Enhanced fat oxidizing mechanisms -
OXPHOS state respiration was also unaffected by 50 μM nitrate; however, both sGC and PKG inhibition decreased this respiration rate in C2C12 cells, whether supplemented with nitrate or not Fig. At μM, nitrate did enhance palmitoyl-carnitine respiration in the OXPHOS state Fig.
Nitrate enhances fatty acid oxidation capacity via NO-cGMP-PKG in C2C12 myotubes. a Muscle differentiation marker expression in C2C12 myoblasts cultured and differentiated over 6 days; b Muscle differentiation marker expression in C2C12 myocytes cultured and differentiated over 6 days in the presence of 0, 50 and μM nitrate.
See also Additional file 1 : Figure S1. LEAK state respiration of palmitoyl-carnitine and malate substrates in permeabilized C2C12 myotubes cultured and differentiated over 6 days in the presence of c 0 or 50 μM nitrate or d 0 or μM nitrate, in the presence or absence of sGC i ODQ, 1 μM or PKG i KT, 1 μM.
OXPHOS state respiration of palmitoyl-carnitine and malate substrates in permeabilized C2C12 myotubes cultured and differentiated over 6 days in the presence of e 0 or 50 μM nitrate or f 0 or μM nitrate, in the presence or absence of sGC i ODQ, 1 μM or PKG i KT, 1 μM. We proceeded to investigate the effects of nitrate supplementation on the expression of FA oxidation enzymes and other mitochondrial proteins in C2C12 cells.
We found that expression of Cpt1b , Acadl and Hadh encoding CPT1B, long-chain acyl-CoA dehydrogenase and HOAD, respectively , was enhanced by μM, but not 50 μM, nitrate supplementation Fig.
The effects of nitrate were time-dependent, reaching significance after 6 days of differentiation Additional file 2 : Figure S2. Notably, inhibition of sGC Figs. Nitrate enhances the expression of fatty acid oxidation enzymes and other mitochondrial proteins via NO-cGMP-PKG in C2C12 myotubes.
a Cpt1b , Acad1 and Hadh , and b Ucp3 , Cycs and Ndufs1 expression in C2C12 myotubes cultured and differentiated over 6 days in the presence of 0, 50 and μM nitrate, c and d in the presence and absence of sGC i ODQ, 1 μM , and e and f in the presence and absence of PKG i KT, 1 μM.
As before, μM nitrate increased expression of Ucp3 , Acadl and Cpt1b , and a similar increase in expression of all three targets was seen with 1 μM sildenafil alone Fig. As expected, μM nitrate and 1 μM sildenafil in combination, acting to both increase cGMP production and decrease cGMP breakdown, produced a greater increase in expression of all three targets than either treatment alone Fig.
Nitrate and sildenafil cumulatively increase the expression of fatty acid oxidation enzymes and other mitochondrial proteins, and increase CPT1B protein levels. a Ucp3 , Acad1 and Cpt1b expression in C2C12 myotubes differentiated in the presence of μM nitrate, 1 μM sildenafil, or both nitrate and sildenafil in combination.
b CPT1B protein levels in C2C12 cells exposed to μM nitrate, 1 μM sildenafil, nitrate and sildenafil in combination, 1 μM ODQ, and nitrate and ODQ in combination.
Finally, we set out to confirm that the changes in Cpt1b expression were reflected at the protein level. Both μM nitrate and 1 μM sildenafil increased CPT1B protein concentration in C2C12 cells, with a greater increase in CPT1B seen when nitrate and sildenafil were administered in combination Fig.
Meanwhile, treatment with the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalinone ODQ , prevented the effect of nitrate on CPT1B concentration Fig. Herein, we report that moderate doses of dietary inorganic nitrate increase the capacity for FA oxidation in skeletal muscle.
Increased mitochondrial volume density was observed at the highest dose of nitrate used in this study, supporting the capacity of muscle for complete FA oxidation. The underlying mechanism involves enhanced NO bioavailability, which activates sGC to increase muscle cGMP concentrations, subsequently activating PKG and leading to PPARα and at higher doses PGC-1α upregulation Fig.
