Association Between Serum Carnosinase Concentration and Activity and Renal Function Impairment in a Type-2 Diabetes Cohort

Introduction: Genetic studies have identified associations of carnosinase 1 (CN1) polymorphisms with diabetic kidney disease (DKD). However, CN1 levels and activities have not been assessed as diagnostic or prognostic markers of DKD in cohorts of patients with type 2 diabetes (T2D).Methods: We established high-throughput, automated CN1 activity and concentration assays using robotic systems. Using these methods, we determined baseline serum CN1 levels and activity in a T2D cohort with 970 patients with no or only mild renal impairment. The patients were followed for a mean of 1.2 years. Baseline serum CN1 concentration and activity were assessed as predictors of renal function impairment and incident albuminuria during follow up.Results: CN1 concentration was significantly associated with age, gender and estimated glomerular filtration rate (eGFR) at baseline. CN1 activity was significantly associated with glycated hemoglobin A1c (HbA1c) and eGFR. Serum CN1 at baseline was associated with eGFR decline and predicted renal function impairment and incident albuminuria during the follow-up.Discussion: Baseline serum CN1 levels were associated with presence and progression of renal function decline in a cohort of T2D patients. Confirmation in larger cohorts with longer follow-up observation periods will be required to fully establish CN1 as a biomarker of DKD

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (≥ 50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function

Selective inhibitors of cardiac ADPR cyclase as novel anti-arrhythmic compounds

ADP-ribosyl cyclases (ADPRCs) catalyse the conversion of nicotinamide adenine dinucleotide to cyclic adenosine diphosphoribose (cADPR) which is a second messenger involved in Ca2+ mobilisation from intracellular stores. Via its interaction with the ryanodine receptor Ca2+ channel in the heart, cADPR may exert arrhythmogenic activity. To test this hypothesis, we have studied the effect of novel cardiac ADPRC inhibitors in vitro and in vivo in models of ventricular arrhythmias. Using a high-throughput screening approach on cardiac sarcoplasmic reticulum membranes isolated from pig and rat and nicotinamide hypoxanthine dinuleotide as a surrogate substrate, we have identified potent and selective inhibitors of an intracellular, membrane-bound cardiac ADPRC that are different from the two known mammalian ADPRCs, CD38 and CD157/Bst1. We show that two structurally distinct cardiac ADPRC inhibitors, SAN2589 and SAN4825, prevent the formation of spontaneous action potentials in guinea pig papillary muscle in vitro and that compound SAN4825 is active in vivo in delaying ventricular fibrillation and cardiac arrest in a guinea pig model of Ca2+ overload-induced arrhythmia. Inhibition of cardiac ADPRC prevents Ca2+ overload-induced spontaneous depolarizations and ventricular fibrillation and may thus provide a novel therapeutic principle for the treatment of cardiac arrhythmias

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling

Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a process which is accompanied by the pumping of four protons across the membrane. Elucidation of the structures of intermediates in these processes is crucial for understanding the mechanism of oxygen reduction. In the work presented here, the reaction of HO with the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical based on comparison with other paramagnetic centers in COX was ∼20%. Recent crystallographic data have shown that one of the CU ligands, His 276 (in the bacterial case), is cross-linked to Tyr 280 and that this cross-linked tyrosine is ideally positioned to participate in dioxygen activation. Here selectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed arise from a tyrosine radical species. It would seem also quite possible that this radical is an intermediate in the mechanism of oxygen reduction

Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru(II)(2,2′-bipyridyl)(3)(2+) was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (τ(1) ≈ 20 μs and τ(2) ≈ 175 μs) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu(A) to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2–3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, τ(2) is increased to 900 μs. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of ≈1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of τ(3) ≈ 2 ms and τ(4) ≈ 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron

Electrical current generation and proton pumping catalyzed by the ba(3)-type cytochrome c oxidase from Thermus thermophilus

AbstractSeveral amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba3-type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus. So far, it has been unclear whether the Th. thermophilus ba3-type cytochrome c oxidase can nevertheless function as an electrogenic proton pump. In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th. thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane. In addition to a `vectorial' consumption of 1.0 H+/e− for water formation, proton pumping with a stoichiometry of 0.4–0.5 H+/e− was observed. The implications of these findings for the mechanism of redox-coupled proton transfer in this unusual cytochrome c oxidase are discussed

Activation of thyroid hormone receptor-β improved disease activity and metabolism independent of body weight in a mouse model of non-alcoholic steatohepatitis and fibrosis

Background and Purpose: Activation of hepatic thyroid hormone receptor β (THR-β) is associated with systemic lipid lowering, increased bile acid synthesis, and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-β agonists decreased hepatic steatosis and circulating lipids, and induced resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-β agonist, as a prototype to investigate the effects of THR-β activation in mice with diet-induced obesity (DIO) and biopsy-confirmed advanced NASH with fibrosis. Experimental Approach: C57Bl/6J mice were fed a diet high in fat, fructose, and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was administered at a daily dose of 3 mg·kg−1 p.o., for 8 weeks. Systemic and hepatic metabolic parameters, histological non-alcoholic fatty liver disease (NAFLD) activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-β activation. Key Results: Treatment with resmetirom did not influence body weight but led to significant reduction in liver weight, hepatic steatosis, plasma alanine aminotransferase activity, liver and plasma cholesterol, and blood glucose. These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, a lower content of α-smooth muscle actin and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis. Conclusion and Implications: Our model robustly reflected clinical observations of body weight-independent improvements in systemic and hepatic metabolism including anti-steatotic activity

Effects of Whole-Body Adenylyl Cyclase 5 (Adcy5) Deficiency on Systemic Insulin Sensitivity and Adipose Tissue

Genome-wide association studies have identified adenylyl cyclase type 5 (ADCY5) as candidate gene for diabetes-related quantitative traits and an increased risk of type 2 diabetes. Mice with a whole-body deletion of Adcy5 (Adcy5–/–) do not develop obesity, glucose intolerance and insulin resistance, have improved cardiac function and increased longevity. Here, we investigated Adcy5 knockout mice (Adcy5–/–) to test the hypothesis that changes in adipose tissue (AT) may contribute to the reported healthier phenotype. In contrast to previous reports, we found that deletion of Adcy5 did not confer any physiological or biochemical benefits. However, this unexpected finding allowed us to investigate the effects of Adcy5 depletion on AT independently of lower body weight and a metabolically healthier phenotype. Adcy5–/– mice exhibited an increased number of smaller adipocytes, lower mean adipocyte size and a distinct AT gene expression pattern with midline 1 (Mid1) as the most significantly downregulated gene compared to control mice. Our Adcy5–/– model challenges previously described beneficial effects of Adcy5 deficiency and suggests that targeting Adcy5 does not improve insulin sensitivity and may therefore limit the relevance of ADCY5 as potential drug target