Advertisement

Deletion of LDLRAP1 Induces Atherosclerotic Plaque Formation, Insulin Resistance, and Dysregulated Insulin Response in Adipose Tissue

  • Tani Leigh
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Tatsuo Kawai
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Kyle Preston
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Sheri Kelemen
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Rachael Okune
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Amanda St Paul
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Cali Corbett
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Amanda M. Peluzzo
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Jun Yu
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Rosario G. Scalia
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
  • Michael V. Autieri
    Correspondence
    Address correspondence to Michael V. Autieri, Ph.D., Temple University School of Medicine, Room 1050, MERB, 3500 N. Broad St., Philadelphia, PA 19140.
    Affiliations
    Department of Cardiovascular Sciences, Lemole Center for Integrated Lymphatics Research, Independence Blue Cross Cardiovascular Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
    Search for articles by this author
      Dyslipidemia, vascular inflammation, obesity, and insulin resistance often overlap and exacerbate each other. Mutations in low density lipoprotein receptor adaptor protein-1 (LDLRAP1) lead to LDLR malfunction and are associated with the autosomal recessive hypercholesterolemia disorder in humans. However, direct causality on atherogenesis in a defined preclinical model has not been reported. The objective of this study was to test the hypothesis that deletion of LDLRAP1 will lead to hypercholesteremia and atherosclerosis. LDLRAP1−/− mice fed a high-fat Western diet had significantly increased plasma cholesterol and triglyceride concentrations accompanied with significantly increased plaque burden compared with wild-type controls. Unexpectedly, LDLRAP1−/− mice gained significantly more weight compared with controls. Even on a chow diet, LDLRAP1−/− mice were insulin-resistant, and calorimetric studies suggested an altered metabolic profile. The study showed that LDLRAP1 is highly expressed in visceral adipose tissue, and LDLRAP1−/− adipocytes are significantly larger, have reduced glucose uptake and AKT phosphorylation, but have increased CD36 expression. Visceral adipose tissue from LDLRAP1−/− mice was hypoxic and had gene expression signatures of dysregulated lipid storage and energy homeostasis. These data are the first to indicate that lack of LDLRAP1 directly leads to atherosclerosis in mice and also plays an unanticipated metabolic regulatory role in adipose tissue. LDLRAP1 may link atherosclerosis and hypercholesterolemia with common comorbidities of obesity and insulin resistance.
      Atherosclerotic vascular syndromes remain a considerable medical and socioeconomic problem, and contribute to the mortality of many conditions such as myocardial infarction, stroke, and peripheral vascular disease. Clarification of the molecular mechanisms of lipoprotein metabolism and the systemic effects of hyperlipidemia is essential to better understand and treat atherosclerosis and other chronic diseases. Metabolic syndrome is a grouping of conditions that includes obesity, high blood pressure, high blood sugar, and high serum triglycerides, and often occurs in concert with hypercholesterolemia and insulin resistance. Clinically, these conditions often overlap and exacerbate each other, implying common cellular and molecular mechanisms in diverse metabolic organs. In the obese animals, expanding visceral fat depots secrete proinflammatory adipokines systemically that exacerbate cardiovascular and other diseases.
      • Grant R.W.
      • Dixit V.D.
      Adipose tissue as an immunological organ.
      Indeed, ApoE−/− mice transplanted with visceral fat have increased levels of circulating proinflammatory cytokines and develop significantly more atherosclerosis compared with mice transplanted with subcutaneous fat.
      • Ohman M.K.
      • Shen Y.
      • Obimba C.I.
      • Wright A.P.
      • Warnock M.
      • Lawrence D.A.
      • Eitzman D.T.
      Visceral adipose tissue inflammation accelerates atherosclerosis in apolipoprotein E-deficient mice.
      Nevertheless, pathways and molecules that may link obesity, insulin resistance, and vascular inflammatory diseases such as atherosclerosis, are not yet clearly elucidated. Recognizing this gap in knowledge, one key to treat these diseases is to identify molecules and cellular pathways in common with each condition.
      One lipoprotein receptor, the low density lipoprotein receptor (LDLR), plays a key role in LDL uptake and regulation of plasma LDL concentration.
      • van de Sluis B.
      • Wijers M.
      • Herz J.
      News on the molecular regulation and function of hepatic low-density lipoprotein receptor and LDLR-related protein 1.
      ,
      • Au D.T.
      • Strickland D.K.
      • Muratoglu S.C.
      The LDL receptor-related protein 1: at the crossroads of lipoprotein metabolism and insulin signaling.
      The LDLR enables endocytosis of LDL by clustering within clathrin-coated regions on the plasma membrane. Accumulating evidence indicates that in the presence of increased plasma fatty acid concentrations, LDLR internalization, endocytoplasmic trafficking, and plasma membrane translocation are dysregulated, with a concomitant association with insulin resistance.
      • Descamps O.
      • Bilheimer D.
      • Herz J.
      Insulin stimulates receptor-mediated uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in adipocytes.
      ,
      • Laatsch A.
      • Merkel M.
      • Talmud P.J.
      • Grewal T.
      • Beisiegel U.
      • Heeren J.
      Insulin stimulates hepatic low density lipoprotein receptor-related protein 1 (LRP1) to increase postprandial lipoprotein clearance.
      Dyslipidemia is associated with indicators of metabolic syndrome such as atherosclerosis, insulin insensitivity, and obesity, indicating that a dysfunctional LDLR can influence more than vascular symptoms.
      • Hurtado-Roca Y.
      • Bueno H.
      • Fernandez-Ortiz A.
      • Ordovas J.M.
      • Ibañez B.
      • Fuster V.
      • Rodriguez-Artalejo F.
      • Laclaustra M.
      Oxidized LDL is associated with metabolic syndrome traits independently of central obesity and insulin resistance.
      For example, LRP1 (LDLR related-protein 1) is translocated to the plasma membrane in response to insulin,
      • Liu C.-C.
      • Hu J.
      • Tsai C.-W.
      • Yue M.
      • Melrose H.L.
      • Kanekiyo T.
      • Bu G.
