C. Lelliott, G. Medina-Gomez, M. Snaith, B. Cannon, and A. Vidal-Puig conceived and designed the experiments. C. Lelliott, G. Medina-Gomez, N. Petrovic, A. Kis, H. Feldmann, M. Bjursell, N. Parker, M. Campbell, P. Hu, D. Zhang, S. Litwin, V. Zaha, K. Fountain, S. Boudina, M. Jimenez-Linan, M. Blount, M. Lopez, A. Meirhaeghe, M. Bohlooly-Y, and M. Strömstedt performed the experiments. C. Lelliott, G. Medina-Gomez, N. Petrovic, A. Kis, H. Feldmann, N. Parker, K. Curtis, K. Fountain, S. Boudina, M. Jimenez-Linan, M. Blount, M. Orešič, E. Abel, B. Cannon, and A. Vidal-Puig analyzed the data. K. Curtis, M. Bohlooly-Y, M. Snaith, and M. Orešič contributed reagents/materials/analysis tools. C. Lelliott, G. Medina-Gomez, M. Snaith, E. Abel, B. Cannon, and A. Vidal-Puig wrote the paper.
The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) has been implicated in important metabolic processes. A mouse lacking PGC-1β (PGC1βKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1βKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1β ablation was partially compensated by up-regulation of PGC-1α in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1βKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1β was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1βKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1βKO mice have impaired mitochondrial function. Lack of PGC-1β also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1β plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.
The authors conduct an in-depth analysis of a PGC-1β knockout mouse; these animals posses specific defects in basal mitochondrial function and adaptation to metabolic stress.
Transcriptional coactivators (TCs) have emerged as key proteins that are capable of regulating entire metabolic networks [
Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α, previously known as PGC-1) [
The gene expression level of PRC is conserved between tissues [
Overexpression studies both in vivo and in vitro have revealed that both PGC-1α and −1β are capable of stimulating mitochondrial biogenesis, increasing mitochondrial oxygen consumption, and elevating gene expression of fatty acid oxidation and mitochondrial electron transport chain (ETC) components [
Despite their functional similarities, it has recently been suggested that PGC-1α and −1β may have differing metabolic roles in liver. PGC-1α stimulates expression of gluconeogenic genes both in vivo and in vitro [
To investigate the function of PGC-1β, we have generated and phenotyped a mouse model lacking PGC-1β (the PGC1βKO mouse). Using a combination of physiological experiments guided by a systems biology approach, we showed that PGC-1β plays a general role in regulating the mitochondrial activity in tissues with active oxidative metabolism. In all tissues studied, lack of PGC-1β was associated with a reduction in the expression of mitochondrial genes, including intermediary subunits and enzymes connected to ETC and oxidative phosphorylation (OxPhos). Despite these mitochondrial defects, the PGC1βKO mouse was able to cope when challenged with a range of physiological stressors including cold acclimatisation and adrenergically-mediated BAT and cardiac stimulation but with abnormal compensatory responses. Altogether, these data suggest that PGC-1β is an important regulator of basal energy homeostasis and essential for the proper metabolic tuning in acute stress situations in multiple organs.
A conditional PGC1βKO was generated using a triple LoxP targeting vector (
(A) Design of the targeting construct for the generation of the PGC1βKO mouse using a phosphoglycerate kinase-neomycin phosphotransferase (PGK-Neo)–based LoxP cassette inserted between exons III and IV and a third LoxP site between exons V and VI. Total collapse of the LoxP sites deletes exons 4 and 5 of the
(B) A Southern blot confirming the generation of the targeted allele in ES cells. WT, wild type ES cells; +/T, ES cells heterozygous for the targeted allele, T.
(C) Schematic showing the restriction digest strategy for the southern blot confirming the generation of the targeted allele in ES cells. P, PsiI; X, XmaI; S, SpeI.
(D) Confirmation of the total LoxP collapse and generation of KO mice by PCR from genomic DNA using primers F1, R1, and R2 shown in (A).
