Increased transport of acetyl‐CoA into the endoplasmic reticulum causes a progeria‐like phenotype

Abstract The membrane transporter AT‐1/SLC33A1 translocates cytosolic acetyl‐CoA into the lumen of the endoplasmic reticulum (ER), participating in quality control mechanisms within the secretory pathway. Mutations and duplication events in AT‐1/SLC33A1 are highly pleiotropic and have been linked to diseases such as spastic paraplegia, developmental delay, autism spectrum disorder, intellectual disability, propensity to seizures, and dysmorphism. Despite these known associations, the biology of this key transporter is only beginning to be uncovered. Here, we show that systemic overexpression of AT‐1 in the mouse leads to a segmental form of progeria with dysmorphism and metabolic alterations. The phenotype includes delayed growth, short lifespan, alopecia, skin lesions, rectal prolapse, osteoporosis, cardiomegaly, muscle atrophy, reduced fertility, and anemia. In terms of homeostasis, the AT‐1 overexpressing mouse displays hypocholesterolemia, altered glycemia, and increased indices of systemic inflammation. Mechanistically, the phenotype is caused by a block in Atg9a‐Fam134b‐LC3β and Atg9a‐Sec62‐LC3β interactions, and defective reticulophagy, the autophagic recycling of the ER. Inhibition of ATase1/ATase2 acetyltransferase enzymes downstream of AT‐1 restores reticulophagy and rescues the phenotype of the animals. These data suggest that inappropriately elevated acetyl‐CoA flux into the ER directly induces defects in autophagy and recycling of subcellular structures and that this diversion of acetyl‐CoA from cytosol to ER is causal in the progeria phenotype. Collectively, these data establish the cytosol‐to‐ER flux of acetyl‐CoA as a novel event that dictates the pace of aging phenotypes and identify intracellular acetyl‐CoA‐dependent homeostatic mechanisms linked to metabolism and inflammation.

Scientific) was used to mount the cover slips. For imaging, Nikon-N-SIM (structured illumination microscopy) was used and data were analyzed by Imaris image analysis software (Bitplane).

Protein extraction, Western blotting, and immunoprecipitation
Protein extracts were prepared in GTIP buffer (10 mM Tris, pH 7.6, 2 mM EDTA, 0.15 M NaCl) supplemented with 1% Triton TM X-100 (Roche Applied Science), 0.25% Nonidet P-40 (Roche Applied Science), complete protein inhibitor mixture (Roche Applied Science), and phosphatase inhibitors (mixture set I and set II; Calbiochem). Detergent-soluble and -insoluble fractions were prepared as described (Gan et al. 2012;Peng et al. 2016). Briefly, cells were lysed in lysis buffer (50 mM pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol), complete with protease inhibitors (Roche Applied Science) and 1% Triton TM X-100 (Buffer A), followed by centrifugation at 100,000 g for 30 min at 4 °C. Supernatants were recovered as Triton-soluble fractions. Pellets were washed with Buffer A three times, then resuspended in lysis buffer containing Buffer A, 1% sodium dodecyl sulfate (SDS), and 0.5% sodium deoxycholate. After sonication and a brief spin down, the lysates were recovered as Triton-insoluble (SDS-soluble) fractions.
The ER isolation was prepared with a commercial ER Enrichment kit (Novus Biologicals), according to manufacturer's protocol. Briefly, 0.5 gram liver tissue were homogenized in isosmotic homogenization buffer using a Dounce Teflon homogenizer. The homogenized tissue was centrifuged at 1,000 g for 10 min at 4 °C and then again at 12,000 g for 15 min at 4 °C. Finally, the supernatants were centrifuged at 90,000 g for 60 min at 4 °C to obtain total ER fraction. The ER fractions were subjected to Western blot analysis or immunoprecipitation.
One microgram of liver extract enriched in either extracellular, intracellular, membrane-associated, or formic acid-soluble proteins was diluted in PBS to a total volume of 3 µL, then dotted onto a nitrocellulose membrane (Bio-Rad) and allowed to dry. Once dry, membranes were rinsed twice in TBS and incubated for one hour at room temperature in 5% BSA (Sigma-Aldrich) in TBST. Membranes were then incubated overnight at 4 °C in 5% BSA in TBST, washed four times in TBST, and incubated for one hour at room temperature in TBST with Alexa Fluor® conjugated anti-mouse secondary antibody (LICOR Biosciences). After secondary incubation, membranes were washed four additional times in TBST, rinsed once in TBS, then imaged using the LICOR Odyssey Infrared Imaging System. Fluorescent signal for each dot was quantified using OptiQuant (Packard Cyclone, Perkin-Elmer Life Sciences, Inc.).

