Comprehensive quantification of metabolic flux during acute cold stress in mice

Bornstein, Marc R.; Neinast, Michael D.; Zeng, Xianfeng; Chu, Qingwei; Axsom, Jessie; Thorsheim, Chelsea; Li, Kristina; Blair, Megan C.; Rabinowitz, Joshua D.; Arany, Zoltan

doi:10.1016/j.cmet.2023.09.002

Cited by 20 publications

(12 citation statements)

References 75 publications

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“…This polar extraction method was refined to 40% methanol, 40% acetonitrile, and 20% water with formic acid for enhanced E. coli metabolite extraction [27], but formic acid was later determined to be dispensable [23]. This AMW method is widely used for metabolomics studies (see [38][39][40][41][42][43][44][45][46][47][48][49][50] for a non-exhaustive list) and was the basis of the current approach. However, despite its widespread use, AMW extraction water content varies widely in the literature [16,22,25,[54][55][56][57][58] and has not been systematically evaluated on a whole-metabolome scale.…”

Section: Discussionmentioning

confidence: 99%

“…We hypothesized that increased extraction water content during extraction would improve recovery these compounds. Using cryopulverized and pooled mouse liver tissue as a model system, we extracted metabolites with a widely used extraction approach of 40% acetonitrile, 40% methanol, and 20% water (AMW20) [38][39][40][41][42][43][44][45][46][47][48][49][50]. Then, we performed a secondary addition of water to the crude extract immediately after homogenization to achieve final water content between 25% (AMW25) and 60% (AMW60) in 5% increments (Figure 1A).…”

Section: Metabolomic Responses To Extraction Water Content Are Not Co...mentioning

confidence: 99%

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A diverse proteome is present and enzymatically active in metabolite extracts

House,

Soper-Hopper,

Vincent

et al. 2023

Preprint

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Metabolomics, a foundational tool in metabolism research, relies on the accurate transmittal of biochemical profiles underlying biological phenotypes. Over the years, workflows used in metabolomics have been assumed to remove enzymes to preserve metabolite levels during processing. Here, we uncover a diverse landscape of over 1,000 proteins, strongly enriched for metabolic enzymes, within metabolite extracts generated using common extraction workflows. Moreover, by combining in-extract stable isotope additions and enzyme inhibitors, we demonstrate transaminase activity, which is preventable by protein removal by 3 kDa filtration. We extend these findings to untargeted metabolomics, revealing that both post-extraction formation of glutamate dipeptide and depletion of total glutathione can also be prevented by removing proteins. Finally, we present a simple yet novel workflow that integrates passive filtration for protein removal of crude metabolite extracts as a superior method for broad-coverage metabolomics. Our findings have broad-reaching experimental implications across all fields that use metabolomics and molecular metabolism, especially cancer, immunology, and diabetes research.

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Section: Discussionmentioning

confidence: 99%

Section: Metabolomic Responses To Extraction Water Content Are Not Co...mentioning

confidence: 99%

A diverse proteome is present and enzymatically active in metabolite extracts

House,

Soper-Hopper,

Vincent

et al. 2023

Preprint

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“…Recently, it was shown that cold exposure promotes uptake of BCAAs in BAT and defects in BCAA catabolism lead to impaired thermogenesis 17 . In a separate study 18 , an extensive analysis of how different tissues use fuels during exposure to cold found that although the contribution of BCAAs to the TCA cycle in BAT and WAT increases in fed mice exposed to cold, it never exceeds 2% of the total TCA cycle metabolism. Our results support a transcriptional and functional link between BCAA catabolism and heme biosynthesis in BAT ( Extended Data Figure 8 ).…”

Section: Main Textmentioning

confidence: 96%

“…Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis 17 . However, BCAAs’ contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux 18 . Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.…”

mentioning

confidence: 97%

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue

Duerre,

Hansen,

John

et al. 2023

Preprint

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With age, people tend to accumulate body fat and reduce energy expenditure1. Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles2,3. The activity of brown and beige depots inversely correlates with BMI and age4–11, suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease12–15. Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function16. Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis17. However, BCAAs’ contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux18. Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.

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“…These temperatures also have a practical application to the study of insulin action, glucose metabolism, and EE in mice housed and studied at standard vivaria and laboratory temperatures (~22°C). Moreover, investigating metabolic phenotypes between RT and TN are more relevant to humans than contrasting RT with more common experimental cold exposures of <15°C (22,23). Results show that at TN, mice have decreased insulin action and EE regardless of whether mice are TN-adapted or short-term transitioned from RT-adapted to TN.…”

Section: Introductionmentioning

confidence: 99%

Insulin at the Intersection of Thermoregulation and Glucose Homeostasis

Winn,

Schleh,

Garcia

et al. 2023

Preprint

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Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28°C) and room (laboratory) temperature (RT, ∼22°C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue specific glucose uptake were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, ‘insulin-independent’ glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.HighlightsEnergy expenditure and insulin-mediated glucose fluxes are reduced in thermoneutral (TN)-adapted mice versus room ‘laboratory’ temperature (RT)-adapted mice.Reduced insulin sensitivity manifests in TN mice regardless of whether they are TN-adapted or short-term transitioned from RT-adapted to TN.TN-adapted mice are resistant to the RT-induced increase in whole-body insulin sensitivity even though metabolic rate is increased.TN-adapted mice switched to RT meets increased thermogenic needs, not by increasing glucose uptake, but by partitioning a greater fraction of glucose from glycogen storage to glycolysis.Brown fat glucose uptake sensitively increases with RT and decreases with TN by an insulin-independent mechanism.

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Comprehensive quantification of metabolic flux during acute cold stress in mice

Cited by 20 publications

References 75 publications

A diverse proteome is present and enzymatically active in metabolite extracts

A diverse proteome is present and enzymatically active in metabolite extracts

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue

Insulin at the Intersection of Thermoregulation and Glucose Homeostasis

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