No differences in food intake, total energy intake, total energy output as faeces, and no change in the ratio of energy in (as food) to energy out (as faeces) (
Assessment of Energy Expenditure on 9-wk-Old Male Chow-Fed PGC1βKO and WT Mice
(A) Growth curves of male (left panel) and female (right panel) mice on normal diet. WT (solid circles) and PGC1βKO (open circles) mice,
(B) Assessment of fat content by DEXA in 8- and 32-wk-old male WT (solid bars) and PGC1βKO mice (open bars),
(C) Representative histological sections of tissues from WAT (
(D) Size distribution of adipocytes from WT and PGC1βKO mice. Two fields from each section from epididymal adipose tissue depot (
(E) Epididymal WAT gene expression from 12-wk-old PGC1βKO (white bars) and WT littermates (black bars). Individual measurements are standardized using 18S, and then the average of the WT group was set to 1.
Body composition analysis using dual energy x-ray absorptiometry (DEXA) confirmed decreased body weight at the expense of fat mass rather than lean mass in both young (8-wk) and older (27-wk) male PGC1βKO mice compared to WT littermates (
Gene expression analysis of gonadal WAT from male PGC1βKO mice and WT littermates showed no significant change in markers of adipogenesis including C/EBPα, PPARγ2,
Ablation of PGC-1β did not result in differences in basal levels of plasma glucose or insulin. Similarly, glucose tolerance tests and insulin tolerance tests (GTTs and ITTs, respectively) performed on 16-wk male and female mice either on chow or after a 13-wk HFD did not reveal differences among genotypes (
Examination of BAT in chow-fed 12-wk-old PGC1βKO male mice and WT littermates showed major changes in the expression of genes involved in intermediary metabolism (
Expression levels of mRNA were assessed on interscapular BAT from 12-wk-old male WT (black bars) and PGC1βKO (white bars) mice. Individual measurements are standardized using 18S, and the average of the WT group set to 1.
(A and B) Expression levels of (A) nuclear-encoded and (B) mitochondrially encoded genes were assessed on interscapular BAT from 12-wk-old male WT (black bars) and PGC1βKO (white bars) mice. Individual measurements are standardized using 18S, and the average of the WT group set to 1.
(C and D) BAT protein levels of ETC and OxPhos components from 15-wk-old female mice were assessed by western blotting of samples from (C) tissues and (D) mitochondrial fractions. WT, black bars and PGC1βKO, white bars. Complex I, α-subcomplex 9 (α-s9); complex II, succinate dehydrogenase subunit B (SDHB); complex III, Fe-S core protein (Fe-S); complex IV, Cox4; complex V, ATP synthase subunit β (ATPβ).
(E) Representative electron micrographs from BAT of WT (left panel) and PGC1βKO (right panel) mice.
A stepwise acclimatisation to cold exposure (4 °C) and towards thermoneutrality (30 °C) was performed on male mice that were 16 wk old at the end of the 3-wk cold-acclimatisation process. The rationale for these experiments was to identify the contribution of PGC-1β to energy expenditure in these two directly opposing thermogenic scenarios. PGC1βKO mice survived at 4 °C after this acclimatisation process. BAT pad weight was similarly increased by cold-acclimatisation in both WT (BAT weight in mg for 30 °C versus 4 °C: WT 117.8 ± 7.1 versus 145.5 ± 9.4,
Male WT and PGC1βKO mice were acclimatised to 30 °C or 4 °C as stated in the
(A) Oxygen consumption per mouse (ml/min). Similar results were obtained when the data were standardised using the body weight (kg)0.75 scaling; WT, black bars; PGC1βKO, white bars.
(B) RER. Solid circles, WT at 4 °C; open circles; PGC1βKO at 4 °C; solid squares, WT at 30 °C; open squares, PGC1βKO at 30 °C.
(C) Oxygen consumption per mouse (ml/min) measured post-administration of 1 mg/kg norepinephrine.
(D) BAT mRNA expression in thermoneutral and cold-adapted mice. WT thermoneutral, black bars; PGC1βKO thermoneutral, white bars; WT cold-adapted, dark gray bars; PGC1βKO cold-adapted, light gray bars. Individual measurements are standardized using 36B4 and the average of the WT, thermoneutral group was set to 1.