      Neuronal LRP1 regulates glucose metabolism and insulin signaling in the brain.
      and hepatocyte-specific LRP1 knockout mice are insulin-resistant.
      • Ding Y.
      • Xian X.
      • Holland W.L.
      • Tsai S.
      • Herz J.
      Low-density lipoprotein receptor-related protein-1 protects against hepatic insulin resistance and hepatic steatosis.
      Proteins involved in regulation of these pathways may represent targets for therapy to treat vascular and metabolic diseases.
      Low density lipoprotein receptor adaptor protein 1 (LDLRAP1) is a cytosolic adaptor protein residing in clathrin-coated pits that interacts with the cytoplasmic tail of the LDL receptor, mediating endocytosis of the LDL–LDLR complex.
      • Brown M.S.
      • Goldstein J.L.
      A receptor-mediated pathway for cholesterol homeostasis.
      LDLRAP1 internalizes LDLR, delivering into plasma endosomes, where it is either recycled into the plasma membrane, or lysosomes, where it is degraded.
      • He G.
      • Gupta S.
      • Yi M.
      • Michaely P.
      • Hobbs H.H.
      • Cohen J.C.
      ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2.
      • Garuti R.
      • Jones C.
      • Li W.-P.
      • Michaely P.
      • Herz J.
      • Gerard R.D.
      • Cohen J.C.
      • Hobbs H.H.
      The modular adaptor protein autosomal recessive hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits.
      • Mishra S.K.
      • Watkins S.C.
      • Traub L.M.
      The autosomal recessive hypercholesterolemia (ARH) protein interfaces directly with the clathrin-coat machinery.
      Proper LDLR homeostasis requires exquisitely regulated endosomal trafficking and sorting transportation machinery to ensure its proper destination when it engages with LDL.
      • Sirinian M.I.
      • Belleudi F.
      • Campagna F.
      • Ceridono M.
      • Garofalo T.
      • Quagliarini F.
      • Verna R.
      • Calandra S.
      • Bertolini S.
      • Sorice M.
      • Torrisi M.R.
      • Arca M.
      Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes.
      Mutations in LDLRAP1 lead to LDL receptor malfunction and cause the autosomal recessive hypercholesterolemia (ARH) disorder in humans.
      • Garcia C.K.
      • Wilund K.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Maioli M.
      • Calandra S.
      • Bertolini S.
      • Cossu F.
      • Grishin N.
      • Barnes R.
      • Cohen J.C.
      • Hobbs H.H.
      Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.
      ,
      • Zuliani G.
      • Arca M.
      • Signore A.
      • Bader G.
      • Fazio S.
      • Chianelli M.
      • Bellosta S.
      • Campagna F.
      • Montali A.
      • Maioli M.
      • Pacifico A.
      • Ricci G.
      • Fellin R.
      Characterization of a new form of inherited hypercholesterolemia: familial recessive hypercholesterolemia.
      ARH patients that lack functional LDLRAP1 in the liver have much lower clearance of LDL from the circulation compared with normal subjects.
      • Garcia C.K.
      • Wilund K.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Maioli M.
      • Calandra S.
      • Bertolini S.
      • Cossu F.
      • Grishin N.
      • Barnes R.
      • Cohen J.C.
      • Hobbs H.H.
      Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.
      This hypercholesterolemia is assumed to be a result of impaired LDL internalization by hepatocytes, because disruption of LDLRAP1 results in 80% reduction in LDL internalization in cultured hepatocytes.
      • Garuti R.
      • Jones C.
      • Li W.-P.
      • Michaely P.
      • Herz J.
      • Gerard R.D.
      • Cohen J.C.
      • Hobbs H.H.
      The modular adaptor protein autosomal recessive hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits.
      ,
      • Sirinian M.I.
      • Belleudi F.
      • Campagna F.
      • Ceridono M.
      • Garofalo T.
      • Quagliarini F.
      • Verna R.
      • Calandra S.
      • Bertolini S.
      • Sorice M.
      • Torrisi M.R.
      • Arca M.
      Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes.
      ,
      • Jones C.
      • Hammer R.E.
      • Li W.-P.
      • Cohen J.C.
      • Hobbs H.H.
      • Herz J.
      Normal sorting but defective endocytosis of the low density lipoprotein receptor in mice with autosomal recessive hypercholesterolemia.
      ,
      • Jones C.
      • Garuti R.
      • Michaely P.
      • Li W.-P.
      • Maeda N.
      • Cohen J.C.
      • Herz J.
      • Hobbs H.H.
      Disruption of LDL but not VLDL clearance in autosomal recessive hypercholesterolemia.
      However, the cellular effects of LDLRAP1 are known to be cell-type specific; for example, hepatocytes and lymphocytes require LDLRAP1 for normal LDLR functioning, whereas fibroblasts cultured from ARH patients have normal internalization of LDL.
      • Arca M.
      • Zuliani G.
      • Wilund K.
      • Campagna F.
      • Fellin R.
      • Bertolini S.
      • Calandra S.
      • Ricci G.
      • Glorioso N.
      • Maioli M.
      • Pintus P.
      • Carru C.
      • Cossu F.
      • Cohen J.
      • Hobbs H.H.
      Autosomal recessive hypercholesterolaemia in Sardinia, Italy, and mutations in ARH: a clinical and molecular genetic analysis.
      Although associated with atherogenesis, surprisingly, no preclinical investigation into direct causality of nonfunctional LDLRAP1 on development of atherogenesis has been reported. The purpose of this study was to test the hypothesis that deletion of LDLRAP1 would lead to hypercholesteremia and increased atherosclerosis. Although deletion of LDLRAP1 led to increased plasma cholesterol and atherosclerotic plaque burden, LDLRAP1−/− mice also surprisingly gained significantly more weight than control mice and were insulin resistant even on a chow diet. LDLRAP1’s effects on obesity, metabolism, and insulin sensitivity have not been previously reported. In wild-type mice, LDLRAP1 was highly expressed in adipocytes and visceral adipose tissue (VAT) in LDLRAP1−/− mice was inflammatory, hypoxic, and contained hypertrophic adipocytes. Insulin signaling and CD36 expression was dysregulated, and glucose uptake in chow-fed LDLRAP1−/− adipocytes was attenuated. VAT in LDLRAP1−/− mice displayed altered gene expression patterns indicative of dysregulated insulin sensitivity and metabolism. Taken together, the results showed that in addition to a recognized function in LDLR and cholesterol internalization, LDLRAP1 plays a central role in adipocyte biology and participates in adipose metabolism and regulation of insulin sensitivity and adipose metabolic processes. This implicates LDLRAP1 as a key molecule and potential therapeutic target in treatment of diseases that collectively drive metabolic syndrome.

