Transport Of Carnosine Research Articles

IDF21-0406 Genetic variants in the Carnosine-Carnosinase system and their impact on diabetic nephropathy

Background: The role of carnosine-carnosinase system in diabetic nephropathy has been established through various studies in animal models. It has been proven that low carnosinase secretion promotes high circulating carnosine levels which acts as a protective factor against adverse effects of hyperglycemia on renal cells. While genetic variants in the carnosinase gene (CNDP1) could influence carnosinase concentration, variants in the carnosine synthase gene (CARNS1) which synthesizes carnosine and variants in the SLC15A4 and SLC15A2 genes which are involved in the renal transport of carnosine could influence carnosine concentration thereby causing nephropathy. Aim: To study the association of genetic variants in the carnosine-carnosinase system (CARNS1, CNDP1, SLC15A4 and SLC15A2) with nephropathy and their influence on carnosinase levels in south Indian population. Method: Study Participants: 580 type 2 diabetes individuals without nephropathy (DM) and 597 type 2 diabetes individuals with nephropathy (DN) from Dr. Mohan’s Diabetes Specialities Centre, Chennai, India. Methodology: Genomic DNA was extracted from blood samples using phenol chloroform method. A total of 27 SNPs from the four genes were genotyped using MassARRAY. The levels of carnosinase in serum was measured in 116 DM and 93 DN participants using commercially available ELISA kit. Results: The D18S880 repeat polymorphism (5-8 repeats of Leucine) in CNDP1 gene and rs1626067 SNP of CARNS1 gene showed significant association with DN. The odds ratio for DN after adjusting for potential confounders such as age, sex, BMI, HbA1c, duration of diabetes and systolic blood pressure for the 6L/XL genotype of D18S880 and the GA genotype of rs1626067 as compared to their respective homozygous normal genotypes was 0.67 (95%CI: 0.48-0.94, p=0.022) and 0.68 (95% CI:0.49-0.93, p=0.016) respectively. The levels of carnosinase (mean±SE in picogram/mL) was significantly higher in DN (3.07±0.19) compared to DM group (2.48±0.13, p=0.038). Discussion: This study gains significance as the role of genetic variants in the carnosine-carnosinase system in DN remains largely unexplored. Most of the studies so far have focussed only on the CNDP1 gene. The association of D18S880 Leucine repeat with DN is significant as it is present in the signal peptide region of CNDP1 gene and may play a role in the secretion of carnosinase. The association of rs1626067 SNP in CARNS1 with DN has not been reported so far. This SNP is positioned in the 3́UTR region of CARNS1 and hence could alter the binding of transcription factors and have a regulatory effect. The significant difference in serum carnosinase levels between DM and DN groups was an interesting finding. When the groups were further stratified based on the genotypes, the sample size was less than 50 in each group and was not sufficiently powered to study the correlation of enzyme levels and genotypes. Measuring the levels and activity of carnosinase in larger number of samples will help identify whether the genetic variations influence the enzyme levels. If found, targeted dietary intervention studies giving carnosine supplements to those carrying the susceptible alleles can be undertaken to alleviate the burden of nephropathy.

An assessment of the transport mechanism and intraneuronal stability of L-carnosine.

L-Carnosine (β-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119μM and a maximum velocity of 0.289nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10-4nmol/mg (prot)/min (with 119μM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.

Carnosine modulates the Sp1-Slc31a1/Ctr1 copper-sensing system and influences copper homeostasis in murine CNS-derived cells.

Carnosine (CAR) is an endogenous dipeptide physiologically present in excitable tissues, such as central nervous system (CNS) and muscle. CAR is acknowledged as a substrate involved in many homeostatic pathways and mechanisms and, due to its biochemical properties, as a molecule intertwined with the homeostasis of heavy metals such as copper (Cu). In CNS, Cu excess and dysregulation imply oxidative stress, free-radical production, and functional impairment leading to neurodegeneration. Here, we report that CAR intercepts the regulatory routes of Cu homeostasis in nervous cells and tissues. Specifically, in a murine neuron-derived cell model, i.e., the B104 neuroblastoma cells, extracellular CAR exposure up to 24 h influenced intracellular Cu entry and affected (downregulated) the key Cu-sensing system, consisting of the gene coding for the Slc31a1 transmembrane Cu importer (alias Ctr1), and the gene coding for the Cu-responsive transcription factor Sp1 ( Sp1). Also, CAR exposure upregulated CAR biosynthesis ( Carns1), extracellular degradation ( Cndp1), and transport ( Slc15a4, alias Pht1) genes and elicited CAR intracellular accumulation, contributing to the outline of functional association between CAR and Cu within the cell. Interestingly, the same gene modulation scheme acting in vitro operates in vivo in brains of mice undergoing dietary administration of CAR in drinking water for 2 wk. Overall, our findings describe for the first time a regulatory interaction between CAR and Cu pathways in CNS and indicate CAR as a novel active molecule within the network of ligands and chaperones that physiologically regulate Cu homeostasis.