To test the maximal BAT-based nonshivering thermogenesis capacity, we used a norepinephrine (NE) challenge (
Gene expression of BAT from cold-acclimatised and thermoneutrally maintained mice was analysed. Comparison of gene expression patterns between WT and PGC1βKO mice housed at 30 °C (
Binomial pathway enrichment analysis using pathways from the Kyoto Encyclopedia Genes and Genomes (KEGG) was performed on gene expression from spotted arrays generated from BAT of PGC1βKO and WT mice to identify alterations in whole genetic pathways (
Major KEGG Pathways Are Changed in PGC1βKO Mouse BAT
We assessed whether reduced expression of ETC genes were associated with functional defects in permeabilised soleus muscle fibres (
Soleus fibres were isolated from WT (black bars) and PGC1βKO mice (white bars) and permeabilised to allow measurement of tissue-associated mitochondrial function.
(A) Mitochondrial respiratory parameters for state 2 (
(B) ATP synthesis rates in permeabilised soleus fibres.
(C) ATP/O ratio in permeabilised soleus fibres. Data are standardised to mg of muscle dry weight (mgdw).
(D) A representative electron micrograph of soleus muscle from WT (left panel) and PGC1βKO (right panel) mice.
To assess whether this oxidative defect could be attributed to the functionality of individual mitochondria, we measured oxidative activity of isolated soleus mitochondria. In all states examined, there was no significant difference between WT and PGC1βKO-derived soleus mitochondria (
We hypothesized that defective PGC-1β may result in electromechanical myocardial dysfunction in the hearts of PGC1βKO mouse. Heart weights from PGC1βKO mice were similar to WT in chow-fed 14-wk-old male mice (
(A) Expression levels of mRNA were assessed on hearts from 24-wk-old male WT (black bars) and PGC1βKO (white bars) mice. Individual measurements are standardized using 18S, and the average of the WT group set to 1.
(B) A representative electron micrograph of mitochondria from WT (left panel) and PGC1βKO (right panel) hearts. The bar indicates a measurement of 200 nm.
(C) mRNA expression of key genes for mitochondrial function in 24-wk-old WT and PGC1βKO mouse hearts.
(D) mRNA expression of key genes for metabolic function in 24-wk-old male WT and PGC1βKO mouse hearts.
Male, 26-wk-old PGC1βKO and WT littermates were challenged with acute infusion of the β1,α1-adrenergic selective agonist dobutamine. Haemodynamic recordings showed that baseline heart rate and left ventricular contractility (+
PGC1βKO and WT littermates (male, 26-wk-old) were treated as stated in
(A) Percentage change in heart rate from basal during dobutamine infusion.
(B and C) Measurement of ventricular performance,
Gene expression analysis of fed-state 12-wk-old male mouse livers showed ablation of PGC-1β but did not result in major changes in mRNA expression levels (
Female 8-wk-old mice were given normal chow or Surwit HFD (Sur) for 24 h. Tissues were then collected for analysis.
(A) Liver weight, as standardized by body weight (LW/BW) for WT (black bars) and PGC1βKO (white bars) after 24 h diets.
(B) Representative histological sections from mice given normal or Surwit diet for 24 h.
Serum Lipid Biochemistry from 24-h Surwit Diet–Fed WT and PGC1βKO Mice
Of interest and contrary to previous reports, we were unable to detect increases in PGC-1α and PGC-1β mRNAs in response to acute HFD in WT liver (
Accumulating evidence indicated that PGC-1β may play a role in energy homeostasis through its effects on substrate metabolism and mitochondrial activity [
The effect of PGC-1β controlling mitochondrial activity was investigated at the mRNA level using spotted array technology and bioinformatics pathway analysis. This approach showed that deletion of PGC-1β results in a significant mitochondrial phenotype. Multi-tissue comparisons demonstrated that PGC-1β controls the level of expression of ETC and OxPhos genes across all the organs studied and that this defect can only be partially compensated for in BAT and WAT by up-regulation of PGC-1α. However, the decrease in mRNA expression of ETC components is not necessarily seen at the protein level. These data suggest that although PGC-1β may be a controller of mitochondrial gene expression, in the absence of PGC-1β, additional factors such as protein degradation may be counter-regulated in specific tissues in an attempt to normalise levels of mitochondrial activity.
