Tetracyclines, a class of antibiotics that target bacterial translation, are commonly used in research for inducible gene expression using Tet-ON/Tet-OFF systems. However, such tetracycline-inducible systems carry a risk. Given that mitochondria have a “bacterial” ancestry, these antibiotics also target mitochondrial translation and impair mitochondrial function. Indeed, treatment with doxycycline—a tetracycline derivative—disturbs mitochondrial proteostasis and metabolic activity, and induces widespread gene expression changes. Together, this affects physiology in well-established model systems ranging from cultured cells to simple organisms and to mice and plants. These changes are observed with doxycycline doses that are widely used to regulate gene expression. In light of these findings, and bearing in mind the conserved role of mitochondria in metabolism and whole organism homeostasis, we caution against the use of tetracyclines in experimental approaches. The use of newly developed tetracycline-based systems that are more sensitive could be an alternative; however, even if no overt mitochondrial toxicity is detected, widespread changes in gene expression may sensitize cells to the intended tetracycline-controlled loss- or gain-of-function, thereby introducing a “two-hit model”. This is highly relevant for cancer research, as mitochondrial metabolism holds a central position in the reallocation of nutrients for biomass production known as the Warburg effect.

The mechanism of insulin resistance in obesity was examined in ten obese (BMI 33 +/- 1 kg/m2) and nine lean (BMI 22 +/- 1 kg/m2) Caucasian women during a hyperglycemic-hyperinsulinemic clamp using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy to measure rates of muscle glycogen synthesis and intramuscular glucose-6-phosphate (G-6-P) concentrations. Under similar steady-state plasma concentrations of glucose (approximately 11 mmol/l) and insulin (approximately 340 pmol/l), rates of muscle glycogen synthesis were reduced approximately 70% in the obese subjects (52 +/- 8 micromol/[l muscle-min]) as compared with the rates in the lean subjects (176 +/- 22 micromol/[l muscle-min]; P < 0.0001). Basal concentrations of intramuscular G-6-P were similar in the obese and lean subjects; but during the clamp, G-6-P failed to increase in the obese group (deltaG-6-P obese 0.044 +/- 0.011 vs. lean 0.117 +/- 0.011 mmol/l muscle; P < 0.001), reflecting decreased muscle glucose transport and/or phosphorylation activity. We conclude that insulin resistance in obesity can be mostly attributed to impairment of insulin-stimulated muscle glycogen synthesis due to a defect in glucose transport and/or phosphorylation activity.

Mitochondrial function is key for maintaining cellular health, while mitochondrial failure is associated with various pathologies, including inherited metabolic disorders and age‐related diseases. In order to maintain mitochondrial quality, several pathways of mitochondrial quality control have evolved. These systems monitor mitochondrial integrity through antioxidants, DNA repair systems, and chaperones and proteases involved in the mitochondrial unfolded protein response. Additional regulation of mitochondrial function involves dynamic exchange of components through mitochondrial fusion and fission. Sustained stress induces a selective autophagy – termed mitophagy – and ultimately leads to apoptosis. Together, these systems form a network that acts on the molecular, organellar, and cellular level. In this review, we highlight how these systems are regulated in an integrated context‐ and time‐dependent network of mitochondrial quality control that is implicated in healthy aging.

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Activation of brown adipose tissue (BAT) contributes to total body energy expenditure through energy dissipation as heat. Activated BAT increases the clearance of lipids and glucose from the circulation, but how BAT accommodates large influx of multiple substrates is not well defined. The purpose of this work was to assess the metabolic fluxes in brown adipocytes during β3-adrenergic receptor (β3-AR) activation.T37i murine preadipocytes were differentiated into brown adipocytes and we used Seahorse respirometry employing a set of specific substrate inhibitors in the presence or absence of β3-AR agonist CL316,243. The main substrate used by these brown adipocytes were fatty acids, which were oxidized equally during activation as well as during resting condition. [U-13C]-glucose tracer-based metabolomics revealed that the flux through the TCA cycle was enhanced and regulated by pyruvate dehydrogenase (PDH) activity. Based on 13C-tracer incorporation in lipids, it appeared that most glucose was oxidized via TCA cycle activity, while some was utilized for glycerol-3-phosphate synthesis to replenish the triglyceride pool. Collectively, we show that while fatty acids are the main substrates for oxidation, glucose is also oxidized to meet the increased energy demand during short term β3-AR activation. PDH plays an important role in directing glucose carbons towards oxidation.

