Fig 1.
Pathway analysis of NT-PGC-1α-responsive genes in brown adipocytes.
(A) Oxygen consumption rates of brown adipocytes expressing NT-PGC-1α or empty vector. Basal and isoproterenol-stimulated mitochondrial respiration (n = 5) was measured as described in Materials and Methods. (B) Ingenuity pathway analysis on the genes whose expression was elevated by NT-PGC-1α compared to controls in brown adipocytes. The statistically significant canonical pathways identified are listed according to their –log (p-value). The number of overlapping genes detected in each pathway was shown. (C) Functional interaction analysis of 65 genes that are enriched in the metabolic and signal transduction pathways identified by the IPA analysis. Genes or gene products are displayed as nodes that represent the functional class of the gene product, and a biological relationship between two genes is represented as a line.
Fig 2.
The effect of NT-PGC-1α on candidate gene expression in brown adipocytes.
(A) Schematic diagram of splicing events of the PGC-1α gene. Splicing between canonical exon 1a and exon 2 produces PGC-1α-a and NT-PGC-1α-a, whereas alternative splicing between distal exon 1b and exon 2 produces PGC-1α-b and NT-PGC-1α-b. AD, transcription activation domain; LxxLL, nuclear receptor interaction motif; RS, arginine/serine-rich domain. (B) Expression of NT-PGC-1α-a and NT-PGC-1α-b proteins in PGC-1α-null brown adipocytes. (C) Quantitative real-time PCR analysis of gene expression in PGC-1α-null brown adipocytes expressing pBABE empty vector, NT-PGC-1α-a or NT-PGC-1α-b (n = 8). Empty vector vs NT-PGC-1α-a or NT-PGC-1α-b: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3.
The effect of NT-PGC-1α on candidate gene expression in 3T3-L1 adipocytes.
Quantitative real-time PCR analysis of gene expression in 7-day-differentiated 3T3-L1 adipocytes expressing pBABE empty vector or NT-PGC-1α-a (n = 6). Empty vector vs NT-PGC-1α: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4.
Gene expression changes in FL-PGC-1α-/- brown adipose tissue in response to CL316243.
(A) Schematic diagram of WT mice expressing PGC-1α and NT-PGC-1α and of FL-PGC-1α-/- mice only expressing NT-PGC-1α254. AD, transcription activation domain; LxxLL, nuclear receptor interaction motif; RS, arginine/serine-rich domain. At the age of 9 to 10 weeks, WT and FL-PGC-1α-/- mice (n = 8 per group) were singly housed and provided a high-fat diet (HFD) ad libitum for 2 weeks, followed by the same diet containing 0.001% CL316243 (HFD + CL316243) for 6 days. (B) Increased expression of NT-PGC-1α254 by β3-AR activation in FL-PGC-1α-/- BAT. NT-PGC-1α-HA in HEK293 cells was used as a positive control. (C) Quantitative real-time PCR analysis of a number of metabolic genes in WT and FL-PGC-1α-/- BAT (n = 7–8 per group). HFD vs HFD+CL316243: *P < 0.05, **P < 0.01. (D) Protein levels of UCP1 in BAT whole cell extracts. Identical amounts of proteins were loaded, and α-tubulin was used as a loading control. (E) Quantitative analysis of mitochondrial biogenesis. The ratio of mitochondrial DNA (mtDNA) relative to nuclear genome (nucDNA) was analyzed in BAT (n = 7–8 per group). **P < 0.01, ***P < 0.001.
Fig 5.
Gene expression changes in FL-PGC-1α-/- inguinal white adipose tissue in response to CL316243.
(A) Increased expression of NT-PGC-1α254 by β3-AR activation in FL-PGC-1α-/- IWAT. NT-PGC-1α-HA in HEK293 cells was used as a positive control. FL-PGC-1α-/- mice were fed a HFD for 2 weeks, followed by the treatment with or without CL316243 for 6 days on HFD. (B, D, E) Quantitative real-time PCR analysis of a number of metabolic genes in WT and FL-PGC-1α-/- IWAT (n = 8 per group). HFD vs HFD+CL316243: *P < 0.05. (C) Western blot analysis of UCP1 expression in IWAT whole cell extracts (100 μg). BAT extracts (10 μg) were added to WT IWAT extract (- CL316243) as a positive control for UCP1. (F) The ratio of mitochondrial DNA (mtDNA) relative to nuclear genome (nucDNA) was analyzed in IWAT (n = 8 per group). *P < 0.05.
Fig 6.
An increase in adipose tissue-driven thermogenesis in FL-PGC-1α-/- mice attenuates adipose tissue expansion under the HFD.
(A, B) Body weight and composition of WT and FL-PGC-1α-/- mice. Mice were measured for body weight and composition prior to introduction of HFD, after 2 weeks on HFD and after additional 6 days on CL 316243+HFD. BW: body weight, LM: lean mass, FM: fat mass (C) Energy expenditure (EE) of WT and FL-PGC-1α-/- mice. Mice were measured for VO2 and VCO2 on HFD for first two days and on HFD + CL316243 for additional four days. EE (kilojoules per hour) was calculated as described in Materials and Methods and expressed per lean mass (LM). (D) Average food intake of WT and FL-PGC-1α-/- mice on HFD. Food intake was expressed as grams per day per mouse. (E) Voluntary activity of WT and FL-PGC-1α-/- mice. Ambulatory activity was monitored at 18 min intervals for 6 days on HFD and HFD + CL316243. (F) Core body temperature of WT and FL-PGC-1α-/- mice (n = 7–9 per group). Mice fed a HFD for 3 weeks were exposed to 4°C and their core body temperature was measured for 5h using a MicroTherma thermometer with the RET-3 mouse rectal probe. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.