Our initial hypothesis was that dysregulation of ETC gene expression would increase the likelihood that the PGC1βKO mouse would become obese. However, despite its mitochondrial phenotype, the PGC1βKO mouse showed elevated resting metabolic rate and lower body weight compared to wild type littermates under ambient room temperature conditions. This phenotype is possibly due to the compensatory increase in expression of PGC-1α and its target genes in BAT. The increase in BAT PGC-1α expression may cause the higher degree of energy expenditure and relatively conserved mitochondria volume fraction despite reductions in ETC gene and protein expression. PGC-1α was also up-regulated in PGC-1β–deficient WAT, but its induction was not robust enough to produce a BAT-like histology or induction of typical BAT genes. Instead, within the reduced WAT content of the PGC1βKO mice, there was a greater proportion of larger, hypertrophic adipocytes. This result suggests that PGC-1β may play an as yet unknown role in white adipocyte biology, and the nature of this role requires further investigation.
Of interest, despite marked elevation of PGC-1α levels in PGC1βKO BAT, we did not observe a full restoration of the expression of mitochondrial genes back to WT levels. However, unlike the heart or skeletal muscle, organs where PGC-1α was not up-regulated, the mitochondrial fraction of BAT tissue was preserved in PGC1βKO mice. Thus, there may be partial functional overlap between PGC-1α and PGC-1β, concerning mitochondrial structure and function, which allows the development of an appropriate mitochondrial BAT content but fails to correct deficiencies in ETC composition. BAT was the only tissue out of three metabolically relevant tissues examined using electron microscopy that displayed relatively normal mitochondrial fraction, suggesting that further tissue function–specific mechanisms may be able to correct for PGC-1β ablation. However, the BAT gene expression pattern in PGC1βKO mice also showed altered expression of genes involved in intermediary metabolism, a pattern of gene expression that may also be primarily the result of PGC-1α up-regulation. Our conclusion from these gene expression analyses is that whereas PGC-1β may be required to set the basal level of mitochondrial ETC gene expression, it is not essential for normal expression of metabolic pathways such as glycolysis, the citric acid cycle, and fatty acid oxidation.
We hypothesized that the elevated resting metabolic rate seen in ambient-temperature housed PGC1βKO mice may be reversed under conditions of thermoneutrality, particularly if it was caused by compensatory up-regulation of PGC-1α. Under thermoneutral conditions, levels of PGC-1α gene expression are suppressed in BAT in response to the lower thermogenic and therefore oxidative demands of life at 30 °C. At thermoneutral conditions, PGC-1α mRNA expression was equivalent in WT and PGC1βKO BAT. Interestingly, under thermoneutral conditions, the PGC1βKO mouse exhibited reduced basal metabolic rate compared to the WT mouse. The energy expenditure measurements with mice acclimatised to 4 °C and 30 °C were performed at 33 °C. This measurement at thermoneutrality gives an indication of the basal metabolic rate. Under these experimental conditions, we can conclude that the PGC1βKO mouse has a reduced basal metabolic rate. The data from mice housed in ambient (22 °C) conditions were also generated at ambient conditions (
It has been suggested that PGC-1β plays a role in BAT accumulation and function during cold exposure [
Studies focused in other metabolically relevant organs where PGC-1β is well expressed also indicated that ablation of PGC-1β resulted in impaired metabolic capacity. For example, mitochondrial volume fraction in soleus of PGC1βKO mice was decreased, and permeabilised soleus muscle fibres from PGC1βKO mice had reduced oxygen consumption and ATP synthesis compared to WT-derived fibres. Similar experiments using isolated soleus mitochondria from PGC1βKO mice failed to show any abnormalities in metabolic performance, indicating that metabolic defects in PGC1βKO soleus tissue relate to lower mitochondrial density relative to WT, although individual mitochondria retain similar metabolic properties.
