Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome, between-genotype metabolite level differences, and 13C-tracing enrichments

OBJECTIVE: Metabolomics as an approach to solve biological problems is exponentially increasing in use. Thus, this a pivotal time for the adoption of best practices. It is well known that disrupted tissue oxygen supply rapidly alters cellular energy charge. However, the speed and extent to which delayed mouse tissue freezing after dissection alters the broad metabolome is not well described. Furthermore, how tissue genotype may modulate such metabolomic drift and the degree to which traced METHODS: By combined liquid chromatography (LC)- and gas chromatography (GC)-mass spectrometry (MS), we measured how levels of 255 mouse liver metabolites changed following 30-second, 1-minute, 3-minute, and 10-minute freezing delays. We then performed test-of-concept delay-to-freeze experiments evaluating broad metabolomic drift in mouse heart and skeletal muscle, differential metabolomic change between wildtype (WT) and mitochondrial pyruvate carrier (MPC) knockout mouse livers, and shifts in RESULTS: Our data demonstrate that delayed mouse tissue freezing after dissection leads to rapid hypoxia-driven remodeling of the broad metabolome, induction of both false-negative and false-positive between-genotype differences, and restructuring of CONCLUSIONS: Our findings provide a previously absent, systematic illustration of the extensive, multi-domain metabolomic changes occurring within the early minutes of delayed tissue freezing. They also provide a novel, detailed resource of mouse liver ex vivo, hypoxic metabolomic remodeling

The mitochondrial pyruvate carrier mediates high fat diet-induced increases in hepatic TCA cycle capacity

Objective: Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. The goal of this investigation was to test whether hepatocyte MPC disruption provides sustained protection from hyperglycemia during long-term HFD and the differential effects of hepatocyte MPC disruption on TCA cycle metabolism in NCD versus HFD conditions. Method: We utilized long-term high fat feeding, serial measurements of postabsorptive blood glucose and metabolomic profiling and 13C-lactate/13C-pyruvate tracing to investigate the contribution of the MPC to hyperglycemia and altered hepatic TCA cycle metabolism during HFD-induced obesity. Results: Hepatocyte MPC disruption resulted in long-term attenuation of hyperglycemia induced by HFD. HFD increased hepatic mitochondrial pyruvate utilization and TCA cycle capacity in an MPC-dependent manner. Furthermore, MPC disruption decreased progression of fibrosis and levels of transcript markers of inflammation. Conclusions: By contributing to chronic hyperglycemia, fibrosis, and TCA cycle expansion, the hepatocyte MPC is a key mediator of the pathophysiology induced in the HFD model of T2D

Mitochondrial citrate metabolism and efflux regulate BeWo differentiation

Abstract Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established BeWo cell culture model of trophoblast differentiation. Differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation

Deficiency of endothelial sirtuin1 in mice stimulates skeletal muscle insulin sensitivity by modifying the secretome

Abstract Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tβ4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tβ4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells

Organelle interactions compartmentalize hepatic fatty acid trafficking and metabolism

Summary: Organelle interactions play a significant role in compartmentalizing metabolism and signaling. Lipid droplets (LDs) interact with numerous organelles, including mitochondria, which is largely assumed to facilitate lipid transfer and catabolism. However, quantitative proteomics of hepatic peridroplet mitochondria (PDM) and cytosolic mitochondria (CM) reveals that CM are enriched in proteins comprising various oxidative metabolism pathways, whereas PDM are enriched in proteins involved in lipid anabolism. Isotope tracing and super-resolution imaging confirms that fatty acids (FAs) are selectively trafficked to and oxidized in CM during fasting. In contrast, PDM facilitate FA esterification and LD expansion in nutrient-replete medium. Additionally, mitochondrion-associated membranes (MAM) around PDM and CM differ in their proteomes and ability to support distinct lipid metabolic pathways. We conclude that CM and CM-MAM support lipid catabolic pathways, whereas PDM and PDM-MAM allow hepatocytes to efficiently store excess lipids in LDs to prevent lipotoxicity