The significance of glutaredoxins for diabetes mellitus and its complications.

Diabetes mellitus is a non-communicable metabolic disease hallmarked by chronic hyperglycemia caused by beta-cell failure. Diabetic complications affect the vasculature and result in macro- and microangiopathies, which account for a significantly increased morbidity and mortality. The rising incidence and prevalence of diabetes is a major global health burden. There are no feasible strategies for beta-cell preservation available in daily clinical practice. Therefore, patients rely on antidiabetic drugs or the application of exogenous insulin. Glutaredoxins (Grxs) are ubiquitously expressed and highly conserved members of the thioredoxin family of proteins. They have specific functions in redox-mediated signal transduction, iron homeostasis and biosynthesis of iron-sulfur (FeS) proteins, and the regulation of cell proliferation, survival, and function. The involvement of Grxs in chronic diseases has been a topic of research for several decades, suggesting them as therapeutic targets. Little is known about their role in diabetes and its complications. Therefore, this review summarizes the available literature on the significance of Grxs in diabetes and its complications. In conclusion, Grxs are differentially expressed in the endocrine pancreas and in tissues affected by diabetic complications, such as the heart, the kidneys, the eye, and the vasculature. They are involved in several pathways essential for insulin signaling, metabolic inflammation, glucose and fatty acid uptake and processing, cell survival, and iron and mitochondrial metabolism. Most studies describe significant changes in glutaredoxin expression and/or activity in response to the diabetic metabolism. In general, mitigated levels of Grxs are associated with oxidative distress, cell damage, and even cell death. The induced overexpression is considered a potential part of the cellular stress-response, counteracting oxidative distress and exerting beneficial impact on cell function such as insulin secretion, cytokine expression, and enzyme activity.

Loss and Recovery of Glutaredoxin 5 Is Inducible by Diet in a Murine Model of Diabesity and Mediated by Free Fatty Acids In Vitro.

Free fatty acids (FFA), hyperglycemia, and inflammatory cytokines are major mediators of ß-cell toxicity in type 2 diabetes mellitus, impairing mitochondrial metabolism. Glutaredoxin 5 (Glrx5) is a mitochondrial protein involved in the assembly of iron-sulfur clusters required for complexes of the respiratory chain. We have provided evidence that islet cells are deprived of Glrx5, correlating with impaired insulin secretion during diabetes in genetically obese mice. In this study, we induced diabesity in C57BL/6J mice in vivo by feeding the mice a high-fat diet (HFD) and modelled the diabetic metabolism in MIN6 cells through exposure to FFA, glucose, or inflammatory cytokines in vitro. qRT-PCR, ELISA, immunohisto-/cytochemistry, bioluminescence, and respirometry were employed to study Glrx5, insulin secretion, and mitochondrial biomarkers. The HFD induced a depletion of islet Glrx5 concomitant with an obese phenotype, elevated FFA in serum and reactive oxygen species in islets, and impaired glucose tolerance. Exposure of MIN6 cells to FFA led to a loss of Glrx5 in vitro. The FFA-induced depletion of Glrx5 coincided with significantly altered mitochondrial biomarkers. In summary, we provide evidence that Glrx5 is regulated by FFA in type 2 diabetes mellitus and is linked to mitochondrial dysfunction and blunted insulin secretion.

Preparation of fatty acid solutions exerts significant impact on experimental outcomes in cell culture models of lipotoxicity.

Free fatty acids are essentially involved in the pathogenesis of chronic diseases such as diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular disease. They promote mitochondrial dysfunction, oxidative stress, respiratory chain uncoupling, and endoplasmic reticulum stress and modulate stress-sensitive pathways. These detrimental biological effects summarized as lipotoxicity mainly depend on fatty acid carbon chain length, degree of unsaturation, concentration, and treatment time. Preparation of fatty acid solutions involves dissolving and complexing. Solvent toxicity and concentration, amount of bovine serum albumin (BSA), and ratio of albumin to fatty acids can vary significantly between equal concentrations, mediating considerable harmful effects and/or interference with certain assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Herein, we studied the impact of commonly used solvents ethanol and dimethyl sulfoxide and varying concentrations of BSA directly and in solution with oleic acid on MTT to formazan conversion, adenosine triphosphate level, and insulin content and secretion of murine ß-cell line MIN6 employing different treatment duration. Our data show that experimental outcomes and assay readouts can be significantly affected by mere preparation of fatty acid solutions and should thus be carefully considered and described in detail to ensure comparability and distinct evaluation of data.

Lipotoxic Impairment of Mitochondrial Function in ß-Cells: A Review.

Lipotoxicity is a major contributor to type 2 diabetes mainly promoting mitochondrial dysfunction. Lipotoxic stress is mediated by elevated levels of free fatty acids through various mechanisms and pathways. Impaired peroxisome proliferator-activated receptor (PPAR) signaling, enhanced oxidative stress levels, and uncoupling of the respiratory chain result in ATP deficiency, while ß-cell viability can be severely impaired by lipotoxic modulation of PI3K/Akt and mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) pathways. However, fatty acids are physiologically required for an unimpaired ß-cell function. Thus, preparation, concentration, and treatment duration determine whether the outcome is beneficial or detrimental when fatty acids are employed in experimental setups. Further, ageing is a crucial contributor to ß-cell decay. Cellular senescence is connected to loss of function in ß-cells and can further be promoted by lipotoxicity. The potential benefit of nutrients has been broadly investigated, and particularly polyphenols were shown to be protective against both lipotoxicity and cellular senescence, maintaining the physiology of ß-cells. Positive effects on blood glucose regulation, mitigation of oxidative stress by radical scavenging properties or regulation of antioxidative enzymes, and modulation of apoptotic factors were reported. This review summarizes the significance of lipotoxicity and cellular senescence for mitochondrial dysfunction in the pancreatic ß-cell and outlines potential beneficial effects of plant-based nutrients by the example of polyphenols.

Pivotal role for alpha1-antichymotrypsin in skin repair.

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.

Investigation of the influence of charge derivatization on the fragmentation of multiply protonated peptides.

The fragmentation of the multiply charged peptides b-chain of bovine insulin and glucagon have been investigated under low energy collision induced dissociation (CID) conditions using an electrospray ion trap mass spectrometer. The influence of charge state, specific amino acids such as aspartate or proline, the location of basic sites, and the derivatization on the fragmentation behavior has been the focus of interest. As a basis for understanding the fragmentation process, the concept of the mobile proton was applied. A set of different derivatives was used to manipulate the sites of protonation of the peptides in order to control and improve the fragmentation behavior. These results can be applied for de novo sequencing, although the sequence-specific fragmentation processes have significant influence on the dissociation behavior of the peptides.