PGC1βKO Hearts Display a Blunted Heart Rate Response to Dobutamine Stimulation In Vivo

<div><p>PGC1βKO and WT littermates (male, 26-wk-old) were treated as stated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040369#s4" target="_blank">Material and Methods</a> and infused with 10 or 40 ng/min/g BW dobutamine to measure in vivo hemodynamic responses WT, solid line; KO, dashed line.</p> <p>(A) Percentage change in heart rate from basal during dobutamine infusion.</p> <p>(B and C) Measurement of ventricular performance, <i>dP</i>/<i>dt</i>, during infusion. The arrow marks the increase in dobutamine concentration in the infusion from 10 to 40 ng/min/g BW. <i>n</i> = 5 mice per genotype.</p></div

PGC1βKO Mice Demonstrate Increased Liver Mass and Development of Fatty Liver after 24 h HFD

<div><p>Female 8-wk-old mice were given normal chow or Surwit HFD (Sur) for 24 h. Tissues were then collected for analysis.</p> <p>(A) Liver weight, as standardized by body weight (LW/BW) for WT (black bars) and PGC1βKO (white bars) after 24 h diets.</p> <p>(B) Representative histological sections from mice given normal or Surwit diet for 24 h. <i>n</i> = 6–7 mice per group.</p></div

The PGC1βKO Mouse Has Altered Metabolism under Standard Environmental Conditions

<div><p>(A) Growth curves of male (left panel) and female (right panel) mice on normal diet. WT (solid circles) and PGC1βKO (open circles) mice, <i>n</i> = 18–21 mice per group.</p> <p>(B) Assessment of fat content by DEXA in 8- and 32-wk-old male WT (solid bars) and PGC1βKO mice (open bars), <i>n</i> = 8–12 mice per group.</p> <p>(C) Representative histological sections of tissues from WAT (<i>n</i> = 6).</p> <p>(D) Size distribution of adipocytes from WT and PGC1βKO mice. Two fields from each section from epididymal adipose tissue depot (<i>n</i> = 4 mice per genotype) were analysed to obtain the mean cell area per animal.</p> <p>(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. <i>n</i> = 5–8 mice per group.</p></div

PGC-1β Ablation Results in Major Changes in BAT Metabolic Gene Expression

<p>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. <i>n</i> = 5–7 mice per group.</p

Isolated Soleus Fibres from PGC1βKO Mice Have Reduced Mitochondrial Activity

<div><p>Soleus fibres were isolated from WT (black bars) and PGC1βKO mice (white bars) and permeabilised to allow measurement of tissue-associated mitochondrial function.</p> <p>(A) Mitochondrial respiratory parameters for state 2 (<i>V</i>₀), state 3 (<i>V</i>_ADP), state 4 (<i>V</i>_oligomycin), and respiratory control ratio (RC).</p> <p>(B) ATP synthesis rates in permeabilised soleus fibres.</p> <p>(C) ATP/O ratio in permeabilised soleus fibres. Data are standardised to mg of muscle dry weight (mgdw). <i>n</i> = 9 WT mice, 11 PGC1βKO mice.</p> <p>(D) A representative electron micrograph of soleus muscle from WT (left panel) and PGC1βKO (right panel) mice.</p></div

Alterations in Gene Expression and Mitochondrial Dimensions in Hearts of PGC1βKO Mice

<div><p>(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. <i>n</i> = 5–7 mice per group.</p> <p>(B) A representative electron micrograph of mitochondria from WT (left panel) and PGC1βKO (right panel) hearts. The bar indicates a measurement of 200 nm.</p> <p>(C) mRNA expression of key genes for mitochondrial function in 24-wk-old WT and PGC1βKO mouse hearts.</p> <p>(D) mRNA expression of key genes for metabolic function in 24-wk-old male WT and PGC1βKO mouse hearts.</p></div

PGC-1β Ablation Reduces ETC Gene and Protein Expression but Mitochondrial Volume Fraction is Unaffected

<div><p>(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. <i>n</i> = 5–7 mice per group.</p> <p>(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). <i>n</i> = 5–6 mice for each protein, with the average value of the WT group set to 1. Representative blots showing two samples from each genotype.</p> <p>(E) Representative electron micrographs from BAT of WT (left panel) and PGC1βKO (right panel) mice.</p></div

Generation of the PGC1βKO Mouse

<div><p>(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 <i>PGC-1</i>β gene.</p> <p>(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.</p> <p>(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.</p> <p>(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).</p></div