Regulation of Brown and White Adipocyte Transcriptome by the Transcriptional Coactivator NT-PGC-1α

The β3-adrenergic receptor (AR) signaling pathway is a major component of adaptive thermogenesis in brown and white adipose tissue during cold acclimation. The β3-AR signaling highly induces the expression of transcriptional coactivator PGC-1α and its splice variant N-terminal (NT)-PGC-1α, which in turn activate the transcription program of adaptive thermogenesis by co-activating a number of transcription factors. We previously reported that NT-PGC-1α is able to increase mitochondrial number and activity in cultured brown adipocytes by promoting the expression of mitochondrial and thermogenic genes. In the present study, we performed genome-wide profiling of NT-PGC-1α-responsive genes in brown adipocytes to identify genes potentially regulated by NT-PGC-1α. Canonical pathway analysis revealed that a number of genes upregulated by NT-PGC-1α are highly enriched in mitochondrial pathways including fatty acid transport and β-oxidation, TCA cycle and electron transport system, thus reinforcing the crucial role of NT-PGC-1α in the enhancement of mitochondrial function. Moreover, canonical pathway analysis of NT-PGC-1α-responsive genes identified several metabolic pathways including glycolysis and fatty acid synthesis. In order to validate the identified genes in vivo, we utilized the FL-PGC-1α-/- mouse that is deficient in full-length PGC-1α (FL-PGC-1α) but expresses a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α254). The β3-AR-induced increase of NT-PGC-1α254 in FL-PGC-1α-/-brown and white adipose tissue was closely associated with elevated expression of genes involved in thermogenesis, mitochondrial oxidative metabolism, glycolysis and fatty acid synthesis. Increased adipose tissue thermogenesis by β3-AR activation resulted in attenuation of adipose tissue expansion in FL-PGC-1α-/- adipose tissue under the high-fat diet condition. Together, the data strengthen our previous findings that NT-PGC-1α regulates mitochondrial genes involved in thermogenesis and oxidative metabolism in brown and white adipocytes and further suggest that NT-PGC-1α regulates a broad spectrum of genes to meet cellular needs for adaptive thermogenesis

Poly(ADPribose)polymerase inhibition counteracts renal hypertrophy and multiple manifestations of peripheral neuropathy in diabetic Akita mice

PURPOSE. This study evaluated the role for poly(ADP-ribose) polymerase (PARP) in diabetes-induced cataractogenesis and early retinal changes. METHODS. Control and streptozotocin (STZ)-diabetic rats were treated with or without the PARP inhibitors 1,5-isoquinolinediol (ISO; 3 mg kg Ϫ1 d Ϫ1 intraperitoneally) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo- [de]anthracen-3-1 (GPI-15427, 30 mg kg Ϫ1 d Ϫ1 orally) for 10 weeks after the first 2 weeks without treatment. Lens clarity was evaluated by indirect ophthalmoscopy and slit lamp examination, and retinal changes were evaluated by immunohistochemistry and Western blot analysis. In in vitro studies, cultured human lens epithelial cells and bovine retinal pericytes and endothelial cells were exposed to high glucose or palmitate. RESULTS. PARP is expressed in lens, and poly(ADP-ribosyl)ated proteins are primarily localized in the 38-to 87-kDa range of the protein spectrum, with several minor bands at 17 to 38 kDa. The 38-to 87-kDa and the 17-to 38-kDa poly(ADPribosyl)ated protein expression increased by 74% and 275%, respectively, after 4 weeks of diabetes and by approximately 65% early after exposure of lens epithelial cells to 30 mM glucose. Both PARP inhibitors delayed, but did not prevent, the formation of diabetic cataract. The number of TUNEL-positive nuclei in flatmounted retinas increased approximately 4-fold in STZ diabetic rats, and this increase was prevented by ISO and GPI-15427. Both PARP inhibitors reduced diabetes-induced retinal oxidative-nitrosative and endoplasmic reticulum stress and glial activation. GPI-15427 (20 M) prevented oxidative-nitrosative stress and cell death in palmitate-exposed pericytes and endothelial cells. CONCLUSIONS. PARP activation is implicated in the formation of diabetic cataract and in early retinal changes. These findings provide a rationale for the development of PARP inhibitors for the prevention of diabetic ocular complications. (Invest Ophthalmol Vis Sci

Gene expression changes in FL-PGC-1α^-/- inguinal white adipose tissue in response to CL316243.

<p>(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: *<i>P</i> < 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). *<i>P</i> < 0.05.</p

Alternative mRNA Splicing Produces a Novel Biologically Active Short Isoform of PGC-1α*

The transcriptional co-activator PGC-1α regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1α (NT-PGC-1α) produced by alternative 3′ splicing that introduces an in-frame stop codon into PGC-1α mRNA. The expressed protein includes the first 267 amino acids of PGC-1α and 3 additional amino acids from the splicing insert. NT-PGC-1α contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1α are dynamically regulated in the context of physiological signals that regulate full-length PGC-1α, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1α is a co-expressed, previously unrecognized form of PGC-1α with functions that are both unique from and complementary to PGC-1α