Abstract 16598: Carnosine Synthetase Overexpression in Skeletal Muscle Promotes Functional Recovery of Ischemic Limb

Carnosine synthetase (ATPGD1) is an ATP grasp enzyme that catalyzes formation of histidyl dipeptides such as carnosine (β-alanine-histidine) and anserine (β-alanine-methyl histidine). These dipeptides are food constituents found in a high concentration in the skeletal muscle (10-20 mM) that have the abilities to chelate metals, scavenge lipid peroxidation products and buffer intracellular pH. These dipeptides are regarded as protective against injury and stress. Despite this knowledge, whether carnosine regulates angiogenesis is unknown. To assess this potential, a model of hindlimb ischemia (HLI) was surgically induced by femoral artery and vein ligation in the C57BL/6J (wild type, WT) mice provided drinking water without or with carnosine (1g/L) for 21 days post-surgery. C57BL/6J WT mice subjected to HLI and supplemented with carnosine had increased skeletal muscle expression of the dipeptide/histidine transporter (PHT1), carnosine transport, improved blood flow recovery, re-vascularization and regeneration of myocytes in the ischemic limb compared with mice without carnosine. To support that elevated skeletal muscle carnosine was beneficial in HLI, transgenic mice overexpressing ATPGD1 in the skeletal muscle were constructed. Transgenic (α-HSA-ATPGD1TG) mice had ~2-2.5-fold increase in carnosine (WT: 2.71±0.54 vs 7.40±0.54 nmoles/mg protein α-HSA-ATPGD1TG; p <0.05) and anserine levels (WT: 3.65±1.12 vs 8.04±0.23 nmoles/mg protein α-HSA-ATPGD1TG p <0.05) in the skeletal muscle than WT mice. At 2 and 3 weeks post-HLI surgery, α-HSA-ATPGD1TG mice had significantly improved blood flow recovery compared with the WT mice (2 weeks: α-HSA-ATPGD1TG: 59±3% vs 45±3% WT; p <0.05; 3 weeks: α-HSA-ATPGD1TG: 65±4% vs 46±2% WT; p <0.05). Because faster recovery of blood flow in the carnosine-supplemented mice was accompanied with increased expression of hypoxia inducible factor (HIF-1α) and vascular endothelial factor (VEGF) in the ischemic limb, we further investigated the mechanistic connection. Skeletal muscle myoblasts C2C12 cells pretreated with octyl-D-carnosine and subjected to 1 h of hypoxia, replacing oxygen with 95% N 2 and 5% CO 2 , had enhanced HIF-1α translocation, VEGF mRNA and secretion compared with untreated hypoxic and normoxic cells. Collectively, our results demonstrate that carnosine improves ischemia-stimulated angiogenesis potentially by stabilizing HIF1-α and promoting VEGF formation suggesting that carnosine may be a useful therapy in critical limb ischemia.

Tocopheryl phosphate mixture (TPM) as a novel lipid-based transdermal drug delivery carrier: formulation and evaluation.

Transdermal drug delivery is a useful route of administration that avoids first-pass metabolism and more invasive delivery options. However, many drugs require enhancers to enable sufficient drug absorption to reach therapeutic effect. Alpha-tocopheryl phosphate (TP) and di-alpha-tocopheryl phosphate (T2P) are two phosphorylated forms of vitamin E which form tocopheryl phosphate mixture (TPM) when combined, and have been proposed to enhance the dermal and transdermal delivery of actives of interest. Here, we report the physicochemical characteristics and morphological properties of TPM formulations, including particle size, deformability and morphology, and its ability to facilitate the transport of carnosine, vitamin D3, CoEnzyme Q10 and caffeine into, and across, the skin. Results demonstrate that TPM self-assembles to form vesicular structures in hydroethanolic solutions ranging in mean size from 101 to 162nM depending on the amount of TPM and ethanol present in the formulation. The ratio of TP to T2P in TPM formulations altered vesicle size and elasticity, with vesicles high in TP found to be more deformable than those rich in T2P. TPM produced a significant (p<0.05) 2.4-3.4-fold increase in the absorption of carnosine, vitamin D3, CoEnzyme Q10 and caffeine into, or through, the skin. The TPM delivery platform was able to deliver a diverse range of actives with differing size and solubility profiles and therefore has significant potential to expand the number and types of drugs available for topical application and transdermal delivery.