      Materials and Methods

      Mice and Study Design

      Mice heterozygous for LDLRAP1 on the B6/129SF1/J background were purchased (stock no. 005212; The Jackson Laboratory, Bar Harbor, ME) and mated to produce a LDLRAP1 homozygous knockout line (LDLRAP1−/−). B6/129SF1/J mice (stock no. 101043) were purchased and used as wild-type controls as indicated by the Jackson Laboratory website. Starting at 8 weeks of age, LDLRAP1−/− and wild-type mice of both sexes were fed a high-fat, high-cholesterol Western diet (40% of total calories as fat, 1% cholesterol; stock no. 5TJT; TestDiet, St. Louis, MO) for 16 weeks. Fasting cholesterol and triglyceride serum levels (mg/dL) from mice on a chow or Western diet were analyzed by the Vanderbilt University Medical College Lipid Core (Nashville, TN). All animal procedures followed protocols approved by the Temple University Institutional Animal Care and Use Committee.
      Bone-marrow–derived macrophages (BMDM) were generated as previously described.
      • Ellison S.
      • Gabunia K.
      • Kelemen S.E.
      • England R.N.
      • Scalia R.
      • Richards J.M.
      • Orr A.W.
      • Traylor Jr., J.G.
      • Rogers T.
      • Cornwell W.
      • Berglund L.M.
      • Goncalves I.
      • Gomez M.F.
      • Autieri M.V.
      Attenuation of experimental atherosclerosis by interleukin-19.
      Briefly, to generate BMDM, mouse femurs and tibiae were flushed with sterile Dulbecco’s modified Eagle medium. Total bone marrow cells were plated at a density of 3.5 × 106 cells per 10-cm Petri dish in macrophage growth medium (complete Dulbecco’s modified Eagle medium with 10% fetal bovine serum and 100 ng/mL M-CSF (PeproTech Inc., Cranbury, NJ) and allowed to differentiate for 7 days, after which cells were detached with Versene 1× solution (Gibco; Thermo Fisher Scientific, Waltham, MA) and replated in macrophage complete medium (Dulbecco’s modified Eagle medium + 10% fetal bovine serum).

      Atherosclerotic Lesion Analysis

      Atherosclerotic plaque formation in the aortic arch was examined by en face staining with Sudan IV. Stained area was calculated as a percentage of the aortic arch as the authors have described.
      • Ellison S.
      • Gabunia K.
      • Kelemen S.E.
      • England R.N.
      • Scalia R.
      • Richards J.M.
      • Orr A.W.
      • Traylor Jr., J.G.
      • Rogers T.
      • Cornwell W.
      • Berglund L.M.
      • Goncalves I.
      • Gomez M.F.
      • Autieri M.V.
      Attenuation of experimental atherosclerosis by interleukin-19.
      ,
      • Gabunia K.
      • Ellison S.
      • Kelemen S.
      • Kako F.
      • Cornwell W.D.
      • Rogers T.J.
      • Datta P.K.
      • Ouimet M.
      • Moore K.J.
      • Autieri M.V.
      IL-19 halts progression of atherosclerotic plaque, polarizes, and increases cholesterol uptake and efflux in macrophages.

      Immunohistochemistry

      Tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, deparaffinized in xylene, and then rehydrated through graded ethanol and counterstained with hematoxylin. Adipose tissue was immunostained with HIF1α antibody (Cat# NB100-479; Novus Biologicals, Centennial, CO). Adipocyte size was quantitated from formalin-fixed, paraffin-embedded tissue using ImageJ software version 1.53a (NIH, Bethesda, MD; http://imagej.nih.gov/ij) from at least three high-power fields from three sections from three different mice were quantitated.

