Systemic Nanoparticle‐Mediated Delivery of Pantetheinase Vanin‐1 Regulates Lipolysis and Adiposity in Abdominal White Adipose Tissue

Abstract Lipolysis in white adipose tissue (WAT) occurs in response to nutritional signals and helps to regulate lipid turnover/adiposity in animals. However, the causal relationships and the mechanisms controlling WAT morphology are not clear. In this report, Vanin‐1, a pantetheinase, is shown to be a novel determinant for lipolysis and adiposity. The expression of Vanin‐1 in the abdominal WAT is positively correlated with lipolysis both in mice and in humans. Mice with global Vanin‐1 deficiency exhibit adipocyte hypertrophy and impaired lipolysis. Use of a nanosystem comprising P3‐peptide, chitosan oligosaccharide lactate, and polyethylene glycol that controls Vanin‐1 expression in the abdominal WAT shows that WAT‐specific Vanin‐1 knockdown blocks fasting‐induced lipolysis and prevents WAT loss. However, WAT‐specific Vanin‐1 mRNA restoration rescues impaired lipolysis and improves glucose/insulin intolerance in diabetic db/db mice. Mechanistically, Vanin‐1 induces PPARγ activity and subsequently facilitates its activation on the proximal promoters of lipolytic genes. Thus, an essential role of Vanin‐1 in the regulation of lipolysis and adiposity is revealed, and a functional RNA delivering strategy for specific intervention of Vanin‐1 expression in WAT is shown. These findings provide a promising approach to treat metabolic diseases caused by dysregulation of Vanin‐1 and lipolysis.

AKT phosphorylation in abdominal WAT, liver and skeletal muscle. * P < 0.05 and ** P < 0.01 vs. Scra siRNA/NPs group. n=5. All values are presented as the mean ± SD. Unpaired Student's t-test was used for comparison between two groups.   Table S2. Lists of primers used for the plasmid construction.

RT-qPCR and western blot analyses
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse  were photographed with a Nikon microscope (ECLIPSE, Ts2R-FL, Tokyo, Japan).

Enzymatic activity
Pantethenase activity was determined as described before [1][2] . In brief, abdominal WAT was

MRI & body composition measurements
To assess the body composition, WT and Vanin-1 -/mice were received micro-MRI examination.

Total GSH and ROS levels
Total GSH and ROS levels in mouse abdominal WAT were quantified by using commercial kits (Beyotime, Shanghai, China for total GSH concentration, Senbeijia Biological Technology, Nanjing, Jiangsu, China for ROS generation) according to manufacturer's instructions.

Mouse primary adipocyte isolation and differentiation
Mouse primary pre-adipocytes were isolated from the mouse abdominal WAT as described previously [3] , and cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Sciencell Research, Carlsbad, CA, USA). For differentiation assays, mouse primary preadipocytes were grown to confluence and then switch to differentiation medium (DMEM with 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 125 nM indomethacin, 20 nM insulin and 1 nM T3). The cells were subsequently maintained in differentiation medium for up to 6 days.

mtDNA content
Total DNA was isolated from white adipose tissues for the quantitation of mtDNA content. RT-qPCR primers for mtDNA (mtND1 and mtCox1) and nuclear DNA (PECAM) are listed in table   S3.

Glucose and insulin tolerance tests
For the glucose tolerance test (GTT), mice were fasted for 16 h and then injected intraperitoneal

Tissue lipid analysis
For lipid measurements in tissues, 50 mg samples were homogenized, TG and TC contents were measured using commercial kits (Jiancheng Institute of Biotechnology, Nanjing, Jiangsu, China) according to the manufacturer's instructions.

Preparation of modified Vanin-1 mRNA
The pGADT7-AD vector carrying the T7 promoter and HA tag was obtained from MiaoLing and used as templates for in vitro transcription. The modified Vanin-1 mRNA was synthesized as described previously [4][5] . Note the templates content was 1.6 μg in our settings.

Physicochemical characterization and stability of PCP NPs in serum condition.
To check the in vitro stability of siRNA or mRNA/PCP NPs in serum conditions, mRNA NPs were incubated in 10% bovine serum containing PBS solution at 37 °C in triplicate for various time periods (0, 3, 6, 12, 24 and 48 h) with 100 rpm shaking. An aliquot of NP solution was taken for particle size measurement using Mastersizer Micro (ZEN3690, Malvern instruments limited, UK).

Hemolysis assay
Hemolysis experiments were performed according to previous reports. Blood samples were freshly obtained from C57BL/6J mice, and centrifuged at 3000 rpm for 10 min. After removing the plasma, red blood cell (RBC) pellet was washed five times with 1 mL of PBS solution, and then diluted in 0.5 mL PBS solution. PCP NPs solutions in PBS at different concentrations were added to 0.5 mL RBC suspension. Note that the positive and negative control samples were prepared by adding 0.5 mL of water and PBS, respectively. The samples were shaken in a shaker incubator at 37℃ for 0.5 h. After centrifugation, images were captured for visual comparison, and the absorbance for each supernatant was measured by using a microplate reader at 541 nm.
Hemolysis percentages of the RBCs were calculated as previously described [6] .

Mechanism of cellular uptake of PCP NPs.
To determine the uptake mechanism of PCP NPs, mouse primary adipocytes were pre-incubated in serum-free medium containing indicated inhibitors for 30 min. Among which, filipin (1 μg/mL), chlorpromazine (10 μg/mL), EIPA (10 μg/mL) were used to block caveolae-mediated endocytosis, clathrin-mediated endocytosis, micropinocytosis, respectively. The cells were then treated with Cy5-labelled PCP NPs at a concentration of 100 μg/mL. After 24 h incubation, the old medium was replaced with fresh complete medium and incubated for an additional 24 h. DAPI (blue) staining for nuclear localization was performed simultaneously. The cells were then fixed to check red fluorescence by a confocal laser scanning microscope (CLSM, LSM700, Zeiss, Germany) and processed using the ZEN imaging software.

Biodistribution of PCP NPs
For the in vivo biodistribution study, C57BL/6J mice received an i.v. injection of PBS, Cy5-

CCK-8 assay
CCK-8 assay was used to qualify the cell toxicity of NPs. In brief, 5×10 3 mouse primary preadipocytes were seeded into each well of a 96-well plate and grown into 100% confluence.
After transformation, mature adipocytes were synchronized with serum-free DMEM, cells were transferred into 100 μL of serum-free DMEM containing either indicated doses of PCP NPs and incubated for 24 h. Then, 10 μL of WST-8 reagent (Jiancheng, Nanjing, Jiangsu, China) was added to each well and incubated at 37°C for 2 h. Finally, a microplate reader was used to measure the absorbance at 450 nm.

Tissue explants
Abdominal WAT (20 mg) was isolated from mice and washed three times with PBS buffer as previously described [3] . After 3-h incubation, NEFAs and glycerol released from the explants were determined using commercial kits (NEFAs: Wako Chemicals, Richmond, VA, USA; Glycerol: Jiancheng Institute of Biotechnology, Nanjing, Jiangsu, China).