Real-time PCR
Real-time PCR was performed as described before (Jonas et al. 2010). Gene expression levels were

Histology and bone histomorphometry
Hematoxylin and eosin staining was done as described before Peng et al. 2014).
Briefly, tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Bone tissue was decalcified using a decalcifier solution (Decalcifier I, Leica Biosystems) and embedded in paraffin.
Paraffin-embedded tissues were sectioned at 5 microns and stained with hematoxylin and eosin.
Hematoxylin and eosin stained slides were examined with a Zeiss Axiovert 200 inverted microscope.
The undecalcified bone histomophometry was performed as described below. Femurs dissected from 3 month-old mice were fixed in 70% ethanol and embedded in prepolymerized polymethyl methacrylate embedding media. The embedded bones were cut at 100 microns using a Saw Microtome (SP1600, Leica Systems). The sections were then subjected to modified Goldner's trichrome staining.

Faxitron radiography and dual-energy X-ray absorptiometry (DEXA)
Bones were fixed in 70% ethanol and soft tissue was removed from the fixed bones. Radiography was performed using a Hewlett Packard Faxitron X-ray system (24 KV for 1.3 min, model 43855A; Hewlett Packard, McMinnville, OR). Bone mineral density (BMD) and total body fat mass were determined using the PIXI-mus small animal dual-energy X-ray absorptiometry (DEXA) device (Lunar) following standard manufacturer protocols. Calibrations were performed with a phantom of known density, and quality assurance measurements were performed prior to BMD measurements.

Whole blood, serum and plasma analytes
Blood was collected transcardially from mice with an insulin syringe. Hematologic parameters were measured on a HemaVet complete blood count (CBC) instrument. For serum, blood was allowed to clot on ice for 15 min, then centrifuged at 1,000 g for 10 min at 4 °C and the supernatant was collected. For plasma, blood was collected in BD Microtainer® tubes with K2EDTA and allowed to clot on ice for 15 min. Following centrifugation at 1,000 g for 10 min at 4 °C, the supernatant was collected. Ferritin and iron levels were assayed in serum using Mouse Ferritin ELISA Kit (FTL) (Abcam) and Iron Assay Kit (Abcam), respectively according to manufacturer's instructions. The lipid serum profile was performed by the UW-Clinical Laboratory. Inflammatory molecules were determined in plasma by Ampersand Biosciences.

Blood and bone marrow smear examination
Fresh whole blood or bone marrow samples were smeared uniformly across a glass slide using another glass slide at a 30 degree smearing angle. The smeared glass slides were then air-dried and stained with PROTOCOL TM Hema 3 TM Stain kit (ThermoFisher Scientific) according to manufacturer's instructions. The Hema 3 TM stained slides were finally examined with a Zeiss Axiovert 200 inverted microscope.

Senescence associated β-galactosidase staining
Primary hepatocytes and cryosections of mouse liver were stained with Senescence β Galactosidase Staining kit (Cell Signaling Technology) according to manufacturer's protocol. Briefly, primary hepatocytes or mouse liver cryosections were fixed with fixation solution for 15 minutes. Fixed sections or cells were then stained with β-galactosidase staining at 37 o C overnight in a dry incubator without carbon dioxide. Primary hepatocytes were counterstained with DAPI to visualize nuclei. The percentage of senescent cells was expressed as the total number of senescent cells divided by the total number of cells counted using immunofluorescence.

OGTT, Glucose, Insulin and Glucagon assays
Oral glucose tolerance tests (oGTT) were performed on 4-hour fasted (6am-10am) mice. Glucose (2g/kg in water) was administered via oral gavage. Blood was collected by retro-orbital collection prior to glucose administration, and at 5, 15, 30, 60, and 120 minutes following the glucose bolus. Plasma was used to determine glucose and insulin levels. Glucose was measured by the glucose oxidase method using a commercially available kit (TR15221, ThermoFisher Scientific). Insulin was measured by radioimmunoassay (RIA; Millipore). Glucagon was measured by radioimmunoassay (RIA; Millipore).

Echocardiography
Transthoracic echocardiography was performed using a Visual Sonics 770 ultrasonography with a 30-MHz transducer (RMV 707B) (Visual Sonics, Toronto) as described previously (Harris et al. 2002). Mice were lightly anesthetized with isoflurane (1%) and maintained on a heated platform. Two-dimensionally guided M-mode images of the LV and Doppler studies were acquired at the tip of the papillary muscles.
LV mass-to-body weight ratio (LV/BW), LV dimension in diastole (LVDd), thickness of the anterior and posterior walls in diastole, and isovolumic relaxation time were recorded. All parameters were measured over at least three consecutive cycles.

Trafficking of newly synthetized glycoproteins
Quantification of trafficking glycoproteins along the secretory pathway was performed as previously described . Briefly, nascent glycoproteins in primary hepatocytes were labeled with Click-It TM ManNAz reagent (tetraacetylated N-Azidoacetyl-D-Mannosamine; ThermoFisher Scientific), and visualized with the Click-It TM Cell Reaction Buffer kit (ThermoFisher Scientific). Primary hepatocytes were counterstained with DAPI (62248, ThermoFisher Scientific) to visualize nuclei. The percentage of MaNAz-labeled cells was expressed as the total number of ManNaz-labeled cells divided by the total number of cells counted, using immunofluorescence.