Indeed, in myocytes, inhibition of sGC or PKG prevented the nitrate-driven upregulation of PPARα-target genes and other mitochondrial proteins. Schematic outlining the proposed mechanism underlying the effect of dietary nitrate on fatty acid FA oxidation and mitochondrial biogenesis in skeletal muscle.
Thus, nitrate dose-dependently increases cellular cGMP, activating protein kinase G. At intermediate doses of nitrate, induction of PPARα augments these changes and increases carnitine levels, further increasing FA oxidation.
At higher doses, increased expression of 3-hydroxyacyl CoA dehydrogenase occurs alongside elevated citrate synthase, indicating mitochondrial biogenesis, which further enhances muscle capacity for FA oxidation.
The excellent agreement in results across different techniques, experimental models and platforms provided a solid foundation for our study and allowed not only a comprehensive description of the effects of nitrate on muscle FA oxidation but also the elucidation of the underlying mechanism.
For instance, the use of isolated rat muscle mitochondria allowed us to consider the changes in intra-mitochondrial FA oxidation pathways, whilst the use of permeabilized muscle fibres, protein levels and enzyme activities provided a picture of FA oxidation capacity at the tissue level.
The finding that nitrate enhanced FA oxidation in cultured myocytes indicates that effects are independent of changes in blood flow, and thus oxygen delivery, secondary to the vasodilatory effects of nitrogen oxides.
As with our previous studies of the effects of dietary nitrate on tissue metabolism and oxygen delivery [ 22 , 23 , 30 ], our use of a SQC diet and deionized water allowed us to acutely manipulate micronutrient concentrations in rats and mice, whilst accurately monitoring nitrate intake without restricting food or water intake.
The nitrate interventions used were relatively short-term in duration 14 — 18 days and although we would expect this to reflect the long-term effects of nitrate supplementation it would be worth confirming this.
Whilst we have shown that nitrate still exerts these effects in hypoxic rats, it would be interesting to additionally investigate and assess the effectiveness of these mechanisms during other metabolic stresses e.
high-fat feeding, diabetes or exercise. The effects of nitrate and hypoxia on FA oxidation and mitochondrial biogenesis appear to be tissue-specific. In the mammalian heart, FA oxidation is the major source of ATP generation under normal conditions [ 32 ], whereas skeletal muscle utilises carbohydrate and FA more evenly [ 33 ], so there is more scope to increase FA oxidation in skeletal muscle.
The relative preservation of FA oxidation in hypoxia may also be a particular feature of the soleus, a highly-oxidative, type 1 muscle, which contrasts with the mixed fibre-type vastus lateralis commonly biopsied for use in human studies.
It is notable, however, that whilst there are variations in mitochondrial function between different muscles of the mouse hindlimb, it is mouse soleus that most closely resembled human quadriceps in terms of the coupling control of electron transport during FA oxidation [ 38 ].
It is possible that hypoxia systemically augments the production of NO from dietary nitrate by sequential reduction, with the expression and nitrate reductase activity of xanthine oxidoreductase increasing under hypoxic conditions [ 39 ].
The increased availability of NO in low oxygen conditions may, however, be offset in skeletal muscle and heart, by the HIF-dependent downregulation of PPARα and PGC-1α [ 34 ], preventing downstream metabolic effects. Indeed, in the absence of hypoxia, there are striking parallels between the effects of nitrate on rat WAT and skeletal muscle, particularly regarding the dose-dependency of the changes observed.
The dose dependent effects of nitrate we report herein may be attributable to differential effects on the expression of PPAR isoforms and PGC-1α. Whilst this dose was not associated with a significant increase in mitochondrial palmitoyl-carnitine respiration or muscle 14 C-palmitate oxidation ex vivo, we saw decreased long-chain FAs in the muscles of these rats, suggesting more subtle effects on in vivo FA oxidation at this dose.