      Metabolic Cage Analysis, Insulin Tolerance Test, and Insulin Levels

      Age-matched LDLRAP1−/− and wild-type male mice were entered into the study at 8 weeks of age where they were either fed a chow or high-fat diet (HFD) for 16 weeks in normal housing before being placed in the cages. After this time, mice were individually housed in TSE PhenoMaster metabolic cages (TSE Systems, Chesterfield, MO) for 96 hours. Measurements taken during the first 24 hours were disregarded because this time was used as an acclimation period. Measurements were taken every 15 minutes, and results for the following parameters were calculated: Carbon dioxide production (mL/hour), respiratory exchange ratio (carbon dioxide/oxygen), energy expenditure (kcal/hour), energy balance (kcal/hour), hourly food consumption (kcal/hour), total food consumed (kcal), total water consumed (mL), locomotor activity (beam breaks), and ambulatory activity (beam breaks). The data analysis was conducted over a 72-hour time frame.
      • Mina A.I.
      • LeClair R.A.
      • LeClair K.B.
      • Cohen D.E.
      • Lantier L.
      • Banks A.S.
      CalR: a web-based analysis tool for indirect calorimetry experiments.
      All data were analyzed and statistics were performed using the CalR software version 1.2 (Banks Lab; https://CalRApp.org)
      • Mina A.I.
      • LeClair R.A.
      • LeClair K.B.
      • Cohen D.E.
      • Lantier L.
      • Banks A.S.
      CalR: a web-based analysis tool for indirect calorimetry experiments.
      as part of the software package. The CalR program implements the most widely accepted method of analysis, which is the analysis of covariance where appropriate, and the generalized linear model where analysis of covariance is not appropriate. In each case, body mass is utilized as a covariate when modeling mass-dependent metabolic parameters. For insulin tolerance, mice as described above were starved for 4 hours, then blood obtained from the tail vain was taken to measure baseline blood glucose. Mice were then injected with 0.75 U of insulin, then blood was obtained at 15, 30, 45, 60, and 120 minutes. Blood glucose test strips were read by the AlphaTRAK Blood Glucose Monitoring System (Zoetis, Parsippany-Troy Hills, NJ).
      Insulin levels were determined at the Vanderbilt University Hormone and Analytical services core facility using a commercially available radioimmunoassay (Millipore Cat. # RI-13K; Sigma-Aldrich, St. Louis, MO). The assay utilizes I125-labeled insulin and a double antibody technique to determine serum insulin levels. The assay was modified by the Vanderbilt Hormone and Analytical Services Core to improve the sensitivity to 0.01 ng/mL. Serum from at least six mice per group were used. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by dividing the product of fasted glucose and insulin by 405 [(glucose × insulin)/405] as previously described.
      • Berglund E.D.
      • Li C.Y.
      • Poffenberger G.
      • Ayala J.E.
      • Fueger P.T.
      • Willis S.E.
      • Jewell M.M.
      • Powers A.C.
      • Wasserman D.H.
      Glucose metabolism in vivo in four commonly used inbred mouse strains.

      Glucose Uptake

      Glucose uptake by adipocytes isolated from male wild-type and LDLRAP1−/− mice on chow diet was quantified using a kit from Promega (#J1342; Promega, Madison, WI) according to the manufacturer's instructions. Briefly, the assay is based on glucose uptake in mammalian cells based on the detection of nonradioactive 2-deoxyglucose-6-phosphate (2DG6P). Reagent containing glucose-6-phosphate dehydrogenase (G6PDH), NADP+, reductase, and recombinant luciferase and proluciferin substrate is added to the sample wells. G6PDH oxidizes 2DG6P to 6-phosphodeoxygluconate and simultaneously reduces NADP+ to NADPH. The reductase uses NADPH to convert the proluciferin to luciferin, which is then used by Ultra-Glo Recombinant Luciferase to produce a luminescent signal that is proportional to the concentration of 2DG6P.

      RNA Isolation, Adipose Fractionation, and Quantitative RT-PCR

      RNA was isolated from tissue using the RNeasy PowerLyzer Tissue & Cells Kit (QIAGEN, Hilden, Germany) and Direct-zol RNA Miniprep Plus (Zymo Research, Irvine, CA), according to the manufacturer's protocol, and 100 ng of RNA from each condition was reverse transcribed as the authors have described.
      • Herman A.B.
      • Vrakas C.N.
      • Ray M.
      • Kelemen S.E.
      • Sweredoski M.J.
      • Moradian A.
      • Haines D.S.
      • Autieri M.V.
      FXR1 is an IL-19-responsive RNA-binding protein that destabilizes pro-inflammatory transcripts in vascular smooth muscle cells.
      ,
      • Ray M.
      • Gabunia K.
      • Vrakas C.N.
      • Herman A.B.
      • Kako F.
      • Kelemen S.E.
      • Grisanti L.A.
      • Autieri M.V.
      Genetic deletion of IL-19 (interleukin-19) exacerbates atherogenesis in Il19−/−×Ldlr−/− double knockout mice by dysregulation of mRNA stability protein HuR (Human Antigen R).
      Target genes were amplified using SYBR green and gene-specific primers, in triplicate, using an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Results were normalized using the housekeeping gene GAPDH. Multiple mRNAs (CT values) were quantitated simultaneously by the StepOne Software version 2.3 (Thermo Fisher Scientific), and relative gene expression was calculated. Primer pairs were purchased from Integrated DNA Technologies (Coralville, IA). The following primer pairs were used:
      GAPDH: F: 5′-GGAGAAACCTGCCAAGTATGA-3′ R: 5′-TCCTCAGTGTAGCCCAAGA-3′
      LDLRAP1: F: 5′-CACTAGCCAGCTCATCGAGA-3′R: 5′-CACCTTGTCGTGCATCTTGT-3′
      HIF1α: F: 5′-CCTGCACTGAATCAAGAGGTTGC-3′ R: 5′-CCATCAGAAGGACTTGCTGGCT-3′
      CD36: F: 5′-GATGACGTGGCAAAGAACAG-3′ R: 5′-TCCTCGGGGTCCTGAGTTAT-3′

