Reliability of resting metabolic rate between and within day measurements using the Vyntus CPX system and comparison against predictive formulas

Background: To detect longitudinal changes of resting metabolic rate (RMR) resulting from the effects of energetic stress, reliable RMR measurements are crucial. The Vyntus CPX is a new automated indirect calorimetry system for which RMR reliability has not been determined. Additionally, its agreement with common predictive RMR formulas is unknown. Aim: To determine the within and between-day reliability of RMR measurements using the Vyntus CPX system and its agreement with predictive RMR formulas. Methods: Young (31 ± 7 years) healthy participants (n = 26, 12 females, 14 males) completed three measurements of RMR, two consecutive measures on the same day, one the day before/after, all under standardised conditions. Reliability was assessed with pairwise comparisons of between-day at the same time (BDST), within day consecutive measurements (WDCM) and between-day different time (BDDT), for parameters of reliability (mean change (MC), intraclass correlation (ICC) and typical error of measurement (TEM)). Measured RMR values (kcal/day) were compared against predictive values of 4 common formulas. Results: Parameters of reliability (mean, (95% confidence interval)) were: -BDST: MC, 0.2(-2.3—2.7)% (p = 0.67); ICC, 0.92(0.84—0.97); TEM, 4.5(3.5—6.2)%. -WDCM: MC, −2.5(-6.2—1.3)% (p = 0.21); ICC, 0.88(0.74—0.88); TEM, 7.0(5.4—9.8)%. -BDDT: MC, −1.5(-4.8—1.9)% (p = 0.57); ICC, 0.90(0.76—0.95); TEM, 6.1(4.8—8.5)%. RMRratios (measured/predicted) were: 1.04 ± 0.14 (Nelson, p = 0.13), 1.03 ± 0.10 (Mifflin, p = 0.21), 0.98 ± 0.09 (Harris-benedict, p = 0.30), 0.95 ± 0.11 (Cunningham1980, p = 0.01), 1.00 ± 0.12 (Cunningham1991, p = 0.90) and 0.96 ± 0.13 (DXA, p = 0.03). Conclusions: The Vyntus CPX is reliable and measured RMR values agreed with four predictive formulas but are lower than Cunningham1980 and DXA RMR estimates for this population.

Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact coculture conditions, and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased OxPhos inhibition-triggered mitochondrial transfer from MSCs to AML cells. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Tumor burden negatively impacts protein turnover as a proteostatic process in non-cancerous liver, heart, and muscle, but not brain

Cancer survivors are more susceptible to pathologies such as hypertension, liver disease, depression, and coronary artery disease when compared to individuals who have never been diagnosed with cancer. Therefore, it is important to understand how tumor burden negatively impacts non-tumor bearing tissues that may impact future disease susceptibility. We hypothesized that the energetic costs of a tumor would compromise proteostatic maintenance in other tissues. Therefore, the purpose of this study was to determine if tumor burden changes protein synthesis and proliferation rates in heart, brain, and liver. One million Lewis Lung Carcinoma (LLC) cells or Phosphate Buffered Saline (PBS, sham) were injected into the hind-flank of female mice at ~4.5 months of age, and the tumor developed for 3 weeks. Rates of proliferation and protein synthesis were measured in heart, brain, liver, and tumor tissue. Compared to Sham, rates of protein synthesis (structural/nuclear, cytosolic, mitochondrial, and collagen) relative to proliferation were lower in the heart and liver of LLC mice, but higher in the brain of LLC mice. In the tumor tissue the ratio of protein synthesis to DNA synthesis was approximately 1.0 showing that protein synthesis in the tumor was used for proliferation with little proteostatic maintenance. We further provide evidence that the differences in tissue responses may be due to energetic stress. We concluded that the decrease in proteostatic maintenance in liver, heart and muscle might contribute to the increased risk of disease in cancer survivors.

The Phosin PptA Plays a Negative Role in the Regulation of Antibiotic Production in Streptomyces lividans

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

Artificial Diets Modulate Infection Rates by Nosema ceranae in Bumblebees

Parasites alter the physiology and behaviour of their hosts. In domestic honey bees, the microsporidia Nosema ceranae induces energetic stress that impairs the behaviour of foragers, potentially leading to colony collapse. Whether this parasite similarly affects wild pollinators is little understood because of the low success rates of experimental infection protocols. Here, we present a new approach for infecting bumblebees (Bombus terrestris) with controlled amounts of N. ceranae by briefly exposing individual bumblebees to parasite spores before feeding them with artificial diets. We validated our protocol by testing the effect of two spore dosages and two diets varying in their protein to carbohydrate ratio on the prevalence of the parasite (proportion of PCR-positive bumblebees), the intensity of parasites (spore count in the gut and the faeces), and the survival of bumblebees. Overall, insects fed a low-protein, high-carbohydrate diet showed the highest parasite prevalence (up to 70%) but lived the longest, suggesting that immunity and survival are maximised at different protein to carbohydrate ratios. Spore dosage did not affect parasite infection rate and host survival. The identification of experimental conditions for successfully infecting bumblebees with N. ceranae in the lab will facilitate future investigations of the sub-lethal effects of this parasite on the behaviour and cognition of wild pollinators.

Tissue-Specific 1H-NMR Metabolomic Profiling in Mice with Adenine-Induced Chronic Kidney Disease

Chronic kidney disease (CKD) results in the impaired filtration of metabolites, which may be toxic or harmful to organs/tissues. The objective of this study was to perform unbiased 1H nuclear magnetic resonance (NMR)-based metabolomics profiling of tissues from mice with CKD. Five-month-old male C57BL6J mice were placed on either a casein control diet or adenine-supplemented diet to induce CKD for 24 weeks. CKD was confirmed by significant increases in blood urea nitrogen (24.1 ± 7.7 vs. 105.3 ± 18.3 mg/dL, p < 0.0001) in adenine-fed mice. Following this chronic adenine diet, the kidney, heart, liver, and quadriceps muscles were rapidly dissected; snap-frozen in liquid nitrogen; and the metabolites were extracted. Metabolomic profiling coupled with multivariate analyses confirm clear separation in both aqueous and organic phases between control and CKD mice. Severe energetic stress and apparent impaired mitochondrial metabolism were observed in CKD kidneys evidenced by the depletion of ATP and NAD+, along with significant alterations in tricarboxylic acid (TCA) cycle intermediates. Altered amino acid metabolism was observed in all tissues, although significant differences in specific amino acids varied across tissue types. Taken together, this study provides a metabolomics fingerprint of multiple tissues from mice with and without severe CKD induced by chronic adenine feeding.

Structural basis for SARM1 inhibition and activation under energetic stress

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1’s own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.