Whilst there was no increase in citrate synthase activity at this dose, PGC-1α levels were increased. Alongside an increase in mitochondrial density at the highest dose, the elevated PPARα levels supported the greatest rate of 14 C-palmitate oxidation and resulted in the lowest levels of total long-chain FAs, although, interestingly, mitochondrial palmitoyl-carnitine respiration representing β-oxidation was no higher than at the medium dose.
These findings show similarities with high-fat fed mice that have undertaken exercise-training to elevate PGC-1α expression [ 14 ]. There was surprisingly good agreement between the effects of nitrate at these doses between rats and mice in the present study. The intermediate dose of nitrate used herein matches that shown to have metabolic effects in studies of human muscle [ 44 ] and which can be achieved in humans via a slight modification of the diet, e.
via a modest increase in consumption of leafy green vegetables such as spinach and beetroot [ 45 ]. In humans, this dose was found to decrease the oxygen cost of exercise [ 46 ], improve mitochondrial efficiency via a lowering of LEAK state respiration [ 44 ], and to lower whole-body resting metabolic rate [ 45 ].
These effects were associated with decreased muscle expression of the putative uncoupler adenine nucleotide translocase, but also a strong trend towards decreased UCP3 expression [ 44 ].
Our findings in rodents do seem to contradict some of these effects in humans. However, beyond the obvious difference in species, some other key differences between the studies should be noted, along with some similarities.
Firstly, our rats were maintained on a standardised version of normal rat chow, which is low in fat content, whereas the humans essentially consumed a regular diet, albeit avoiding foodstuffs high in nitrate [ 44 , 45 ].
Therefore, rats would have conceivably had a greater scope to increase their capacity for FA oxidation compared to humans as PPAR activation via the diet would have been lower at baseline, whilst the rats were also sedentary and would therefore be expected to have relatively low muscle mitochondrial contents at baseline.
In addition, the mitochondrial respiration studies of human vastus lateralis employed pyruvate and malate as substrates [ 44 ], as opposed to the FA derivative palmitoyl-carnitine that we used here, and as such they were not optimised to see differences in FA oxidation. Finally, we have not measured whole-body oxygen consumption in these rats, and it would be interesting to do so, but previous work from our group with the livers of rats receiving the same doses of nitrate showed a dose-dependent fall in HIF-1 activation, suggesting a better matching of oxygen supply and demand despite decreased circulating haemoglobin, and thus possibly indicating decreased oxygen consumption or improved mitochondrial efficiency, at least in this tissue [ 30 ].
Our finding of enhanced skeletal muscle FA oxidation capacity with moderate consumption of dietary nitrate now deserves further investigation in healthy human subjects, as well as those with metabolic disease, with further investigation in animal models of metabolic syndrome.
In combination with our previous finding that nitrate stimulates the browning of WAT, however, our work suggests that nitrate supplementation holds promise as a potential therapy for metabolic disease. We have found that a moderate dose of dietary nitrate enhances skeletal muscle FA oxidation capacity by promoting intra-mitochondrial pathways of FA oxidation and, at higher doses, mitochondrial biogenesis.
The underlying mechanism is dose-dependent, and occurs via enhanced NO bioavailability and increased muscle cGMP levels, leading to the upregulation of PPARα and PGC-1α. All procedures involving live animals were carried out by a licence holder in accordance with UK Home Office regulations, and underwent review by the University of Cambridge Animal Welfare and Ethical Review Committee.
Some, but not all, of the rats used in this work were also used in separate, but parallel studies of oxygen consumption by other tissues [ 22 , 23 ] and oxygen delivery [ 30 ], with a view to reducing the total numbers of animals used in accordance with UK Home Office best practice.
Where relevant data e. nitrate intakes and plasma [NO x ] has been reported previously, we refer to the previous paper and thus have not duplicated data. After 12 days acclimatization, animals received either 0. After 12 days acclimatization, rats received distilled water or water supplemented with NaNO 3 0.
