(a) WB for Perilipin 5 in nuclear and cytoplasmic fractions prepared from heart (H), soleus (S), gastrocnemius (G), white adipose tissue (W), liver (L), BAT (B) and quadriceps (Q), of wild-type mice fed (left) or fasted (right) for 16 h. Data are representative of two independent experiments (each sample represents pooled fractions of three mice). (b,c) Time-dependent nuclear enrichment of Perilipin 5 during isoproterenol (Iso, 10 μM) and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM) treatment of C2C12 myotubes (b) and MLTC-1 cells (c). (d) Subcellular distribution of Perilipin 5 in nuclear, cytoplasmic and fat cake fractions after treatment of MLTC-1 cells with albumin-bound oleate (1.2 mM oleate for 16 h), vehicle or Iso/IBMX (2 h). (e) Subcellular distribution of Perilipin family proteins, Perilipins 1–5, in nuclear (N), cytoplasmic (C) and fat cake (FC) fractions after treatment of MLTC-1 cells with vehicle or Iso/IBMX for 2 h. (f) Localization of mCherry-Perilipin 5 or mCherry-Perilipin 2 in C2C12 myoblasts after treatment with vehicle or Iso/IBMX for 2 h. Nucleus stained with DAPI (blue). (g) Mouse Perilipin 5 PKA substrate consensus sequence is conserved among mammalian species. Amino acids identified by MS are shown in blue, and phosphoserine site is shown in red. (h) Immunofluorescence localization of myc-Perilipin 5 in C2C12 myoblasts transfected with wild-type myc-Perilipin 5, myc-Perilipin 5 S155A or myc-Perilipin 5 S155E after treatment with vehicle or Iso/IBMX for 2 h. Myc-Perilipin 5 wild-type and mutant proteins are shown in red, and the nucleus in blue (DAPI). Scale bar, 10 μm.

(a) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. (b) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. (c,d) IP of Perilipin 5 (c) or PGC-1α (d) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. (e) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. (f) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). (g) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. (h) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.

(a) qPCR to detect expression of indicated genes in C2C12 myoblasts individually transfected with the following plasmids: EV, wild-type myc-Perilipin 5, S155A Perilipin 5 and S155E Perilipin 5, followed by treatment with vehicle or Iso/IBMX for 2 h. Values are mean±s.e.m. n=3/group. Data are representative of two independent experiments. *P<0.05 compared with vehicle and, ^#P<0.05 compared with EV and ^&P<0.05 compared with wild type using ANOVA followed by Tukey post-test. (b) qPCR to detect expression of the indicated genes in C2C12 myoblasts transfected with siControl or siPerilipin 5 duplexes with subsequent reconstitution with EV, wild-type myc-Perilipin 5 or S155A myc-Perilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. Values are mean±s.e.m. n=3/group. Data are representative of two independent experiments. *P<0.05 compared with the vehicle and, ^#P<0.05 compared with siControl and ^&P<0.05 compared with wild type using ANOVA followed by Tukey post-test. (c) qPCR to detect expression of indicated genes in brown adipocytes transfected with siControl or siPerilipin 5 followed by vehicle or Iso/IBMX treatment for 2 h. For all qPCR data in , values are expressed as mean±s.e.m. n=3 biological replicates/group with two technical replicates for each biological replicate. Each biological replicate represents an independent transfection and treatment. These data were replicated in a second independent experiment. Comparative Ct method (ΔΔ Ct) was used to analyse all qPCR data. Expression of each gene was normalized to that of the 18S ribosomal subunit as endogenous control and the relative expression was calculated in comparison with reference samples as follows: (a) EV, vehicle (b) siControl + EV, vehicle, (c) siControl, vehicle. ANOVA, analysis of variance; EV, empty vector; qPCR, quantitative PCR.

(a) P1 – 4 represent different regions of the PGC-1α promoter; P3 corresponds to the PPRE. (b) ChIP-qPCR assay demonstrates specific binding of Perilipin 5 and PGC-1α-containing complexes to the PGC-1α promoter region at the PPRE in C2C12 myotubes treated with Iso/IBMX or vehicle for 2 h. Control IgGs (anti-guinea pig and anti-rabbit) served as the negative controls for ChIP. (c) ChIP-qPCR the P1, P2 and P4 regions of the PGC-1α promoter. The results were analysed using qPCR and are shown as the percentage of input. Values are expressed as means±s.e.m. n=3 biological replicates/experimental group with each biological replicate representing an independent immunopreciptation for each treatment. Student t-test *P<0.05. Data are representative of two independent experiments (n=3). These data were replicated in a second independent experiment. PPRE, PPAR response element; qPCR, quantitative PCR.

C2C12 myoblasts were co-transfected with the indicated plasmids (25–100 ng each, as labelled) (a,b,d,f) or the indicated plasmids and siRNA duplexes (c,e) together with the 2 kb PGC-1α promoter luciferase plasmid. Forty-eight hours after the transfection, cells were treated with vehicle or Iso/IBMX for 2 h and luciferase activity was measured. In all experiments Renilla luciferase vector was co-transfected to normalize for transfection efficiency. Values are expressed as mean±s.e.m. n=6 biological replicates for each data point with each biological replicate representing an independent transfection and treatment of C2C12 myoblasts. The data shown were replicated in two additional independent experiments. *P<0.05: Iso/IBMX compared with vehicle treatment of the same transfected vectors or siRNA. ^#P<0.05: the indicated transfection/treatment compared with control group. Control groups are (a) 50 ng PGC-1α, Iso/IBMX, (b) 50 ng Perilipin 5, Iso/IBMX, (c) Perilipin 5 + siCon, Iso/IBMX, (d) Perilipin 5 WT, Iso/IBMX, (e) Perilipin 5 + siCon, Iso/IBMX, and (f) PGC-1α + Perilipin 5, vehicle. Statistical analysis was done by ANOVA followed by Tukey post-test. ANOVA, analysis of variance.

(a) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD⁺ levels with Iso/IBMX or siPerilipin 5 treatments (right). (b) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. (c) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). (d) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. (e) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n=3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P<0.05 compared with vehicle and ^#P<0.05 compared with siControl. The data were analysed using one-way ANOVA followed by Tukey post-test. ANOVA, analysis of variance; mtDNA, mitochondrial DNA; qPCR, quantitative PCR.

During stimulation of cells or tissues by catecholamines, fasting and perhaps other stimuli such as cold exposure and exercise, Perilipin 5 is phosphorylated by protein kinase A and enriches in the nucleus to form transcriptional complexes with PGC-1α and SIRT1. Perilipin 5 activates the deacetylase activity of SIRT1 by displacing DBC1, an inhibitor of SIRT1, thereby promoting PGC-1α co-activator activity via deacetylation. In this PGC-1α/SIRT1-dependent manner, nuclear Perilipin 5 augments expression of genes that promote mitochondrial biogenesis and function.