NT-PGC-1α protein is sufficient to link β3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis

Abstract

PGC-1α is an inducible transcriptional coactivator that regulates cellular energy metabolism and adaptation to environmental and nutritional stimuli. In tissues expressing PGC-1α, alternative splicing produces a truncated protein (NT-PGC-1α) corresponding to the first 267 amino acids of PGC-1α. Brown adipose tissue also expresses two novel exon 1b-derived isoforms of PGC-1α and NT-PGC-1α, which are 4 and 13 amino acids shorter in the N termini than canonical PGC-1α and NT-PGC-1α, respectively. To evaluate the ability of NT-PGC-1α to substitute for PGC-1α and assess the isoform-specific role of NT-PGC-1α, adaptive thermogenic responses of adipose tissue were evaluated in mice lacking full-length PGC-1α (FL-PGC-1(-/-)) but expressing slightly shorter but functionally equivalent forms of NT-PGC-1α (NT-PGC-1α(254)). At room temperature, NT-PGC-1α and NT-PGC-1α(254) were produced from conventional exon 1a-derived transcripts in brown adipose tissue of wild type and FL-PGC-1α(-/-) mice, respectively. However, cold exposure shifted transcription to exon 1b, increasing exon 1b-derived mRNA levels. The resulting transcriptional responses produced comparable increases in energy expenditure and maintenance of core body temperature in WT and FL-PGC-1α(-/-) mice. Moreover, treatment of the two genotypes with a selective β(3)-adrenergic receptor agonist produced similar increases in energy expenditure, mitochondrial DNA, and reductions in adiposity. Collectively, these findings illustrate that the transcriptional and physiological responses to sympathetic input are unabridged in FL-PGC-1α(-/-) mice, and that NT-PGC-1α is sufficient to link β(3)-androgenic receptor activation to adaptive thermogenesis in adipose tissue. Furthermore, the transcriptional shift from exon 1a to 1b supports isoform-specific roles for NT-PGC-1α in basal and adaptive thermogenesis.

FIGURE 1.

The FL-PGC-1α^−/− mice expressing a cold-inducible NT-PGC-1α²⁵⁴ are cold-tolerant. A, schematic diagram of NT-PGC-1α and NT-PGC-1α²⁵⁴ mRNAs and proteins. B, quantitative real-time PCR analysis of full-length PGC-1α and NT-PGC-1α mRNA in BAT from WT mice and NT-PGC-1α²⁵⁴ mRNA in BAT from FL-PGC-1α^−/− mice, respectively, placed at room temperature (22 °C) or 4 °C for 5 h. Data represent mean ± S.E. **, p < 0.01; ***, p < 0.001. C, Western blot analysis of full-length PGC-1α/NT-PGC-1α and NT-PGC-1α²⁵⁴ proteins in brown fat extracts from WT and FL-PGC-1α^−/− mice, respectively, placed at room temperature (RT; 22 °C) or 4 °C (4C) for 5 h. Asterisk represents nonspecific bands. Lysates containing full-length PGC-1α and NT-PGC-1α were used as a positive control. D, body temperature of 10-week-old WT (n = 4) and FL-PGC-1α^−/− (n = 4) mice exposed to cold (4 °C). Core body temperature was measured with a rectal thermometer every hour. Data represent mean ± S.E. *, p < 0.05. E, recruitment of NT-PGC-1α²⁵⁴ onto the UCP1 enhancer in BAT. The amount of the UCP1 enhancer immunoprecipitated with IgG or PGC-1α antibody from WT and FL-PGC-1α^−/− BAT was analyzed by quantitative PCR. A nonspecific intragenic region of the UCP1 gene was used as an additional negative control. Data represent mean ± S.E. *, p < 0.05.

FIGURE 2.

Cold-inducible exon 1b-derived isoforms of PGC-1α and NT-PGC-1α. A, schematic structure of the 5′ region of the murine PGC-1α gene. The schematic structure was slightly adapted from the Miura et al. (14). Boxes indicate exons, and lines indicate introns. The coding regions and untranslated regions are shown in gray and white, respectively. An alternative exon 1b and a canonical exon 1a are spliced to the common exon 2 by three different splicing events. Three different isoforms of NT-PGC-1α are also produced with combination of alternative 3′ splicing at the intron between exons 6 and 7. B, mRNA and protein sequences of exon 1a- and exon 1b-derived isoforms of PGC-1α/NT-PGC-1α. The 5′ region sequences of exon 1a- and exon 1b-derived transcripts are shown with isoform-specific primers underlined (top). Amino acid sequences of exon 1a- and exon 1b-derived isoforms. The gray box represents the common coding sequences of exon 2 (bottom). C, cold induction of exon 1b-derived transcripts in BAT. Using isoform-specific primers, quantitative real-time PCR analysis was carried out to examine expression of exon 1a- and exon 1b-derived transcripts in BAT from WT and FL-PGC-1α^−/− mice, respectively. Mice were placed at room temperature (22 °C) or cold-exposed for 5 h. Data represent mean ± S.E. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. rev, reverse; fwd, forward.

