Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). Here we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8+ T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8+ T cells caused morphological and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8+ T cell response and required T cell–intrinsic type 1 interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8+ T cells in IAC.

Genipin, a natural compound from Gardenia jasminoides, is a well-known compound in Chinese medicine that is used for the treatment of cancer, inflammation, and diabetes. The use of genipin in classical medicine is hindered because of its unknown molecular mechanisms of action apart from its strong cross-linking ability. Genipin is increasingly applied as a specific inhibitor of proton transport mediated by mitochondrial uncoupling protein 2 (UCP2). However, its specificity for UCP2 is questionable, and the underlying mechanism behind its action is unknown. Here, we investigated the effect of genipin in different systems, including neuroblastoma cells, isolated mitochondria, isolated mitochondrial proteins, and planar lipid bilayer membranes reconstituted with recombinant proteins. We revealed that genipin activated dicarboxylate carrier and decreased the activity of UCP1, UCP3, and complex III of the respiratory chain alongside with UCP2 inhibition. Based on competitive inhibition experiments, the use of amino acid blockers, and site-directed mutagenesis of UCP1, we propose a mechanism of genipin's action on UCPs. At low concentrations, genipin binds to arginine residues located in the UCP funnel, which leads to a decrease in UCP's proton transporting function in the presence of long chain fatty acids. At concentrations above 200 mM, the inhibitory action of genipin on UCPs is overlaid by increased nonspecific membrane conductance due to the formation of protein-genipin aggregates. Understanding the concentration-dependent mechanism of genipin action in cells will allow its targeted application as a drug in the above-mentioned diseases. SIGNIFICANCE Genipin is a well-known natural cross-linking agent for proteins, collagen, gelatin, and chitosan. However, the mechanism of its multiple effects on cells and mitochondria is under dispute. Mitochondrial UCP2 was previously revealed as an important target for genipin. We show that inhibition of UCP2 by genipin at submillimolar concentrations depends on the presence of three positively charged arginines in the funnel of the protein. This mechanism is similar to the UCP inhibition by purine nucleotides such as ATP/ADP and GTP/GDP. The effect of genipin is not specific to UCP2, which can be explained by the presence of arginines in homologous UCP1 and UCP3. This insight is crucial for the design of specific inhibitors of UCPs. FIGURE 9 Extracted ion chromatograms of genipin-modified UCP1 tryptic peptides. Signals at m/z 919.47 and 914.14 correspond to UCP1 tryptic peptide 184 NVIICTELVTYDLMKGALVNNK 206 carrying K198 (919.47; tR ¼ 21.4) and C188 (919.14; tR ¼ 24.2), respectively, modified with genipin adduct of 158 amu after incubation with 50 mM (A) and 1 mM (B) genipin. Corresponding CID spectra are provided in Fig. S1. (C) Positions of cysteine C188 and lysine K198 in UCP1, at which genipin modifications were detected. Three-dimensional structure of UCP1 was computed based on the crystallographic structure of ANT (PDB: 1OKC) using PyMol. To see this figure in color, go...

The prevalence and progression of many illnesses, such as neurodegenerative and cardiovascular diseases, obesity, and cancer, vary between women and men, often in an age-dependent manner. A joint hallmark of these diseases is some type of mitochondrial dysfunction. While several mitochondrial proteins are known to be regulated by sex hormones, the levels of those proteins have not been systematically analyzed with regard to sex and age, and studies that consider sex and/or age differences in the protein expression are very rare. In this study, we compared the expression patterns of physiologically important mitochondrial proteins in female and male C57BL/6N mice of age cohorts frequently used in experiments. We found that sex-related differences in the expression of uncoupling proteins 1 and 3 (UCP1 and UCP3) occur in an age-dependent manner. The sex-specific expression of UCP1 and UCP3 in brown adipose tissue (BAT) was inversely correlated with differences in body weight. Expression of UCP4 in the brain, Complex I in the spleen, and Complex II in the brain and BAT was least affected by the sex of the mouse. We further demonstrated that there are serious limitations in using VDAC1 and actin as markers in western blot analyses, due to their sex- and age-specific fluctuations. Our results confirm that sex and age are important parameters and should be taken into account by researchers who examine the mechanistic aspects of diseases.Highlights The levels of UCP1 and UCP3 protein expression differ between females and males in an age-dependent manner.Pre-pubertal expression of almost all proteins tested in this study does not depend on the sex of the mouse.Expression of VDAC1 and actin, which are often used as loading control proteins in western blot analysis, is tissue-specifically influenced by sex and age.

Mitochondria play a key role in regulating and buffering intracellular [Ca 2þ ]. Ca 2þ transport across the mitochondrial membrane occurs through the mitochondrial Ca 2þ uniporter (MCU), ryanodine receptor (mRyR), Na þ /Ca 2þ exchanger (mNCE), and putative Ca 2þ /H þ exchanger (mCHE). Exposure to excess Ca 2þ leads to opening of the mitochondrial permeability transition pore (mPTP), releasing Ca 2þ and triggering apoptosis. The molecular components of mPTP are currently unknown, but cyclosporine A (CsA) and ADP both delay its opening. In this study, we isolated mitochondria from rat hearts, energized them with Na þ-free K þ succinate (to block contribution of mNCE), and measured extramatrix [Ca 2þ ] with spectrophotometry to monitor matrix Ca 2þ uptake. By blocking Ca 2þ uptake through the MCU with Ru360, we uncovered a persistent efflux of Ca 2þ from mitochondria at [Ca 2þ ] lower than those that induced mPTP opening. The rate of Ca 2þ efflux was proportional to the concentration of added CaCl 2. There was no significant difference in the rate of efflux between Ru360 and ruthenium red. Since we eliminated the contributions of MCU, mRyR, and mNCE, this efflux is likely through mCHE. ADP greatly delayed mPTP opening and significantly (p=0.016) slowed the rate of efflux. CsA delayed opening of mPTP and marginally slowed the rate of efflux. Thus, the rate of Ca 2þ efflux appeared to depend on the matrix Ca 2þ binding capacity, as it slowed slightly in response to CsA and significantly in response to ADP. Our results suggest that mCHE plays a pivotal role in Ca 2þ efflux. Under conditions of increased Ca 2þ , mCHE may facilitate mPTP opening or possibly be a component of mPTP.

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