      PCR Array

      Gene expression in VAT from wild-type or LDLRAP1−/− mice was performed using the RT2 Profiler PCR Array Mouse Adipogenesis, Cat# PAMM-049Z from QIAGEN as described by the manufacturer. The results are graphed as SD from independent experiments. For separation of adipocytes from the stromal vascular fraction (SVF), RNA was isolated from the adipocytes separated from the stromal vascular layer from wild-type mice as described.
      • Kilroy G.
      • Dietrich M.
      • Wu X.
      • Gimble J.M.
      • Floyd Z.E.
      Isolation of murine adipose-derived stromal/stem cells for adipogenic differentiation or flow cytometry-based analysis.
      Briefly, epididymal adipose tissue was removed, cut into small pieces, digested with collagenase, shaken for 2 hours at 37°C, and then poured through a 100-μm filter and centrifuged at 600 × g for 15 minutes. The top layer consisting of adipocytes and the bottom pellet consisting of stromal vascular cells were collected and RNA isolated as described.

      Western Blot Analysis

      Protein extracts were made from VAT collected from male LDLRAP1−/− and wild-type mice, cut into pieces. Tissue was homogenized in a lysis buffer using TissueLyser LT (QIAGEN) for 5 minutes at 50 Hz. Supernatants after centrifugation (14,000 × g, 15 minutes at 4°C) were used for Western blotting. After centrifugation, supernatant was collected and added to 5× SDS running buffer and double-distilled water to obtain a protein concentration of 100 μg. Protein extracts were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and equal loading of protein extracts on gels was verified by Ponceau S staining of the membrane and blotting with the housekeeping protein anti-GAPDH; reactive proteins were visualized using enhanced chemiluminescence. The following antibodies were used for immunoblotting: CD36 Invitrogen, Cat# PA5-27236 (Thermo Fisher Scientific), ARH (LDLRAP1) LSBio, Cat# LS-C377809 (Lifespan Biosciences, Seattle, WA), GAPDH Cell Signaling, Cat# 2118S (Cell Signaling Technology, Danvers, MA), AKT Cell Signaling (cs9272) (Cell Signaling Technology), pAKT (Ser 473) Cell Signaling (cs9271s) (Cell Signaling Technology). Relative intensity of bands was quantitated by scanning image analysis and the ImageJ densitometry software program version 1.53a (NIH, Bethesda, MD; http://imagej.nih.gov/ij) and normalized to internal control proteins.

      Statistical Analysis

      Results are expressed as means ± SEM. Differences between groups were determined by t-test or two-way analysis of variance where appropriate using GraphPad Prism version 8.4.3 (GraphPad Software, San Diego, CA). Differences were considered statistically significant when P < 0.05. For all metabolic cage experiments, differences between groups were determined with one- or two-way analysis of variance with multiple comparisons, analysis of covariance, or the generalized linear model where appropriate using the CalR software version 1.2 as part of the cage software package.

      Results

      LDLRAP1 Knockout Mice Are Dyslipidemic and Develop Atherosclerotic Plaque on HFD

      Heterozygous mice obtained from The Jackson Laboratory were crossed to obtain homozygous LDLRAP1 knockout mice. Figure 1A demonstrates absence of the LDLRAP1 protein. To test the hypothesis that mice lacking LDLRAP1 would develop atherosclerotic plaque, mice were fed an atherogenic, Western diet (40% fat, 1% cholesterol) for 16 weeks, and aortic arches from these mice were removed and examined by en face staining of the aortic arch. Figure 1, B and C, show that whereas the wild-type and LDLRAP1+/− mice did not develop plaque, aortic arches from LDLRAP1−/− mice had significantly increased lesion area compared with those in the wild-type and LDLRAP1+/− mice. A wide variation in lesion areas in the LDLRAP1−/− cohort was observed, which upon further analysis mirrored sex of the mice. Although plaque accumulation was seen in both male and female LDLRAP1−/− mice, male mice developed significantly more plaque compared with female mice (13.0% ± 4.8% vs 3.0% ± 1.5%, respectively) (Figure 1D).
      Figure thumbnail gr1
      Figure 1Global knockout of LDLRAP1 increases atherosclerotic plaque formation. A: Western blot analysis of liver tissue from LDLRAP1−/− and wild-type mice immunoblotted with anti-LDLRAP1 and anti-GAPDH antibodies. B: Representative images of aortic arches from wild-type (left), LDLRAP1+/− (middle), and LDLRAP1−/− (right) male mice fed high-fat diet for 16 weeks; plaques were identified by en face Sudan IV staining and analysis. C: Quantification of percent plaque formation in aortic arches from wild-type, homozygous knockout, and heterozygous LDLRAP1 knockout mice. D: Quantification of percent plaque formation in aortic arches from male and female wild-type mice compared with male and female LDLRAP1−/− knockout mice. n = 14 wild-type mice (C); n = 19 homozygous knockout mice (C); n = 6 knockout mice (C). ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
      LDL receptor internalization participates in plasma cholesterol clearance, therefore, the hypothesis that global LDLRAP1 knockout would lead to an increase in plasma cholesterol levels was tested. Figure 2A shows that fasting LDLRAP1−/− mice had significantly higher total cholesterol on a chow diet (90.9 ± 5.8 vs 169.0 ± 6.8 mg/dL, P < 0.001, for wild-type and LDLRAP1−/− mice, respectively). LDLRAP1−/− mice also had significantly higher total cholesterol after 16 weeks on HFD (124.2 ± 14.6 vs 433.2 ± 31.4, P < 0.001 for wild-type and LDLRAP1−/− mice, respectively) (Figure 2A). Interestingly, there were no significant differences in plasma cholesterol between male and female LDLRAP1−/− mice under either dietary condition (data not shown). Fasting serum triglyceride levels were also measured. LDLRAP1−/− mice displayed significantly higher levels of serum triglycerides compared with the wild-type mice on both a chow (42.6 ± 5.1 vs 91.7 ± 13.8 mg/dL, P < 0.01, for wild-type and LDLRAP1−/− mice, respectively) and 16 weeks of HFD (58.0 ± 8.8 vs 147.0 ± 14.3 mg/dL, P < 0.001, for wild-type and LDLRAP1−/−, respectively) (Figure 2B). LDLRAP1−/− mice had significantly higher cholesterol and triglycerides than their wild-type counterparts regardless of sex (Figure 2, C and D).
      Figure thumbnail gr2
      Figure 2LDLRAP1−/− mice have significantly higher levels of serum cholesterol and triglycerides, and gain more weight compared with wild-type mice. A: Fasting (16 hours) serum cholesterol levels of LDLRAP1−/− and wild-type mice on chow and after 16 weeks of a HFD diet. B: Fasting serum triglyceride levels of LDLRAP1−/− and wild-type mice on 16 weeks of HFD. C: Cholesterol levels in male and female LDLRAP1−/− and wild-type mice on 16 weeks of HFD. D: Triglyceride levels in male and female LDLRAP1−/− and wild-type mice on 16 weeks of HFD. E: LDLRAP1−/− mice gain significantly more weight during 16 weeks of HFD compared with wild-type mice. F: Both male and female LDLRAP1−/− mice gain more weight on 16 weeks of HFD compared with their wild-type male and female counterparts. Asterisks indicate significant differences between LDLRAP1−/− and wild-type control mice. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. HFD, high-fat diet; WT, wild-type.