Differentiation medium was changed daily for the first 3 days, and every 12 h for the remaining 3 days. After 2 days of differentiation, cells were also supplemented with 0, 50 or μM nitrate. In inhibitor experiments, cells were additionally incubated with either 1 μM ODQ sGC inhibitor for the final 2 days of differentiation, 1 μM KT PKG inhibitor; Santa Cruz Biotechnology Inc.
Cells were lysed with 1 mL radio-immunoprecipitation buffer Sigma-Aldrich supplemented with protease inhibitor Complete mini protease inhibitor cocktail, Roche, Germany and the protein concentration quantified using the Pierce BCA Protein Assay kit Thermo Scientific.
For respirometry analysis, the medium was removed and 0. From the microcentrifuge tubes, μL of solution were added to μL PBS and the cell number counted. Nitrate was quantified using a dedicated HPLC system ENO, Eicom; Tokyo, Japan , employing sequential ion chromatography, online reduction to nitrite using a cadmium column, and post-column-derivatization with a modified Griess reagent, as described in previously published protocols [ 47 ].
Aqueous and organic metabolites were extracted from soleus muscle as described previously [ 23 ]. Aqueous extracts were reconstituted in μL acetonitrile:water before metabolites were measured using hydrophilic interaction liquid chromatography-mass spectrometry.
Multiple reaction monitoring in positive ion mode was used with the following optimised mass transitions: malonyl-CoA, Malonyl-CoA, cGMP, cAMP, AMP, ADP and ATP had separately optimised declustering potentials and collision energies as follows: 41 V, 23 eV; 81 V, 41 eV; 71 V, 31 eV; Other optimisation parameters common to each species were ion spray voltage 5.
A 5-μL injection volume was analysed on a QTRAP mass spectrometer AB Sciex, Toronto, Canada attached to an Acquity ultra-performance liquid chromatography system UPLC; Waters Corporation, MA, USA. The column was re-equilibrated for a further 3 min at cessation of the gradient.
Malonyl-CoA was normalised to a universally 13 C- and 15 N-labelled glutamate internal standard Cambridge Isotope Laboratories Inc. cGMP data were normalised to the same internal standard and compared with an 8-point cGMP calibration line with concentrations ranging from 1 nM to 50 μM.
cAMP data were normalised to the same internal standard and compared with an 8-point cAMP calibration line with concentrations ranging from 1 nM to 50 μM. Organic extracts were reconstituted in μL chloroform:methanol and mixed thoroughly.
When fully dissolved, μL of each sample were transferred to a 3. This derivatisation procedure converts FAs to fatty acid methyl esters. The vials were then vortexed, and incubated for 90 min at 80 °C. After cooling, μL HPLC-grade water and 1 mL hexane were added, and the vials thoroughly mixed by vortexing.
The upper, organic, layer was transferred to a 2 mL GC vial and allowed to dry overnight in a fume hood. FAME samples were reconstituted in μL hexane and a 2 μL injection run on a Trace GC Ultra coupled to a Trace DSQ II single quadrupolar mass spectrometer Thermo Scientific.
The transfer line from the oven to the mass spectrometer was heated to °C and the inlet to °C. Chromatograms were processed using the Xcalibur software suite version 2.
Individual peaks were integrated and subsequently normalised to total peak intensity. Peaks were assigned based on fragmentation patterns and matched to the National Institute of Standards and Technology NIST, USA library.
Respirometry was performed on 2—5 mg saponin- permeabilized soleus muscle fibre bundles at 37 °C using Clark-type oxygen electrodes Strathkelvin Instruments, Strathkelvin, UK , essentially as described [ 23 ]. Briefly, 0. Muscle fibres were recovered from the electrode chamber to allow normalisation of oxygen consumption rates to dry muscle mass.
For C2C12 studies, cells suspended in μL of respiration medium were added to the Clark-type electrode chambers for analysis, along with 0. Mitochondria were isolated from soleus muscle from rats according to published protocols [ 48 ].
CPT1 activity was determined in soleus muscle mitochondrial isolates using 3 H-carnitine according to previously published protocols [ 49 ]. The assay buffer contained mM Tris—HCl, 0.
