Validation of Nutritional Approaches to Modulate Cardiovascular and Diabetic Risk Factors in Patients with Hypertriglyceridemia or Prediabetes-The MoKaRi II Randomized Controlled Study.

Hypertriglyceridemia and diabetes mellitus type 2 are among the most important metabolic diseases globally. Diet plays a vital role in the development and progression of both clinical pictures. For the 10-week randomized, controlled, intervention study, 67 subjects with elevated plasma triglyceride (TG) concentrations (≥1.7 mmol/L) and 69 subjects with elevated fasting glucose concentrations (≥5.6 < 7.0 mmol/L) were recruited. The intervention groups received specially developed, individualized menu plans and regular counseling sessions to lower (A) TG or (B) fasting glucose and glycated hemoglobin A1c as well as other cardiovascular and diabetic risk factors. The hypertriglyceridemia intervention group was further supplemented with fish oil (3.5 g/d eicosapentaenoic acid + docosahexaenoic acid). The two control groups maintained a typical Western diet. Blood samples were taken every 2 weeks, and anthropometric data were collected. A follow-up examination was conducted after another 10 weeks. In both intervention groups, there were comparable significant reductions in blood lipids, glucose metabolism, and anthropometric parameters. These results were, with a few exceptions, significantly more pronounced in the intervention groups than in the corresponding control groups (comparison of percentage change from baseline). In particular, body weight was reduced by 7.4% (6.4 kg) and 7.5% (5.9 kg), low-density lipoprotein cholesterol concentrations by 19.8% (0.8 mmol/L) and 13.0% (0.5 mmol/L), TG concentrations by 18.2% (0.3 mmol/L) and 13.0% (0.2 mmol/L), and homeostatic model assessment for insulin resistance by 31.8% (1.1) and 26.4% (0.9) (p < 0.05) in the hypertriglyceridemia and prediabetes intervention groups, respectively. Some of these changes were maintained until follow-up. In patients with elevated TG or fasting glucose, implementing individualized menu plans in combination with regular counseling sessions over 10 weeks led to a significant improvement in cardiovascular and diabetic risk factors.

Evaluation of Influencing Factors on Metabolism of Land-Based n-3 Poly Unsaturated Fatty Acids-The KoALA Study.

This study aimed to investigate the impact of influencing factors (sex, eicosapentaenoic acid (EPA) status at baseline, linoleic acid (LA) intake, milk fat intake) on the conversion of α-linolenic acid (ALA) obtained from linseed oil into its long-chain metabolites. In addition, the effect of ALA on cardiovascular risk markers was investigated. This study used a parallel design approach by randomly assigning the 134 subjects to one of four diets (high in LA (HLA); low in LA (LLA); high in milk fat (MF); control (Western diet)) each enriched with linseed oil (10 en%, 22-27 mL ≙ 13-16 g ALA). Blood samples were taken at baseline and after 4, 8, and 12 weeks of dietary intervention. The study was fully completed by 105 subjects (57.4 ± 12.1 years; 65.7% female). Results showed that ALA (296-465%), C-20:4n3 (54-140%), and EPA (37-73%) concentrations in erythrocytes increased in all groups (p < 0.01). In contrast, docosahexaenoic acid (19-35%, p < 0.01) and n-3 index (10-21%, p < 0.05) dropped in the HLA, LLA, and control groups. An increase in C-22:5n3 was only observed in the MF (36%) and control groups (11%) (p < 0.05). In addition, an increase in LA (7-27%) was found in the HLA, LLA, and control groups, whereas C-20:3n6 (16-22%), arachidonic acid (10-16%), C-22:4n6 (12-30%), and C-22:5n6 (32-47%) decreased (p < 0.01). The conversion into EPA was higher in men than in women (69 vs. 39%, p = 0.043) and in subjects with low EPA status compared to participants with high EPA status (79 vs. 29%, p < 0.001). A high LA status attenuates the conversion rate. In line with the literature, no clear effects on blood lipids and parameters of glucose metabolism were found in relation to ALA supplementation.

A Combined Western and Bead-Based Multiplex Platform to Characterize Extracellular Vesicles.

In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 µg for all nine antibodies, compared with ∼10 µg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field.

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes.

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.

Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC-ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

Intracellular Protein-Lipid Interactions Studied by Rapid-Scan Electron Paramagnetic Resonance Spectroscopy.

Protein-membrane interactions play key roles in essential cellular processes; studying these interactions in the cell is a challenging task of modern biophysical chemistry. A prominent example is the interaction of human α-synuclein (αS) with negatively charged membranes. It has been well-studied in vitro, but in spite of the huge amount of lipid membranes in the crowded environment of biological cells, to date, no interactions have been detected in cells. Here, we use rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study αS interactions with negatively charged vesicles in vitro and upon transfection of the protein and lipid vesicles into model cells, i.e., oocytes of Xenopus laevis. We show that protein-vesicle interactions are reflected in RS spectra in vitro and in cells, which enables time-resolved monitoring of protein-membrane interaction upon transfection into cells. Our data suggest binding of a small fraction of αS to endogenous membranes.

Functionally different α-synuclein inclusions yield insight into Parkinson's disease pathology.

The formation of α-synuclein (α-S) amyloid aggregates, called Lewy bodies (LBs), is a hallmark of Parkinson's disease (PD). The function of LBs in the disease process is however still unclear; they have been associated with both neuroprotection and toxicity. To obtain insight into this contradiction, we induced the formation of α-S inclusions, using three different induction methods in SH-SY5Y cells and rat-derived primary neuronal cells. Using confocal and STED microscopy we observed induction-dependent differences in α-S inclusion morphology, location and function. The aggregation of α-S in functionally different compartments correlates with the toxicity of the induction method measured in viability assays. The most cytotoxic treatment largely correlates with the formation of proteasome-associated, juxta-nuclear inclusions. With less toxic methods cytosolic deposits that are not associated with the proteasome are more prevalent. The distribution of α-S over at least two different types of inclusions is not limited to cell models, but is also observed in primary neuronal cells and in human mesencephalon. The existence of functionally different LBs, in vivo and in vitro, gives important insights in the impact of Lewy Body formation on neuronal functioning and may thereby provide a platform for discovering therapeutics.