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1mM AAPH and 20nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

Role of l-carnosine in the control of blood glucose, blood pressure, thermogenesis, and lipolysis by autonomic nerves in rats: involvement of the circadian clock and histamine

L-carnosine (β-alanyl-L-histidine; CAR) is synthesized in mammalian skeletal muscle. Although the physiological roles of CAR have not yet been clarified, there is evidence that the release of CAR from skeletal muscle during physical exercise affects autonomic neurotransmission and physiological functions. In particular, CAR affects the activity of sympathetic and parasympathetic nerves innervating the adrenal glands, liver, kidney, pancreas, stomach, and white and brown adipose tissues, thereby causing changes in blood pressure, blood glucose, appetite, lipolysis, and thermogenesis. CAR-mediated changes in neurotransmission and physiological functions were eliminated by histamine H1 or H3 receptor antagonists (diphenhydramine or thioperamide) and bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian clock. Moreover, a carnosine-degrading enzyme (carnosinase 2) was shown to be localized to histamine neurons in the hypothalamic tuberomammillary nucleus (TMN). Thus, CAR released from skeletal muscle during exercise may be transported into TMN-histamine neurons and hydrolyzed. The resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of CAR on neurotransmission and physiological function. Thus, CAR appears to influence hypoglycemic, hypotensive, and lipolytic activity through regulation of autonomic nerves and with the involvement of the SCN and histamine. These findings are reviewed and discussed in the context of other recent reports, including those on carnosine synthetases, carnosinases, and carnosine transport.

Transport Mechanisms of Carnosine in SKPT Cells: Contribution of Apical and Basolateral Membrane Transporters

The aim of this study was to investigate the transport properties of carnosine in kidney using SKPT cell cultures as a model of proximal tubular transport, and to isolate the functional activities of renal apical and basolateral transporters in this process. The membrane transport kinetics of 10 microM [3H]carnosine was studied in SKPT cells as a function of time, pH, potential inhibitors and substrate concentration. A cellular compartment model was constructed in which the influx, efflux and transepithelial clearances of carnosine were determined. Peptide transporter expression was probed by RT-PCR. Carnosine uptake was 15-fold greater from the apical than basolateral surface of SKPT cells. However, the apical-to-basolateral transepithelial transport of carnosine was severely rate-limited by its cellular efflux across the basolateral membrane. The high-affinity, proton-dependence, concentration-dependence and inhibitor specificity of carnosine supports the contention that PEPT2 is responsible for its apical uptake. In contrast, the basolateral transporter is saturable, inhibited by PEPT2 substrates but non-concentrative, thereby, suggesting a facilitative carrier. Carnosine is expected to have a substantial cellular accumulation in kidney but minimal tubular reabsorption in blood because of its high influx clearance across apical membranes by PEPT2 and very low efflux clearance across basolateral membranes.

Characterization of carnosine uptake and its physiological function in human intestinal epithelial Caco‐2 cells

Carnosine (beta-Ala-L-His) is known to have the physiological functions of an antioxidant. Although dietary carnosine is thought to be absorbed across intestinal epithelial cells, the mechanism for this absorption is not yet well understood and its function in the intestinal tract is also obscure. The intestinal transport of carnosine was characterized in the present study by using human intestinal Caco-2 cells, and its physiological function in these cells was further examined. The carnosine uptake was proton-dependent, being activated by lowering the apical pH value. Its uptake was significantly inhibited by other dipeptides, whereas it was not inhibited by other amino acids. These characteristics of the carnosine uptake strongly suggest its transport into the cells via peptide transporter 1 (PepT1). Since carnosine has antioxidative activity, we studied its effect on the H2O2-induced secretion of inflammatory cytokines in Caco-2 cells. The H2O2 induced increase in IL-8 secretion was inhibited by a pretreatment with carnosine for 3 h, this inhibition being presented in a dose-dependent manner. These results suggest that carnosine had a protective effect against oxidative stress in intestinal epithelial cells.

Dipeptide uptake and transport characteristics in rabbit tracheal epithelial cell layers cultured at an air interface.

To determine the functional presence ofa H+/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 microM. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by beta-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids beta-Ala and L-His. Unexpectedly. transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced ab transport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H+-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.