The reaction was initiated with the addition of 20 μL of mitochondrial homogenates and incubated for 8 min at 37 °C. The reaction was then terminated by the addition of 60 μL of HCl.
The palmitoyl- 3 H-carnitine formed during the reaction was separated according to previously published protocols [ 50 , 51 ] and the radioactivity counted to determine CPT1 activity. Palmitate oxidation rates were measured in soleus muscle mitochondrial isolates using 14 C-palmitate according to previously published methods [ 52 , 53 ].
Briefly, modified Krebs-Ringer buffer mM NaCl, 2. A suspended microcentrifuge tube containing μL of benzethonium hydroxide were placed inside the vial to trap the 14 CO 2 produced during the reaction. Mitochondria were added to the system, which was then sealed with a rubber cap.
The reaction was initiated by the addition of palmitate:BSA complex containing 1 μCi of 14 C-palmitate to a final palmitate concentration of μM via a syringe through the rubber cap.
The vial was incubated for 30 min at 37 °C before termination with 50 μL of HClO 4. The microcentrifuge tube containing the benzethonium hydroxide and trapped 14 CO 2 was then removed, transferred to a scintillation vial, and the radioactivity counted.
Frozen tissues were powdered under liquid nitrogen with a mortar and pestle and homogenized in potassium phosphate buffer mM KH 2 PO 4 , 5 mM EDTA, 0.
HOAD activity was assayed according to a published protocol [ 54 ]. The assay buffer contained 50 mM imidazole pH 7. NADH absorbance was monitored at nm for 3 min.
CS was assayed according to a previously published protocol [ 55 ]. The assay buffer contained 20 mM Tris pH 8. The reaction was initiated by the addition of 0. Frozen tissue was crushed using a liquid nitrogen-cooled pestle and mortar, and homogenates prepared from which the nuclear subcellular fraction was isolated using a commercial kit Cayman Chemical Company, MI, USA.
Ice-cold hypotonic buffer was added to crushed tissue in a 5 μL:1 mg ratio. The solution was homogenised using a polytron before being incubated on ice for 15 min. The pellet was resuspended in μL hypotonic buffer and incubated on ice.
The pellet was resuspended in 50 μL ice-cold extraction buffer, vortexed vigorously for 15 s and subsequently rocked on a shaking platform for 15 min on ice. A small aliquot was used to quantify protein concentration using a spectrophotometer. The wells of a plate were coated by a double-stranded DNA dsDNA sequence containing the peroxisome proliferator response element.
By utilising nuclear extracts, only binding of proteins within the nucleus are quantified, which effectively represents activated PPARs.
Nuclear extracts and all kit reagents were allowed to equilibrate to room temperature before use. To the blank and non-specific binding wells μL of transcription factor assay buffer were added.
In the competitor dsDNA wells, 80 μL transcription factor assay buffer were added followed by 10 μL PPAR competitor dsDNA.
In the positive control wells, 90 μL of transcription factor assay buffer were added followed by 90 μL of positive control. Finally, 90 μL of transcription factor assay buffer were added to each sample well followed by 10 μL nuclear extract.
The plate was covered and incubated overnight at 4 °C without agitation. The wells were then emptied and washed five times with μL of wash buffer, with care taken on the final wash to remove residual buffer. The wash step was repeated as above and any residual wash buffer carefully removed.
To each well, except the blanks, μL of horseradish peroxidase HRP -conjugated secondary antibody were added and the plate covered and incubated for 1 h at room temperature without agitation.
The wells were washed with μL of wash buffer as above, before μL of developing solution was added and the plate incubated for 30 min at room temperature with gentle shaking.
After incubation, μL stop solution were added to each well and the absorbance read at nm within 5 min. RNA concentration was quantified at nm using a SmartSpecPlus spectrophotometer Bio-Rad. For analysis of steady-state mRNA levels, the relative abundance of transcripts of interest was assessed by quantitative-PCR in SYBR Green FastStart Universal Master Mix Applied Biosystems with a StepOnePlus detection system Applied Biosystems.