The heart is the third oxidative tissue that is a major site of PGC-1β expression. Absence of PGC-1β in heart also led to a reduction of mitochondrial fraction, consistent with reduced cardiac expression of ETC genes. However, despite these potential bioenergetic defects, PGC1βKO mice have normal basal heart rates and ventricular contractility. Left ventricular function was also maintained after dobutamine treatment, suggesting that despite their mitochondrial defect, the PGC-1β–deficient hearts are able to adapt normally to imposed hemodynamic loads. The only difference in adrenergic stress that we observed between the PGC1βKO mice and WT was a blunting of the expected increase in heart rate in the PGC1βKO mice. A similar defect in heart rate regulation was also found in the PGC-1α knockout mice [
To this point, we have discussed exclusively a role for PGC-1β in oxidative metabolism. Nonetheless, PGC-1β may also play an important role in regulating hepatic lipid production [
Our results indicate that PGC-1β has a well-defined role in controlling mitochondrial gene expression and function in many different organs. Despite this, PGC-1β can be ablated without overt metabolic failure, at least in unstressed conditions. This may be due in part to robust compensatory mechanisms, as demonstrated by the up-regulation of PGC-1α expression in WAT and BAT. Interestingly, other organs such as liver, muscle, or heart did not show up-regulation of PGC-1α under the conditions investigated. Following from these differences in PGC-1α expression, it is likely that the lean phenotype of this mouse model at ambient temperature should be considered the result of overcompensation mediated by up-regulation of PGC-1α, at least in BAT and WAT. Conversely, defects observed in skeletal muscle, heart, and liver are more likely to be the result of the absence of PGC-1β given the lack of PGC-1α induction in those tissues. When considering the roles played by PGC-1α and PGC-1β, our results show that there are specific effects of PGC-1β that cannot be compensated for by PGC-1α. Taken altogether, our results indicate that PGC-1β seems to cover basal bioenergetic needs whereas PGC-1α provides the extra bioenergetic support required under conditions of increased energy demand.
All reagents used in this paper were supplied from Sigma-Aldrich (St. Louis, Missouri, United States), unless stated.
Animals were housed in a temperature-controlled room with a 12-h light/dark cycle. Food and water were available ad libitum unless noted. All animal protocols used in this study were approved by the UK Home Office, The Institutional Animal Care and Use Committee of the University of Utah, United States, and the Animal Ethics Committees of Gothenburg and North Stockholm, Sweden. Mice were cared for according to the Guiding Principles for Research Involving Animals and Human Beings.
A triple LoxP strategy was used to target the PGC-1β locus in order to generate mice with both standard and conditional KO alleles at this locus. The targeting vector was a ~8 kilobase (kb) 129/SvJ mouse genomic subclone containing a floxed neomycin phosphotransferase selectable marker cassette inserted into intron 3 and a single LoxP site inserted into intron 5 (
Mice were placed at weaning (3-wk-old) on normal chow diet (12% fat, 62% carbohydrates, and 26% protein with a total energy content of 12.6 kJ/g) (R3 diet, Lactamin AB, Stockholm, Sweden). Mouse weights were taken at the same time each week, until the end of the specific protocol period. Mice were routinely housed at 22 °C, except for those used for the BAT activity and cold acclimatisation experiments (see below).
To examine 48-h food intake, cages (23 × 16 cm) were prepared with normal chow and incubated at 80 °C for 1 h to correct for any differences in humidity. After 2 h at room temperature, the cages were accurately weighed. 12-h-fasted mice were put in preweighed cages with free access to food and water. After 48 h, the mice were removed and all faecal matter was collected. The cages were reincubated at 80 °C in order to dry out waterspill and urine, and then reweighed after 2 h cooling. The difference in weights of the cage before and after the 48-h assessment produced the weight of food consumed. For measurement of energy content of faeces and food, samples were dried at 55 °C overnight and stored in airtight containers at −20 °C until assayed. The gross energy content of the dried samples was determined using a bomb calorimeter (C5000, IKA Werke GmbH & Co., KG, Germany). To assess the effect of 24-h high-fat feeding, 8-wk-old female mice were assigned into two groups: ad libitum normal food or ad libitum Surwit diet (58% of calories derived from fat, predominantly hydrogenated coconut oil; D12331, Research Diets, New Brunswick, New Jersey, United States). Start of the 24-h period was 9 am. At the end of the time period, mice were killed and dissected as above. Water was freely available during all procedures.