      LDLRAP1−/− Mice Have a Modified Metabolic Phenotype

      Mice were weighed prior to and upon completion of the atherosclerosis study. Mice entered the study with approximately similar weights (22.5 ± 4.0 g vs 24.5 ± 4.4 g for wild-type and LDLRAP1−/− mice, respectively). As expected, all mice gained weight during the 16-week study. However, Figure 2E shows that LDLRAP1−/− mice gained significantly more weight after HFD compared with wild-type mice (10.9 ± 0.9 g vs 17.1 ± 1.2 g, P < 0.01 for wild-type mice, n = 32, and LDLRAP1−/− mice, n = 28, respectively). Male and female LDLRAP1−/− mice gained significantly more weight compared with their wild-type counterparts (12.3 ± 1.0 vs 19.7 ± 1.4 g, P < 0.001, for wild-type and LDLRAP1−/− mice, respectively). Male LDLRAP1−/− mice gained significantly more weight compared with female LDLRAP1−/− mice (19.7 ± 1.4 g vs 13.9 ± 1.5 g, P < 0.01 for male and female mice, respectively) (Figure 2F).
      These significant differences in weight gain suggested that LDLRAP1−/− mice might have a modified metabolic phenotype. To determine whether LDLRAP1−/− mice display changes in insulin sensitivity, insulin tolerance tests were performed on LDLRAP1−/− and wild-type mice both before and after administering a HFD. For the insulin tolerance test, male mice were administered 0.75 U/kg of insulin after 16 hours fasting. On a chow diet, male LDLRAP1−/− mice had significantly higher blood glucose levels before and after insulin bolus injection compared with the wild-type mice (n = 9 and 11 for wild-type and LDLRAP1−/− mice, respectively) (Figure 3A). After 16 weeks of HFD, glucose levels were not significantly different between the LDLRAP1−/− and wild-type mice (n = 11 and 16 for wild-type and LDLRAP1−/− mice, respectively) (Figure 3B). This may be a result of the wild-type mice becoming obese and developing insulin resistance over the course of the HFD. On chow diet, female LDLRAP1−/− mice trended, but were not significantly insulin resistant compared with female wild-type mice. Importantly, on a chow diet, male mice had fasting glucose levels significantly higher than control mice (Figure 3C). There were no differences in female mice; further metabolic studies were performed on male mice (Figure 3D).
      Figure thumbnail gr3
      Figure 3LDLRAP1 knockout mice are insulin resistant on chow diet and HFD. A: Blood glucose levels in male LDLRAP1−/− and wild-type mice after i.p. injection of 0.75 U/kg insulin of mice on a chow diet. B: Blood glucose levels in male mice after 16 weeks of HFD diet after i.p. injection of 0.75 U/kg insulin. No significant differences between knockout and wild-type mice were noted. C: Fasting glucose levels in male mice on chow and HFD after being starved for 16 hours. D: Fasting glucose levels in female mice on chow and HFD after being starved for 16 hours. E: Serum insulin levels in male mice. LDLRAP1−/− mice trended higher. There were no significant differences in insulin levels between knockout and wild-type mice on chow diet, but insulin levels in LDLRAP1−/− mice were significantly higher compared with those in wild-type mice after 16 weeks on HFD. F: Mice are insulin resistant. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin levels as described in . Data were analyzed by two-way analysis of variance with multiple comparisons, or t-test where appropriate ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. HFD, high-fat diet; WT, wild-type.
      Serum insulin levels were checked in male mice starved for 16 hours. On a chow diet, although LDLRAP1−/− mice trended higher, there were no significant differences in insulin levels between knockout and wild-type mice (0.10 ± 0.04 ng/mL vs 0.25 ± 0.11 ng/mL for wild-type and LDLRAP1−/− mice, respectively). However, insulin levels in LDLRAP1−/− mice were significantly higher compared with those in wild-type mice after 16 weeks on HFD (0.48 ± 0.10 ng/mL, and 1.29 ± 0.32 ng/mL for wild-type and LDLRAP1−/− mice, respectively, P < 0.05) (Figure 3E). HOMA-IR is a mathematical model that describes insulin sensitivity. High HOMA-IR values indicate less sensitivity to insulin than low HOMA-IR values.
      • Parks B.W.
      • Sallam T.
      • Mehrabian M.
      • Psychogios N.
      • Hui S.T.
      • Norheim F.
      • Castellani L.W.
      • Rau C.D.
      • Pan C.
      • Phun J.
      • Zhou Z.
      • Yang W.P.
      • Neuhaus I.
      • Gargalovic P.S.
      • Kirchgessner T.G.
      • Graham M.
      • Lee R.
      • Tontonoz P.
      • Gerszten R.E.
      • Hevener A.L.
      • Lusis A.
      Genetic architecture of insulin resistance in the mouse.
      As expected, Figure 3F shows statistically significant differences in HOMA-IR index between chow- and HFD-fed mice, showing development of insulin resistance with HFD. Importantly, significant differences were noted between wild-type and LDLRAP1−/− mice on HFD, and a trend (P = 0.057) between wild-type and LDLRAP1−/− mice on the chow diet. These data suggest that the increased insulin level in LDLRAP1−/− mice is likely compensatory and indicative of insulin resistance. On a chow diet, although insulin resistant, the LDLRAP1−/− mice were not yet hyperinsulinemic, but became so after 16 weeks of HFD. Together, these data indicate that male LDLRAP1−/− mice are insulin-resistant, both on chow and on 16 weeks of HFD.
      To further characterize the metabolic effects of LDLRAP1 deletion, male LDLRAP1−/− and wild-type mice were individually housed in metabolic cages for a total of 96 hours (24 hours for acclimatization, 72 hours for data collection) either on a normal chow diet or after 16 weeks of consumption of a HFD. Every 15 minutes, data were recorded for carbon dioxide produced (mL/hour), energy expenditure (kcal/hour), energy balance (kcal/hour), food consumed (kcal), water consumed (mL), locomotor activity (beam breaks), and ambulatory activity (beam breaks). Weight was also quantitated each time the mouse entered a hanging glass tube. Male mice were used because female mice did not show any significant differences in insulin tolerance. Male LDLRAP1−/− mice gained more weight both on chow and HFD (Figure 4), although food consumption was not significantly different. LDLRAP1−/− mice showed increased energy expenditure as demonstrated by increased carbon dioxide production. However, it should be noted that under these experimental conditions, LDLRAP1−/− mice had increased ambulatory and locomotor activity compared with that of wild-type mice (n = 8). Because the relationship between locomotor activities and metabolic rate in mice is linear, it is possible that the observed changes in energy expenditure were due to the energetic cost of locomotion and not primary changes in the activity of metabolic organs.
      • Schefer V.
      • Talan M.I.
      Oxygen consumption in adult and AGED C57BL/6J mice during acute treadmill exercise of different intensity.
      Figure thumbnail gr4
      Figure 4LDLRAP1 global knockout leads to altered metabolic profile both on chow and on a high-fat diet. A: Mice on chow diet. B: Mice on HFD for 16 weeks. Mice were housed in metabolic cages as described in . After 24 hours’ acclimation, measurements were taken every 15 minutes for 72 hours. Data were analyzed and statistics were performed using the CalR software version 1.2 as part of the software package. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. HFD, high-fat diet; WT, wild-type.