QuantiTect primer assays for rat Ppara and Pparbd were obtained from QIAgen. For gene analysis in C2C12 cells, RNA extraction was performed as above, with the single difference being that μL of lysis buffer were added directly to the wells, and subsequently pipetted onto the spin columns for purification.
Production of cDNA and RT-qPCR analysis of Myod , Tnni1 , Tnni2 , CptIb , Acadl , Hadh , Ucp3 , Cycs , and Ndufs1 expression proceeded as above, using QuantiTect primer assays obtained from QIAgen. Immunoblotting for citrate synthase, PGC-1α and malonyl-CoA decarboxylase was performed on soleus muscle lysates according to published protocols [ 56 ].
Membranes were incubated in primary antibody solution containing rabbit polyclonal IgG raised against CS, PGC-1α or MCD all Abcam, UK for 2 h at room temperature CS and PGC-1α or overnight at 4 °C MCD.
After washing with TBS-T for 2 h with a solution change every 15 min, membranes were incubated in secondary antibody solution containing goat anti-rabbit IgG, conjugated to HRP for 1 h, before visualisation using ECL-plus and quantification as previously described [ 56 ].
Samples diluted 20 times and standards were incubated in duplicate on a well plate containing an immobilised antibody to mouse CPT1B for 2 h at 37 °C.
After unbound substances were washed away, biotin conjugated to a secondary antibody raised against CPT1B was added, and the plate incubated for 1 h at 37 °C. After further washes, a streptavidin-HRP conjugate was added and incubated for 1 h at 37 °C, followed by further washes to remove unbound conjugate.
A substrate solution was then added to the wells and incubated, protected from light, for 30 min at 37 °C. Diluted HCl was added to stop the enzymatic reaction, and finally the optical density was measured at nm with readings at nm subtracted to account for optical imperfections in the plate.
Data were collated and normalised to total protein in the original sample. Analysis of variance ANOVA was used to determine significant differences across the four groups of the hypoxia and dose—response studies. Data were collated in Excel before 1- or 2-way analysis of variance ANOVA was used to determine significant differences across experimental groups Graphpad, Instat.
Bonferroni post-hoc analysis was used for multiple analysis of selected groups, where appropriate. All data supporting the results of this article are available in an online Additional file 3.
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Jacobs RA, Diaz V, Meinild AK, Gassmann M, Lundby C. Jansson EA, Huang L, Malkey R, Govoni M, Nihlen C, Olsson A, et al. A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis. Aleshin S, Grabeklis S, Hanck T, Sergeeva M, Reiser G. Surprisingly, activated brown adipocytes also take up large amounts of glucose from the circulation while primarily utilizing FA to fuel thermogenesis 19 , 41 — Therefore, it has been suggested that, while oxidizing FA to fuel thermogenesis, brown adipocytes concurrently increase glycolysis and de novo FAS to replenish intracellular TAG pool in lipid droplets 15 — However, it is currently unclear how brown adipocytes concurrently perform FAO and FAS in the same cell because these processes are two mutually exclusive pathways in healthy cells.
More interestingly, recent studies have shown that cold-activated BAT in rodents and humans utilizes additional substrates such as branched-chain amino acids BCAA 50 , 51 , glutamate 44 , and succinate 52 to support thermogenesis.
Extracellular succinate contributes to thermogenic respiration in BAT by the succinate dehydrogenase SDH -mediated oxidation in the TCA cycle 52 , although its relative contribution to thermogenesis is unclear.
It is also possible that carbons from these additional substrates replenish TCA cycle intermediates that leave the cycle for biosynthetic pathways e. In most mammalian cells, mitochondrial FAO suppresses glycolysis, pyruvate oxidation, and de novo FAS The resulting decrease in acetyl-CoA reduces the production of pyruvate-derived citrate that exits the mitochondria to serve as the precursor for FAS.