FIGURE 3.

Exon 1b-derived NT-PGC-1α-b and NT-PGC-1α-c are functional transcriptional coactivators. A, identical localization of NT-PGC-1α-b and NT-PGC-1α-c to NT-PGC-1α-a. Three different isoforms of NT-PGC-1α-HA were expressed in CHO-K1 cells, and their localization was analyzed by immunocytochemistry using anti-HA antibody. B, transcriptional coactivation assay. pcDNA3.1 and NT-PGC-1α-HA (a, b, c) were co-transfected in COS-1 cells with a GAL4-responsive luciferase reporter, GAL4-PPARγ, and a Renilla luciferase reporter, respectively. 24 h after transfection, cells were treated with BRL49653. Luciferase activity was determined 48 h after transfection, and the relative luciferase units (RLU) were normalized using Renilla luciferase activity. Data represent mean ± S.E. of at least five independent experiments. ****, p < 0.0001 and ns, not significant. DBD represents DNA-binding domain. The same lysates were used for protein expression of three different isofoms of NT-PGC-1α-HA using anti-PGC-1α antibody. α-Tubulin was used as a loading control. ns, not significant; RLU, relative luciferase units.

FIGURE 4.

Three isoforms of NT-PGC-1α²⁵⁴ retain the functional properties of NT-PGC-1α. A, localization of three different isoforms of NT-PGC-1α²⁵⁴. NT-PGC-1α²⁵⁴-HA (a, b, and c) were expressed in CHO-K1 cells, and their localization was analyzed by immunocytochemistry using anti-HA antibody. B, transcriptional coactivation assay. Each isoform of NT-PGC-1α²⁵⁴-HA and NT-PGC-1α-HA was co-transfected in COS-1 cells with a GAL4-responsive luciferase reporter, GAL4-PPARγ, or GAL4-ERRα-LBD. Luciferase activity was determined 48 h after transfection, and the relative luciferase units (RLU) were normalized using Renilla luciferase activity. Data represent mean ± S.E. of at least three independent experiments. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. The same lysates were used for protein expression of each isoform of NT-PGC-1α²⁵⁴-HA and NT-PGC-1α-HA using anti-PGC-1α antibody. α-Tubulin was used as a loading control. C, transcriptional coactivation assays were performed as described above. PPARγ/(PPRE)₃-TK-luc and ERR/pERRα-promoter luc were used, respectively. ns, not significant.

FIGURE 5.

Change in energy expenditure of FL-PGC-1α^−/− mice after treatment with selective β₃-adrenergic receptor agonist. WT (n = 8) and FL-PGC-1α^−/− mice (n = 8) were provided a low fat diet for 2 weeks prior to measurement of O₂ consumption for a total of 5 days, the first three days while consuming the low fat diet containing vehicle followed by 2 days of consuming the same diet containing 0.001% CL316,243, a selective β₃-adrenergic receptor agonist. Energy expenditure was calculated as described under “Materials and Methods” and expressed per unit body weight (BW) because weight and body composition did not differ between the genotypes.

FIGURE 6.

Morphological and transcriptional remodeling of adipose tissue in FL-PGC-1α^−/− mice after treatment with selective β₃-adrenergic receptor agonist. A, CL316,243-mediated induction of morphological changes and mitochondrial biogenesis in adipose tissue. Brown and white adipose tissues of wild-type (n = 4) and FL-PGC-1α^−/− (n = 4) mice given the low fat diet (LF) (control) or LF+CL diet for 6 days were stained with Mitotracker Red and imaged by confocal microscopy. B, quantitative analysis of mitochondrial biogenesis. The ratio of mitochondrial to nuclear DNA was analyzed in BAT, EWAT, IWAT, and RPWAT from WT (n = 8) and FL-PGC-1α^−/− (n = 8) mice given the LF+CL diet for 4 days. C and D, quantitative real time PCR analysis of gene expression in BAT and IWAT from WT (n = 8) and FL-PGC-1α^−/− (n = 8) mice given the LF+CL diet for 4 days, respectively. Data represent mean ± S.E. E, CL316,243-mediated induction of UCP1 protein in IWAT. Immunoblotting of UCP1 protein in IWAT from WT (n = 3) and FL-PGC-1α^−/− (n = 3) mice given the LF+CL diet for 4 days. The BAT lysates from WT mice given the LF+CL diet were used as a positive control. β-Actin was used as a control for equal loading of the samples. ns, not significant.