Cloning and Functional Expression of a Brain Peptide/Histidine Transporter

Here we report the cloning and functional characterization of a rat novel peptide/histidine transporter (PHT1), which was expressed in the brain and the retina. The cDNA encodes the predicted protein of 572 amino acid residues with 12 putative membrane-spanning domains. The amino acid sequence has moderate homology with a nonspecific peptide transporter found in the plant. When expressed in Xenopus laevis oocytes, PHT1 cRNA induced high affinity proton-dependent histidine transport activity. This transport process was inhibited by dipeptides and tripeptides but not by free amino acids such as glutamate, glycine, leucine, methionine, and aspartate. Dipeptide carnosine transport activity was also confirmed by direct uptake measurement. By in situ hybridization analysis, PHT1 mRNA was widely distributed throughout whole brain. Especially, intense hybridization signals were found in the hippocampus, choroid plexus, cerebellum, and pontine nucleus. Signals were located in both the neuronal and small nonneuronal cells in these areas. PHT1 protein could contribute to uptake of oligopeptides, which function as neuromodulators, and clearance of degraded neuropeptides and be a new member in the growing superfamily of proton-coupled peptide and nitrate transporters, although its structure, localization, and pharmacological characteristics are unique among these members.

The characteristics of carnosine transport and carnosine-induced electrical phenomena by the everted intestine of guinea pig.

The properties of carnosine (Car) and glycylglycine (Gly-Gly), transported across the mucosal border, were studied in isolated guinea pig everted ileum. The initial influxes of both dipeptides could be described by single Michaelis-Menten kinetics, having a nearly equal value of maximum influx. Mutual inhibition studies showed that the inhibition observed between Car and Gly-Gly was fully competitive, indicating that both Car and Gly-Gly share a common carrier. Although carrier-mediated influxes of the dipeptides were independent of Na+, the addition of the dipeptides into the mucosal solution evoked sudden and sustained increments of mucosal negativity. The changes in short-circuit current (delta Isc) evoked by the peptides increased as the Na+ concentration in the solution was increased, although both dipeptides evoked small increases in Isc, even in the absence of Na+. In spite of these common properties of transport and transport-related electrical phenomena, it was seen that the maximum change in transmural potential difference (delta PDt max) evoked by Car was about half that of Gly-Gly. Such a discrepancy between coincident Jmax values and values of delta PDt max suggests that the mechanism of induction of ionic flow is different for these two dipeptides.

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (β-alanyl- l-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na +, K + or choline + gradient conditions (extravesicular > intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na +-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a K m of 9.6 ± 1.4 mM and a V max of 2.9 ± 0.2 nmol / mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl- l-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na +-independent process.

3-O-methyl- d-glucose and β-alanine: Rapid axoplasmic transport of metabolically inert low molecular weight substances

The dynamics of the axoplasmic transport of free β-alanine and 3-O-methyl- d-glucose were quantitatively characterized in vivo in the pike olfactory nerve following intranasal application of the tritiated substances. Both compounds were found to be taken up by the epithelium but not metabolized to a significant extent. Axoplasmic transport showed the same dynamics as rapid transport of protein and free leucine in this nerve. No synthesis or transport of carnosine was detectable. The results are in agreement with the view that rapid axoplasmic transport is capable of conveying free, low molecular weight molecules in a non-specific manner.

Axoplasmic transport of carnosine (?-alanyl-L-histidine) in the mouse olfactory pathway

Carnosine in the chemoreceptor neurons of the olfactory epithelium can be labeled in vivo by intranasal irrigation with either(14)C-β-alanine or(14)C-L-histidine. This newly synthesized carnosine (but not the precursor amino acids) is translocated to the olfactory bulb, where the olfactory chemoreceptor axons synapse with the dendrites of mitral cells and other second-order neurons. Labeled carnosine arrives in the bulb several hours after intranasal administration of precursor. Similar arrival time is seen for macromolecules after intranasal administration of [(3)H]L-fucose, [(14)C]L-proline, or [(14)C]L-histidine. Macromolecules labeled with [(3)H]uridine take much longer to reach the bulb. Carnosine is also labeled after [(3)H]uridine administration. No labeling of macromolecules is observed after administration of 1-[(14)C]-β-alanine. Oral administration of the same dose of [(14)C]-β-alanine gives almost no labeled carnosine in bulb or epithelium. This method has permitted us to estimate that the half-life of labeled carnosine in both the bulb and epithelium is about 20 h. This method provides a means of selectively prelabeling the olfactory chemoreceptor neurons in the olfactory epithelium and their synapses in the olfactory bulb prior to cellular and subcellular separation procedures, and may also enable us to monitor the influences of olfactory stimulation on synthesis and transport of carnosine.

Carnosine transport by hamster jejunum in vitro and its inhibition by other di- and tripeptides.

Carnosine transport by hamster jejunum in vitro and its inhibition by other di- and tripeptides.