Thus, FAO-dependent inhibition of PDH activity in the mitochondria is the primary mechanism preventing both pyruvate oxidation and de novo FAS from glucose.
Additionally, FAO can inhibit glycolysis. A portion of excess citrate produced from FA-derived acetyl-CoA is exported to the cytosol, where it in turn inhibits glycolytic enzymes, such as phosphofructokinases PFK1, PFK2 and pyruvate kinase PK 20 , 55 — Conversely, when extracellular glucose increases, enhanced glycolysis provides more pyruvate to the mitochondria.
The conversion of pyruvate to acetyl-CoA by PDH and to OAA by pyruvate carboxylase PC increases citrate production in the mitochondria.
Under high glucose, excess citrate is exported to the cytosol and hydrolyzed back to acetyl-CoA and OAA by ATP-citrate lyase ACLY. Acetyl-CoA is then carboxylated to malonyl-CoA by two acetyl-CoA carboxylases, ACC1 and ACC2 Malonyl-CoA is the precursor of de novo synthesized FA.
Remarkably, malonyl-CoA produced by ACC2 allosterically inhibits CPT1 59 , 60 that controls the entry of long-chain fatty acids from the cytosol into mitochondria. By this mechanism, glucose-derived malonyl-CoA prevents the oxidation of newly synthesized and pre-existing FAs.
Thus, malonyl-CoA is a key metabolite regulating the balance between FAS and FAO. ACC1 is cytosolic and directs malonyl-CoA toward de novo FA synthesis catalyzed by fatty acid synthase FAS. In contrast, ACC2 is associated with the OMM and regulates FAO through malonyl-CoA-mediated CPT1 inhibition 59 — 61 , While lipogenic WAT predominantly expresses ACC1, BAT expresses similar amounts of ACC1 and ACC2 In addition, BAT expresses CPT1β, an isoform with high sensitivity to malonyl-CoA 65 — Despite the expression of ACC2 and CPT1β, BAT mitochondria have the highest CPT1 activity among the tissues expressing CPT1β 65 — High FAO in BAT is surprising in light of the inhibitory effect of malonyl-CoA produced by ACC2 on CPT1β-mediated FA transport.
It is unclear whether ACC2 activity or association to the mitochondria is negatively regulated by cold in brown adipocytes. It is interesting to note that concurrent FAO and FAS have also been observed in a subset of cancer cells 68 — Glycolytic colorectal cancer cells recruit FAO as an adaptive response to extracellular acidification associated with increased pyruvate to lactate conversion A selective decrease in the transcription of ACACB gene under acidosis was in part the mechanism permitting mitochondrial FAO.
However, it is unlikely that the selective decrease in ACACB gene expression provides a mechanism by which brown adipocytes concurrently perform FAO and FAS because BAT upregulates the expression of both ACACA and ACACB genes encoding ACC1 and ACC2, respectively, in response to cold As another example, a subset of highly proliferating B-cell lymphomas concurrently stimulates mitochondrial FAO while increasing glycolysis and FAS 69 ; however, the underlying mechanism remains unknown.
Single-cell and single-nucleus RNA sequencing of BAT has uncovered the existence of multiple brown adipocyte subpopulations with large variability in their transcriptomes and with different degrees of thermogenic capacities 71 — Compared with the high-thermogenic brown adipocytes, low-thermogenic brown adipocytes express lower levels of Ucp1 along with reduced mitochondrial respiration The co-existence of functionally different brown adipocytes within the BAT may in part explain how BAT performs FAO and FAS simultaneously.
Further studies are required to delineate the location, functional specialization, and substrate utilization of these brown adipocyte subpopulations and their ratios in response to environmental stimuli.
In addition to heterogeneity of brown adipocytes, recent studies have demonstrated the presence of metabolically distinct populations of mitochondria within the same brown adipocyte: cytosolic mitochondria CM and peridroplet mitochondria PDM 74 — PDM are found to be anchored to the lipid droplets and have reduced motility and fusion-fission dynamics that segregate PDM from the rest of the mitochondrial population 79 , While CM preferentially oxidize FA for theromgenesis, PDM have a higher capacity for pyruvate oxidation and ATP synthesis 74 Figure 2.