For body composition analysis, dual energy x-ray absorptiometry (DEXA, GE Medical Systems Lunar Corporation, Madison, Wisconsin, United States) was performed on isoflurane anaesthetized mice as previously described [
GTTs and ITTs on mice fed chow and HFDs were performed as previously described [
PGC1βKO mice and WT littermates were exposed to 30 °C for 3 wk, placed at 18 °C for 1 wk, and then exposed to 4 °C for 3 wk or kept at 30 °C for the duration of the protocol. For both 30 °C and 4 °C acclimated animals, resting metabolic rate was measured in awake animals at 30 °C. In addition, NE-stimulated (1 mg NE/kg body weight in saline, (−) arterenol bitartrate, intraperitoneally administered) energy expenditure was evaluated in anaesthetized (pentobarbital, 90 mg/kg) animals at 33 °C. Animals were allowed to recover from NE administration for 2–3 h at 30 °C. The mice where then rehoused at their acclimatisation temperature for 1 wk prior to tissue collection for gene and protein expression analysis to avoid effects of NE on gene expression. Oxygen consumption in conscious animals was followed for 3 h using an open circuit system with a chamber volume of 3 l and a flow rate of 1 l/min (Somedic, Hörby, Sweden). This system allowed the ambient temperature of the instruments to be adjusted between 5 °C and 40 °C, together with the volume and flow rates to optimise the system for the particular investigation. Oxygen consumption, carbon dioxide release, and ambient temperature data were collected every second minute via MacLab/2e (AD Instruments Pty. Ltd., Castle Hill, Australia). Resting metabolic rate was defined as the average of the lowest metabolic rates observed at three time points. Determinations in WT and PGC1βKO mice were performed in alternating order.
Mice were anesthetized with isoflurane and underwent endotracheal intubation. The airway was connected to mouse ventilator (Model 687, Harvard Apparatus, Holliston, Massachusetts, United States) to control breathing. The oxygen flow rate was 1 l/min. The left jugular vein was identified and accessed by cut down method using a 25 G needle connected to a syringe with dobutamine hydrochloride (Sigma) that was mounted on a Standard Infuse/Withdraw Harvard 33 Twin Syringe Pump (Harvard Apparatus). A micromanometer-tipped catheter (Millar Instruments, Houston, Texas, United States) was then inserted into the left ventricle via right carotid artery, and hemodynamic measurements was obtained as described [
Mice were anaesthetised using isoflourane. Blood, tissues used for RNA, protein extraction, and histology were prepared as previously published [
Enzymatic assay kits were used for determination of basic blood biochemical parameters as described in [
Tissue samples for morphological analysis were prepared according to published protocols [
Stereological assessments of mitochondrial volume fractions (
Isolation of mitochondria was performed in hearts and skeletal muscle from 10-wk-old male PGC-1β mice and their WT littermates. Mice were killed by cervical dislocation. The hearts or hind limb skeletal muscle of four mice per genotype were pooled and immediately placed in ice-cold isolation medium (for hearts, 250 mM sucrose, 5 mM Tris, 2 mM EGTA at pH 7.4; for skeletal muscle, 100 mM KCl, 50 mM Tris, 2 mM EGTA at pH 7.4). Mitochondria were prepared essentially as described in [
The kinetics of proton conductance and ETC function were measured in mitochondria in the presence of oligomycin (1 μg/ml), where that rate of respiration is directly proportional to the leak of protons across the mitochondrial inner membrane rather than a combination of ADP phosphorylation and proton leak. Mitochondrial function was assessed as described previously [
Total RNA was isolated from tissues as described and reverse-transcribed using the SuperscriptII kit (Invitrogen, Frederick, Maryland, United States) or Hi-Capacity cDNA archive kit (Applied Biosystems, Foster City, California, United States), following the manufacturers protocol. Oligonucleotide primers and TaqMan probe were designed using Primer Express, version 2.0 (Applied Biosytems). Primer and probe sequences, together with gene abbreviations, can be found in
