% of people. The development of diabetic neuropathy is regulated by blood arteries that nourish the peripheral nerves and metabolic problems such as increased stimulation of polyol pathway, loss of myo-inositol and enhanced non-enzymatic glycation. It's divided into four types based on where neurons are most affected: autonomic, peripheral, proximal, and focal, with each kind presenting different symptoms like numbing, gastrointestinal disorders, and heart concerns. Pharmacotherapy for neuropathic pain is complex and for many patients, effective treatment is lacking; as a result, scientific proof recommendations are crucial. As a result, the current demand is to give the most vital medications or combinations of drugs that work directly on the nerves to help diabetic neuropathy patients feel less pain without causing any adverse effects. In diabetic neuropathy research, animal models are ubiquitous, with rats and mice being the most typically chosen for various reasons. This review covers the epidemiology, clinical features, pathology, clinical symptom, mechanism of diabetic neuropathy development, diagnosis, screening models of animals, diabetic neuropathy pharmacotherapy.

Mapping Glucose Uptake, Transport and Metabolism in the Bovine Lens Cortex.

Purpose: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions. Methods: Ex vivo bovine lenses were incubated in artificial aqueous humour containing normoglycaemic stable isotopically-labelled (SIL) glucose (5 mM) for 5 min-20 h. Following incubations, lenses were frozen for subsequent matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) analysis using high resolution mass spectrometry. Manually dissected, SIL-incubated lenses were subjected to gas chromatography-mass spectrometry (GC-MS) to verify the identity of metabolites detected by MALDI-IMS. Normal, unincubated lenses were manually dissected into epithelium flat mounts and fibre cell fractions and then subjected to either gel-based proteomic analysis (Gel-LC/MS) to detect facilitative glucose transporters (GLUTs) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Indirect immunofluorescence and confocal microscopy of axial lens sections from unincubated fixed lenses labelled with primary antibodies specific for GLUT 1 or GLUT 3 were utilised for protein localisation. Results: SIL glucose uptake at 5 min was concentrated in the equatorial region of the lens. At later timepoints, glucose gradually distributed throughout the epithelium and the cortical lens fibres, and eventually the deeper lens nucleus. SIL glucose metabolites found in glycolysis, the sorbitol pathway, the pentose phosphate pathway, and UDP-glucose formation were mapped to specific lens regions, with distinct regional signal changes up to 20 h of incubation. Spatial proteomic analysis of the lens epithelium detected GLUT1 and GLUT3. GLUT3 was in higher abundance than GLUT1 throughout the epithelium, while GLUT1 was more abundant in lens fibre cells. Immunohistochemical mapping localised GLUT1 to epithelial and cortical fibre cell membranes. Conclusion: The major uptake site of glucose in the bovine lens has been mapped to the lens equator. SIL glucose is rapidly metabolised in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres, with low levels of metabolic activity observed in the nucleus.

Biallelic mutations in sord are a common cause of potentially treatable genetic neuropathy

IntroductionAs opposed to CMT1, for which over 90% of cases have mutations in known genes, only 20–30% of CMT2/dHMN patients receive a genetic diagnosis. Since up to 70% of CMT2 and dHMN cases are sporadic, identifying candidate pathogenic genes from single cases remains challenging also for next-generation sequencing techniques.MethodsWe took advantage of the largest collection of over 1,100 CMT patients in whom WES and/or WGS has been performed in the GENESIS analysis platform. We specifically looked for genes for which significant DNA variants are present in multiple families as well as for individual alleles overrepresented in CMT cases.ResultsWe identified 45 cases from 38 families across multiple ethnicities, carrying a particular nonsense mutation in SORD, c.753delG;p.Ala253GlnfsTer27, either in homozygous or compound heterozygous state with a second variant. With an allele frequency of 0.004 in healthy controls, the p.Ala253GlnfsTer27 variant represents one of the most common pathogenic alleles in humans. SORD is an enzyme that converts sorbitol into fructose, in the two-step polyol pathway that has been implicated in diabetic neuropathy. In patient-derived fibroblasts, we find a complete loss of SORD protein and increased intracellular sorbitol. Also, as a biomarker, serum fasting sorbitol level was over 100 times higher in patients compared to healthy individuals. In Drosophila, we show that loss of SORD orthologues causes synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibi- tors normalized intracellular sorbitol levels in patient fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes.ConclusionsWe demonstrate biallelic mutations in sorbitol dehydrogenase (SORD) as the most frequent recessive form of hereditary neuropathies. These findings establish a potentially treatable cause in a sig- nificant fraction of patients with inherited neuropathies and may contribute to a better understanding of the pathophysiology of diabetic neuropathy.andrea.cortese@ucl.ac.uk

Shabyar Ameliorates High Glucose Induced Retinal Pigment Epithelium Injury Through Suppressing Aldose Reductase and AMPK/mTOR/ULK1 Autophagy Pathway.

Shabyar (SBA) is a traditional medicine formula for relieving vision loss caused by factors including diabetic retinopathy (DR) in clinics. However, the mechanism of it on retina protective effect still unclear. The present study aimed to investigate whether its protective effect was related to aldose reductase (AR) inhibition and retinal pigment epithelial cell injury mediated by autophagy or not. Human retinal pigment epithelial cells (ARPE-19) induced by high glucose was used as a model in vitro, with Epalrestat (EPL, AR inhibitor) and Difrarel (DFR, DR therapeutic drug) as positive controls. Western blotting and Polyol pathway products assay showed that SBA reduced the expression of AR protein and the content of ROS, and sorbitol, increased the level of Na+-K+-ATPase and alleviated cell edema. Western blotting and DCFH-DA probe assay showed that SBA decreased pAMPK/AMPK and pULK1/ULK1 which associated with autophagy initiation, down-regulated Beclin-1, Atg3, Atg5, Atg7, LC3 II and Bax/Bcl2 ratio, and up-regulated pmTOR/mTOR, SQSTM1/p62 and mitochondrial membrane potential (MMP), reduces intracellular autophagosomes. Real-Time PCR assay showed that SBA had no significant effect on mRNA expression of AR and mTOR. These data demonstrated that SBA treatment inhibits the autophagy of ARPE-19 through the AMPK/mTOR/ULK1 signaling pathway, and reduced early-stage apoptosis occurred by high glucose. These findings reveal the protective role and mechanism of SBA on retinal pigment epithelium, and provide experimental basis for the clinical application of SBA in the treatment of DR.