In line with increased oxidative phosphorylation, PDM is enriched with ATP synthase compared to CM 74 although UCP1 levels are comparable in PDM and CM 74 , More interestingly, an increase in PDM is associated with lipid droplet expansion in brown adipocytes Given that coupled respiration is dependent on ADP availability, excess citrate produced from pyruvate-derived acetyl-CoA in the PDM may exit the mitochondria and be converted to malonyl-CoA by ACC1 and ACC2, thus contributing to de novo FAS for TAG synthesis and concurrently preventing FA entry into these specific subpopulations of mitochondria Figure 2.
On the contrary, in CM preferentially oxidizing FA 74 , FA-derived acetyl-CoA could inhibit PDH activity, resulting in a decrease in pyruvate-derived citrate production and subsequent malonyl-CoA accumulation in close vicinity of CM Figure 2.
It is unclear whether there is a difference in ACC2 levels between PDM and CM. CM could maximize UCP1-mediated thermogenesis by producing high levels of NADH and FADH 2 from FAO.
The resulting rapid oxidation of FA-derived citrate through the TCA cycle may prevent citrate export to the cytosol for inhibition of glycolytic enzymes. Figure 2 The co-existence of two functionally different mitochondria within the brown adipocyte. A scheme of two types of mitochondria identified in the brown adipocyte: cytosolic mitochondria CM and peridroplet mitochondria PDM 74 — PDM are anchored to the lipid droplets and segregated from the pool of CM.
CM preferentially oxidize FA and are more thermogenic compared to PDM. FA-derived citrate would be rapidly oxidized through the TCA cycle to support UCP1-mediated thermogenesis.
On the contrary, PDM have a higher capacity for pyruvate oxidation and ATP synthesis compared to CM. Given that coupled respiration is dependent on ADP availability, excess citrate would escape from the mitochondria and be converted to malonyl-CoA by ACC1 and ACC2, thus contributing to de novo lipogenesis and simultaneously preventing CPT1β-mediated FA entry into PDM.
The co-existence of two functionally different mitochondria within the brown adipocyte may in part explain the concurrence of glycolysis, FA synthesis, and FA oxidation in brown adipocytes. FA, fatty acids; ETC, electron transport chain; CPT, carnitine palmitoyltransferase; UCP1, uncoupled protein 1; TCA, the tricarboxylic acid cycle; OAA, oxaloacetate; MPC, mitochondrial pyruvate carrier; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; ACLY, ATP-citrate lyase; ACC, acetyl-CoA carboxylases; FAS, fatty acid synthase; TAG, triacylglycerol.
In contrast to the lipogenic role of PDM in brown adipocytes, several studies reported conflicting results that PDM promotes the oxidation of FA released from lipid droplets 77 , This discrepancy may imply that the role of PDM is differently regulated by the cell type, nutritional status, or cellular stress.
Proteome profiling of PDM and CM in BAT has identified a subset of mitochondrial proteins differentially expressed between PDM and CM although their impact on the functional difference has not been explored Additional studies are required to quantitatively characterize PDM and CM mitochondrial proteins e.
Brown adipocytes have two unique features: 1 UCP1-mediated dissipation of the PMF, which provides a mechanism for maximal substrate oxidation in the mitochondria and 2 concurrence of glycolysis, de novo FAS, and FAO.
Upon activation, brown adipocytes increase glycolysis and de novo FAS to replenish intracellular TAG pools that are depleted due to increased lipolysis and FAO. The co-existence of FA-oxidizing and lipogenic mitochondria within the brown adipocyte in addition to heterogeneity of brown adipocytes may in part explain the unique capacity of brown adipocytes to be involved simultaneously in FAO and FAS.
The author confirms being the sole contributor of this work and has approved it for publication. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Cannon B, Nedergaard J. Brown adipose tissue: Function and physiological significance.
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