Expression of the RNA-binding protein Csde1 (Cold shock domain protein e1) is strongly upregulated during erythropoiesis compared to other hematopoietic lineages. Csde1 expression is impaired in the severe congenital anemia Diamond Blackfan Anemia (DBA), and reduced expression of Csde1 in healthy erythroblasts impaired their proliferation and differentiation. To investigate the cellular pathways controlled by Csde1 in erythropoiesis, we identified the transcripts that physically associate with Csde1 in erythroid cells. These mainly encoded proteins involved in ribogenesis, mRNA translation and protein degradation, but also proteins associated with the mitochondrial respiratory chain and mitosis. Crispr/ Cas9-mediated deletion of the first cold shock domain of Csde1 affected RNA expression and/or protein expression of Csde1-bound transcripts. For instance, protein expression of Pabpc1 was enhanced while Pabpc1 mRNA expression was reduced indicating more efficient translation of Pabpc1 followed by negative feedback on mRNA stability. Overall, the effect of reduced Csde1 function on mRNA stability and translation of Csde1-bound transcripts was modest. Clones with complete loss of Csde1, however, could not be generated. We suggest that Csde1 is involved in feed-back control in protein homeostasis and that it dampens stochastic changes in mRNA expression.RNA binding proteins (RBP) regulate transcript stability and translation. RBPs can cooperate with protein complexes of the general mRNA translation machinery, or with protein complexes that control mRNA location and/ or degradation. Deregulated expression of such RBPs affects protein synthesis from a set of transcripts particularly dependent on that specific RBP. This has been referred to as an RNA regulon 1 . The RNA regulon may be cell-type specific, because it depends on the available transcriptome in these cells. The RNA regulon may also define a set of ubiquitously expressed transcripts whose translation is modified by cell type specific expression of RBP. For instance, hematopoietic stem-and progenitor cells (HSPC) express the RBP Musashi-2, which is crucial for maintenance and repopulation ability of the HSPC 2 . The RBP Znf36l2 (Zinc finger protein 36-like 2), a member of the Tristetraproline family, is expressed in early erythroblasts and mediates glucocorticoid-mediated expansion of the erythroid compartment 3 . We observed that the RBP Csde1 is strongly upregulated in expanding erythroblasts (>100-fold) compared to other hematopoietic cell types 4 . In Diamond Blackfan Anemia (DBA), a ribosomopathy due to haploinsufficiency of ribosomal proteins, this upregulation was impaired 4 . This raised our interest in the role of Csde1 in erythropoiesis.Csde1 was first described as Unr (upstream of N-ras) in Drosophila melanogaster 5 . It binds an AG-rich domain in the 3′UTR of Msl (Male sex lethal) and suppresses translation 6,7 . Its expression is highly conserved across species and Csde1 binds its own IRES to repress translation in mammalian cells 8 . Csde1 also ...

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Brown adipose tissue (BAT) catabolizes glucose and fatty acids to produce heat and thereby contributes to energy expenditure. Long-term high-fat diet (HFD) feeding results in so-called ‘whitening’ of BAT characterized by increased lipid deposition, mitochondrial dysfunction, and reduced fat oxidation. The aim of the current study was to unravel the rate and related mechanisms by which HFD induces BAT whitening and insulin resistance. Wild-type mice were fed a HFD for 0, 1, 3, or 7 days. Within 1 day of HFD, BAT weight and lipid content were increased. HFD also immediately reduced insulin-stimulated glucose uptake by BAT, indicating rapid induction of insulin resistance. This was accompanied by a tendency toward a reduced uptake of triglyceride-derived fatty acids by BAT. Mitochondrial mass and Ucp1 expression were unaltered, whereas after 3 days of HFD, markers of mitochondrial dynamics suggested induction of a more fused mitochondrial network. Additionally, HFD also increased macrophage markers in BAT after 3 days of HFD. Counterintuitively, the switch to HFD was accompanied by an acute rise in core body temperature. We showed that a single day of HFD feeding is sufficient to induce the first signs of whitening and insulin resistance in BAT, which reduces the uptake of glucose and triglyceride-derived fatty acids. BAT whitening and insulin resistance are likely sustained by reduced mitochondrial oxidation due to changes in mitochondrial dynamics and macrophage infiltration, respectively. Likely, the switch to HFD swiftly induces thermogenesis in other metabolic organs, which allows attenuation of BAT thermogenesis.