Advanced glycation end-products (AGEs) are non-enzymatic protein and amino acid adducts as well as DNA adducts which form from dicarbonyls and glucose. AGE formation is enhanced in diabetes and is associated with the development of diabetic complications. In the current review, we discuss mechanisms that lead to enhanced AGE levels in the context of diabetes and diabetic complications. The methylglyoxal-detoxifying glyoxalase system as well as alternative pathways of AGE detoxification are summarized. Therapeutic approaches to interfere with different pathways of AGE formation are presented.

The reactive metabolite methylglyoxal (MG) has been identified as mediator of pain. Scavenging of free MG and the prevention of MG-derived post-translational modifications may provide a useful therapeutic treatment. An arginine-rich, fatty acid coupled, cyclic peptide (CycK(Myr)R4E) with high proteolytic stability and prolonged circulation was developed for the scavenging of MG. It was shown to reduce the formation of albumin-MG adducts in vitro and prevented MG-induced pain by reducing plasma MG levels through the formation of peptide-MG adducts in vivo. CycK(Myr)R4E therefore presents a promising option for the treatment of pain and other diabetic complications associated with high MG levels.

The determination of individual prostaglandins (PG) in humans is mainly performed in urine samples. The quantification of PGs in human plasma could improve the understanding of particular PG species under various physiological and pathological conditions. 15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is a dehydrated downstream product of PGD₂ and is of high interest due to its recently discovered anti-inflammatory effects. Increasing availability of highly sensitive mass spectrometry allows the quantification of low abundant biomarkers like 15d-PGJ₂ in human plasma samples. Herein, a sensitive LC-MS/MS method for the determination of 15d-PGJ₂ was established. The method was validated according to the guidance of the American Food and Drug Administration and tested in plasma samples from patients with poorly controlled diabetes, considered to be a pro-inflammatory condition. Extraction of 15d-PGJ₂ was achieved with an easy-to-use liquid-liquid extraction by ethyl acetate following a methanol precipitation. The lower limit of quantification was 2.5 pg mL⁻¹ and linearity (R ² = 0.998) was guaranteed between 2.5 and 500 pg mL⁻¹ for 15d-PGJ₂. Selectivity was assured by the use of two individual mass transitions (qualifier and quantifier). Precision and accuracy were validated in an inter- and intraday assay with a coefficient of variation below 11.8% (intraday) and 14.7% (interday). In diabetic patients with an HbA_1C > 9%, increased plasma concentrations of 15d-PGJ₂ compared to control plasma were measured. 15d-PGJ₂ correlated negatively with the inflammation marker C-reactive protein. The developed LC-MS/MS method represents a new possibility to quantify 15d-PGJ₂ with high specificity in human plasma samples. This may contribute to a better understanding of the potential anti-inflammatory effects of 15d-PGJ₂ in severe long-term pro-inflammatory disorders like diabetes, cancer, or cardiovascular disease.

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of amino acids. and are associated with diabetic complications. One proposed pathomechanism is the impaired processing of AGE-modified proteins or peptides including prohormones. Two approaches were applied to investigate whether substrate modification with AGEs affects the processing of substrates like prohormones to the active hormones. First, we employed solid phase peptide synthesis to generate unmodified as well as AGE-modified protease substrates. Activity of proteases towards these substrates was quantified. Second, we tested the effect of AGE-modified proinsulin on the processing to insulin. Proteases showed the expected activity towards the unmodified peptide substrates containing arginine or lysine at the C-terminal cleavage site. Indeed, modification with N^ε-carboxymethyllysine (CML) or methylglyoxal-hydroimidazolone 1 (MG-H1) affected all proteases tested. Cysteine cathepsins displayed a reduction in activity by around 50 % towards CML and MG-H1 modified substrates. The specific proteases trypsin, proprotein convertases subtilisin-kexin type proteases (PCSKs) and carboxypeptidase E (CPE) were completely inactive towards modified substrates. Proinsulin incubation with methylglyoxal at physiological concentrations for 24 h resulted in the formation of MG-modified proinsulin. The formation of insulin was reduced by up to 80 % in a concentration dependent manner. Here we demonstrate the inhibitory effect of substrate-AGE modifications on proteases. The finding that PCSKs and CPE, which are essential for prohormone processing are inactive towards modified substrates could point to a yet unrecognized pathomechanism resulting from AGE-modification relevant for the etiopathogenesis of diabetes and the development of obesity.

Introduction: Pathological levels of methylglyoxal (MG), a reactive dicarbonyl product of glucose, contribute to major neurological complications associated with type II diabetes, including chronic neuropathic pain. Strategies to target elevated MG have included small molecule MG scavengers, but scavenger deficiencies in proteolytic stability and onset of scavenging activity have precluded clinical translation. To address this gap, we developed a long-lasting and highly reactive cyclic peptide CycK(Myr)R₄E, and here evaluated its antihyperalgesic efficacy in the db/db mouse model of type II diabetes painful diabetic neuropathy. Objectives: To test the hypothesis that CycK(Myr)R₄E can reduce behavioral and molecular signs of painful diabetic neuropathy. Methods: We assessed heat hypersensitivity as an index of hyperalgesia, and touch-evoked expression of phosphorylated extracellular signal–regulated kinase as a measure of neuronal activity in spinal cord dorsal horn. Results: We report that a single systemic injection of CycK(Myr)R₄E (3 mg/kg) reversed heat hypersensitivity. Repeated systemic injection of CycK(Myr)R₄E (0.125 mg/kg, 3 times per week, 6–12 weeks of age) prevented heat hypersensitivity and reduced stimulus-evoked phosphorylated extracellular signal–regulated kinase. Conclusion: These studies promote CycK(Myr)R₄E as the most promising MG scavenger for the prevention and treatment of hyperalgesia in type 2 diabetic neuropathic pain.