Male 14-wk-old mice were killed and dissected as above. The tissues were extracted for RNA as above and purified using the RNA clean-up protocol from the RNeasy Mini Kit (Qiagen Ltd, Crawley, UK). RNA was quantified spectroscopically at 260 nm using a GeneQuant Nucleotide calculator (Amersham Biosciences, Little Chalfont, UK) and checked for integrity on a 1% TBE gel using ethidium bromide staining. cDNA was amplified from total RNA using template-switching PCR and labeled with Cy3 or Cy5 dyes as previously described [
Female 15-wk-old chow-fed mice were anaesthetised using isoflurane and killed by cervical dislocation and heart removal. BAT, heart, and soleus muscle tissue was removed and either snap-frozen in liquid nitrogen or used to prepare a mitochondrial extract as described in
The data presented here were analysed using the Student
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Two fields from each section from (A) omental WAT, (B) subcutaneous WAT, and (C) female gonadal WAT depots (age 24 wk,
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Sections are as follows: BAT (A and B), liver (C and D), soleus muscle (E and F), pancreatic sections stained for glucagon (G and H), or pancreatic sections stained for insulin (I and J). A representative section from WT (A, C, E, G, and I) or PGC1βKO (B, D, F, H, and J) are shown,
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Mice were placed on normal diet or HFD for 13 wk. GTTs (left panels) or ITTs (right panels) were performed on the mice at 16 wk of age. Plasma glucose levels of WT mice (open circles) and PGC1βKO (solid circles) were measured during the protocol.
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Individual measurements were standardised using 18S, and the average of the WT group set to 1.
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Soleus tissue was pooled from four WT mice (black bars) or four PGC1βKO mice (white bars) and mitochondria isolated on five separate occasions (
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Gene expression was analysed in 12-wk-old fed-state male WT (black bars) and PGC1βKO (white bars) livers. Individual measurements are standardised using 18S and then the average of the WT group was set to 1.
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WT chow, black bars; WT Surwit HFD, white bars; PGC1βKO chow, dark gray bars; PGC1βKO Surwit HFD, light gray bars. Individual measurements are standardised using 36B4 and then the average of the WT chow group was set to 1.
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The food intake and energetic content of faeces from 12-wk-old male mice was determined over a 48-h period.
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The data are presented both comparing raw weights and as tissue weight/body weight. Statistical comparison between WT and PGC1βKO mice was performed using
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RNA was isolated from 14-wk-old normal diet-fed male PGC1βKO and WT mice and then analysed using spotted arrays as described in the
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Sybr as a probe indicates the use of a Sybr-Green system instead of Taqman.
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Sybr as a probe indicates the use of a Sybr-Green system instead of Taqman.
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Sybr as a probe indicates the use of a Sybr-Green system instead of Taqman.
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The authors would like to thank all lab staff who performed mouse breeding and biochemical analyses at AstraZeneca and the University of Cambridge for this project. We wish to thank Jane Löfvenmark for invaluable help during the early stages of the project. We would like to thank the animal care staff at the institutions involved for their work on this project. We also thank Ian McFarlane, Lyn Carter and Jeremy Skepper for providing technical help.
brown adipose tissue
cytochrome oxidase subunit
embryonic stem cell
electron transport chain
high-density lipoprotein
high-fat diet
kyoto encyclopedia genes and genomes
low-density lipoprotein
norepinephrine
not significant
oxidative phosphorylation
peroxisome proliferator-activated receptor-gamma coactivator
respiratory exchange ratio
very low-density lipoprotein
white adipose tissue
wild-type