      Glucose Uptake and Insulin Signaling Are Dysregulated in Adipose Tissue in LDLRAP1−/− Mice

      It is important to identify the molecular mechanisms of LDLRAP1 metabolic effects. LDLRAP1 mRNA is detected at low levels in most tissues. Its expression has been previously shown to be enhanced in liver, but mRNA abundance in adipose tissue has not been reported.
      • Garcia C.K.
      • Wilund K.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Maioli M.
      • Calandra S.
      • Bertolini S.
      • Cossu F.
      • Grishin N.
      • Barnes R.
      • Cohen J.C.
      • Hobbs H.H.
      Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.
      As a first step toward understanding why LDLRAP1 mice were obese and insulin-resistant, glucometabolic tissue (adipose, skeletal muscle, and liver) were isolated from wild-type mice before and after HFD to determine LDLRAP1 abundance. Figure 5A shows that the expression of LDLRAP1 is significantly greater in VAT compared with liver and skeletal muscle. This suggests that although each of these tissues may be impacted by LDLRAP1 deletion, effects on insulin resistance may be more pronounced because of increased expression in adipose tissue, and prompted further studies in VAT.
      Figure thumbnail gr5
      Figure 5Expression of LDLRAP1 in adipose tissue. A: Expression of LDLRAP1 is significantly greater in VAT compared with liver and skeletal muscle. Tissue was isolated from glucometabolic tissue and LDLRAP1 expression quantitated by quantitative RT-PCR. B: LDLRAP1 is primarily expressed in adipocytes compared with stromal vascular cells. VAT isolated from wild-type and LDLRAP1−/− mice was separated into adipocytes and stromal cells, and LDLRAP1 expression was quantitated by quantitative RT-PCR. C: Adipocytes from LDLRAP1−/− mice are significantly hypertrophic compared with wild-type adipocytes. VAT from chow- and HFD-fed wild-type and LDLRAP1−/− mice were stained, and adipocyte size was quantitated by ImageJ version 1.53a software. D: VAT from LDLRAP1−/− mice are hypoxic. VAT from chow- and HFD-fed wild-type and LDLRAP1−/− mice were isolated and HIF1α expression quantitated by quantitative RT-PCR. E: VAT was also immunostained with HIF1α antibody; red-brown staining indicates positive reaction. F: Quantitation of nuclear HIF1α in VAT shown in E. Three sections from three mice each were counted and quantitated. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 50 μm. Original magnification: ×600 (C); ×200 (E). HFD, high-fat diet; SVF, stromal vascular fraction; VAT, visceral adipose tissue; WT, wild-type.
      Adipose tissue is composed of adipocytes as well as a SVF composed of, but not limited to, mesenchymal progenitor cells, leukocyte subtypes, and endothelial cells. To determine in which compartment LDLRAP1 was preferentially expressed, visceral adipose tissue was separated into SVF and adipocyte fractions by digestion and differential centrifugation. Figure 5B shows that LDLRAP1 mRNA is over 13-fold more abundant in the adipocyte fraction compared with the SVF. Little to no LDLRAP1 was detected in SVF or adipocytes from knockout mice. Adipocyte hypertrophy is considered a key event associated with a loss of insulin sensitivity both in lean and obese conditions.
      • Goralski K.B.
      • Sinal C.J.
      Type 2 diabetes and cardiovascular disease: getting to the fat of the matter.
      Morphological analysis of adipose tissue in chow- and HFD-fed mice determined that the adipocytes from LDLRAP1−/− mice were significantly hypertrophic compared with wild-type adipocytes (Figure 5C), which is indicative of insulin insensitivity. Hypoxic conditions are recognized to contribute to insulin resistance.
      • Hosogai N.
      • Fukuhara A.
      • Oshima K.
      • Miyata Y.
      • Tanaka S.
      • Segawa K.
      • et al.
      Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation.
