Fat and Sugar—A Dangerous Duet. A Comparative Review on Metabolic Remodeling in Rodent Models of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a common disease in Western society and ranges from steatosis to steatohepatitis to end-stage liver disease such as cirrhosis and hepatocellular carcinoma. The molecular mechanisms that are involved in the progression of steatosis to more severe liver damage in patients are not fully understood. A deeper investigation of NAFLD pathogenesis is possible due to the many different animal models developed recently. In this review, we present a comparative overview of the most common dietary NAFLD rodent models with respect to their metabolic phenotype and morphological manifestation. Moreover, we describe similarities and controversies concerning the effect of NAFLD-inducing diets on mitochondria as well as mitochondria-derived oxidative stress in the progression of NAFLD

Patient listening on social media for patient-focused drug development: a synthesis of considerations from patients, industry and regulators

Patients, life science industry and regulatory authorities are united in their goal to reduce the disease burden of patients by closing remaining unmet needs. Patients have, however, not always been systematically and consistently involved in the drug development process. Recognizing this gap, regulatory bodies worldwide have initiated patient-focused drug development (PFDD) initiatives to foster a more systematic involvement of patients in the drug development process and to ensure that outcomes measured in clinical trials are truly relevant to patients and represent significant improvements to their quality of life. As a source of real-world evidence (RWE), social media has been consistently shown to capture the first-hand, spontaneous and unfiltered disease and treatment experience of patients and is acknowledged as a valid method for generating patient experience data by the Food and Drug Administration (FDA). While social media listening (SML) methods are increasingly applied to many diseases and use cases, a significant piece of uncertainty remains on how evidence derived from social media can be used in the drug development process and how it can impact regulatory decision making, including legal and ethical aspects. In this policy paper, we review the perspectives of three key stakeholder groups on the role of SML in drug development, namely patients, life science companies and regulators. We also carry out a systematic review of current practices and use cases for SML and, in particular, highlight benefits and drawbacks for the use of SML as a way to identify unmet needs of patients. While we find that the stakeholders are strongly aligned regarding the potential of social media for PFDD, we identify key areas in which regulatory guidance is needed to reduce uncertainty regarding the impact of SML as a source of patient experience data that has impact on regulatory decision making

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+) at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells

Effects of TMPD/ascorbate on mitoBK_Ca channel activity.

<p><b>A.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 and −40 mV under control conditions, after the addition of 250 µM TMPD with 500 µM Ascorbate (Asc) and after perfusion. <b>B.</b> Analysis of the probability of channel opening under the conditions described in A. *P<0.01 vs. the control.</p

Schematic of the proposed model and the applied substrates/inhibitors of the respiratory chain and activators/blockers of the mitoBK_Ca channel.

<p>Complexes of the respiratory chain are shown, including NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinol cytochrome c oxidoreductase (complex III), and cytochrome c oxidase (complex IV). All of the substances (i.e., those that interact with the respiratory chain and with the mitoBK_Ca channel) that were used in this study are shown.</p

Cyanide abolishes the inhibitory effect of the respiratory chain substrates succinate and NADH.

<p><b>A.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 mV under control conditions, after the addition of 30 µM KCN and 5 mM succinate plus 30 µM KCN, and after perfusion. <b>B.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 mV under control conditions, after the addition of 30 µM KCN and 200 µM NADH plus 30 µM KCN, and after perfusion. <b>C.</b> Distribution of P_o at different voltages under the conditions described in A and B.</p

2D BN/SDS-PAGE separation of native astrocytoma mitochondria protein extracts.

<p>Two-dimensional separation was performed as described in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068125#s2" target="_blank">Materials and Methods</a></i>, and the PVDF membrane was first immunoblotted for the BK_Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK_Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK_Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc₁ and Sc₂, complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.</p