Detection of follistatin-based inhibitors of the TGF-ß signaling pathways in serum/plasma by means of LC-HRMS/MS and Western blotting.

Cytokines of the transforming growth factor beta (TGF-ß) superfamily such as myostatin and activin A are considered as key regulators of skeletal muscle mass. In vivo, their activity is controlled by different binding proteins such as follistatin (FST), whose interaction with the circulating growth factors prevents activation of the activin type II receptors. FST-based protein therapeutics are therefore not only promising drug candidates for the treatment of muscular diseases but also potential performance-enhancing agents in sports. Within this study, two complementary detection assays for FST-based inhibitors of the TGF-ß signaling pathways in doping control serum and plasma samples were developed by using both monomeric FST and dimeric FST-Fc fusion proteins as model compounds. The initial testing procedure is based on immunoaffinity purification, tryptic digestion, and LC-HRMS/MS, offering high specificity by targeting tryptic signature peptides of FST. As the glycoprotein is also produced endogenously, the confirmation method employs immunoaffinity purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blotting in order to detect the intact proteins and differentiate synthetic FST-Fc constructs from naturally occurring FST isoforms. Both assays were found to be highly specific with an estimated detection limit of 10 ng/ml. Moreover, a commercial sandwich enzyme-linked immunosorbent assay was used to determine endogenous FST values. The detected FST serum levels of healthy volunteers were found below 5 ng/ml, which is in accordance with reference values from the literature and below the doping control detection methods' limit of detection (LOD). The presented assays expand the range of available tests for emerging doping agents, and the initial testing procedure can readily be modified to include further protein drugs.

A Global Cndp1-Knock-Out Selectively Increases Renal Carnosine and Anserine Concentrations in an Age- and Gender-Specific Manner in Mice.

Carnosinase 1 (CN1) is encoded by the Cndp1 gene and degrades carnosine and anserine, two natural histidine-containing dipeptides. In vitro and in vivo studies suggest carnosine- and anserine-mediated protection against long-term sequelae of reactive metabolites accumulating, e.g., in diabetes mellitus. We have characterized the metabolic impact of CN1 in 11- and 55-week-old Cndp1-knockout (Cndp1-KO) mice and litter-matched wildtypes (WT). In Cndp1-KO mice, renal carnosine and anserine concentrations were gender-specifically increased 2- to 9-fold, respectively in the kidney and both most abundant in the renal cortex, but remained unchanged in all other organs and in serum. Renal oxidized/reduced glutathione concentrations, renal morphology and function were unaltered. In Cndp1-KO mice at week 11, renal asparagine, serine and glutamine levels and at week 55, renal arginine concentration were reduced. Renal heat-shock-protein 70 (Hspa1a/b) mRNA declined with age in WT but not in Cndp1-KO mice, transcription factor heat-shock-factor 1 was higher in 55-week-old KO mice. Fasting blood glucose concentrations decreased with age in WT mice, but were unchanged in Cndp1-KO mice. Blood glucose response to intraperitoneal insulin was gender- but not genotype-dependent, the response to intraperitoneal glucose injection was similar in all groups. A global Cndp1-KO selectively, age- and gender-specifically, increases renal carnosine and anserine concentrations, alters renal amino acid- and HSP70 profile and modifies systemic glucose homeostasis. Increase of the natural occurring carnosine and anserine levels in the kidney by modulation of CN1 represents a promising therapeutic approach to mitigate or prevent chronic kidney diseases such as diabetic nephropathy.

Carnosine Activates Cellular Stress Response in Podocytes and Reduces Glycative and Lipoperoxidative Stress.

Carnosine improves diabetic complications, including diabetic nephropathy, in in vivo models. To further understand the underlying mechanism of nephroprotection, we studied the effect of carnosine under glucose-induced stress on cellular stress response proteins in murine immortalized podocytes, essential for glomerular function. High-glucose stress initiated stress response by increasing intracellular heat shock protein 70 (Hsp70), sirtuin-1 (Sirt-1), thioredoxin (Trx), glutamate-cysteine ligase (gamma-glutamyl cysteine synthetase; γ-GCS) and heme oxygenase-1 (HO-1) in podocytes by 30-50% compared to untreated cells. Carnosine (1 mM) also induced a corresponding upregulation of these intracellular stress markers, which was even more prominent compared to glucose for Hsp70 (21%), γ-GCS and HO-1 (13% and 20%, respectively; all p < 0.001). Co-incubation of carnosine (1 mM) and glucose (25 mM) induced further upregulation of Hsp70 (84%), Sirt-1 (52%), Trx (35%), γ-GCS (90%) and HO-1 (73%) concentrations compared to untreated cells (all p < 0.001). The glucose-induced increase in 4-hydroxy-trans-2-nonenal (HNE) and protein carbonylation was reduced dose-dependently by carnosine by more than 50% (p < 0.001). Although podocytes tolerated high carnosine concentrations (10 mM), high carnosine levels only slightly increased Trx and γ-GCS (10% and 19%, respectively, compared to controls; p < 0.001), but not Hsp70, Sirt-1 and HO-1 proteins (p not significant), and did not modify the glucose-induced oxidative stress response. In podocytes, carnosine induced cellular stress tolerance and resilience pathways and was highly effective in reducing high-glucose-induced glycative and lipoperoxidative stress. Carnosine in moderate concentrations exerted a direct podocyte molecular protective action.

CNDP1 knockout in zebrafish alters the amino acid metabolism, restrains weight gain, but does not protect from diabetic complications.

The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1-/- zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1-/- zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala-His and Ser-His. Under diabetic conditions increased carnosine levels in CNDP1-/- embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications.

Protective Actions of Anserine Under Diabetic Conditions.

BACKGROUND/AIMS: In rodents, carnosine treatment improves diabetic nephropathy, whereas little is known about the role and function of anserine, the methylated form of carnosine. METHODS: Antioxidant activity was measured by oxygen radical absorbance capacity and oxygen stress response in human renal tubular cells (HK-2) by RT-PCR and Western-Immunoblotting. In wildtype (WT) and diabetic mice (db/db), the effect of short-term anserine treatment on blood glucose, proteinuria and vascular permeability was measured. RESULTS: Anserine has a higher antioxidant capacity compared to carnosine (p < 0.001). In tubular cells (HK-2) stressed with 25 mM glucose or 20⁻100 µM hydrogen peroxide, anserine but not carnosine, increased intracellular heat shock protein (Hsp70) mRNA and protein levels. In HK-2 cells stressed with glucose, co-incubation with anserine also increased hemeoxygenase (HO-1) protein and reduced total protein carbonylation, but had no effect on cellular sirtuin-1 and thioredoxin protein concentrations. Three intravenous anserine injections every 48 h in 12-week-old db/db mice, improved blood glucose by one fifth, vascular permeability by one third, and halved proteinuria (all p < 0.05). CONCLUSION: Anserine is a potent antioxidant and activates the intracellular Hsp70/HO-1 defense system under oxidative and glycative stress. Short-term anserine treatment in diabetic mice improves glucose homeostasis and nephropathy.

Carnosine Catalyzes the Formation of the Oligo/Polymeric Products of Methylglyoxal.

BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.

Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift.

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.