      Further characterization of VAT from LDLRAP1−/− mice showed significantly increased expression of HIF1α mRNA and protein in chow conditions, and trended (P = 0.057) higher in HFD-fed LDLRAP1−/− mice (Figures 5D-5F), suggesting increased hypoxic conditions in VAT in LDLRAP1−/− mice.
      Insulin signal transduction utilizes the protein kinase AKT, which is phosphorylated and activated in normal adipocytes upon insulin exposure.
      • Sharma M.
      • Dey C.S.
      AKT ISOFORMS-AS160-GLUT4: the defining axis of insulin resistance.
      ,
      • Karlsson H.K.R.
      • Zierath J.R.
      • Kane S.
      • Krook A.
      • Lienhard G.E.
      • Wallberg-Henriksson H.
      Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects.
      The phosphorylation state of AKT in adipose tissue from wild-type and LDLRAP1 knockout mice was determined by Western blot analysis using phospho-specific antibody. Figure 6A indicates a representative Western blot, and Figure 6B indicates densiometric quantification from four mice showing that on HFD, AKT phosphorylation is significantly decreased in VAT from LDLRAP1 knockout mice compared with that in wild-type mice (P < 0.05, n = 4 mice). This is noteworthy considering the hyperinsulinemic state of the LDLRAP1−/− mice. Decreased AKT phosphorylation is a molecular indicator suggesting insulin signaling in adipose tissue from LDLRAP1 knockout mice is impaired.
      Figure thumbnail gr6
      Figure 6Dysregulated insulin signaling and CD36 expression in VAT in LDLRAP1−/− mice. A: Decreased AKT phosphorylation in LDLRAP1−/− mice. Proteins were extracted from VAT from chow- and HFD-fed wild-type and LDLRAP1−/− mice, and immunoblotted with the antibodies shown. B: Densiometric analysis of immunoblot in A from four different mice for each condition. C: CD36 mRNA expression. VAT from chow- and HFD-fed wild-type and LDLRAP1−/− mice were isolated, and CD36 mRNA expression was quantitated by quantitative RT-PCR. D: Representative Western blot of CD36 protein expression. Proteins were extracted from VAT from chow- and HFD-fed wild-type and LDLRAP1−/− mice, and immunoblotted with the antibodies shown. E: Expression of HIF1α and CD36 is increased in VAT in LDLRAP1−/− mice compared with wild-type and LDLR−/− mice. VAT isolated from three mice in each in group were pooled, and mRNA expression was quantitated by quantitative RT-PCR. Expression is significantly higher in LDLRAP1−/− VAT compared with wild-type and LDLR−/−. F: Adipocytes from LDLRAP1−/− mice take up less glucose compared with wild-type mice. Primary adipocytes were isolated from chow-fed male wild-type and LDLRAP1−/− mice, and glucose uptake was determined. Experiment was performed in triplicate from at least three independent isolations. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. HFD, high-fat diet; VAT, visceral adipose tissue; WT, wild-type.
      CD36 is an oxLDL receptor as well as a fatty acid transporter whose activation, leading to increased fatty acid uptake, requires AKT phosphorylation.
      • Karlsson H.K.R.
      • Zierath J.R.
      • Kane S.
      • Krook A.
      • Lienhard G.E.
      • Wallberg-Henriksson H.
      Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects.
      Because hypercholesteremia-induced insulin resistance is associated with increased CD36 abundance, and CD36 has been implicated as a mediator of lipid-induced insulin resistance,
      • Liu Y.
      • Neumann D.
      • Glatz J.F.C.
      • Luiken J.J.F.P.
      Molecular mechanism of lipid-induced cardiac insulin resistance and contractile dysfunction.
      ,
      • Kashyap S.R.
      • Ioachimescu A.G.
      • Gornik H.L.
      • Gopan T.
      • Davidson M.B.
      • Makdissi A.
      • Major J.
      • Febbraio M.
      • Silverstein R.L.
      Lipid-induced insulin resistance is associated with increased monocyte expression of scavenger receptor CD36 and internalization of oxidized LDL.
      it was logical to examine CD36 abundance in VAT from LDLRAP1−/− mice. Figure 6C shows significantly more CD36 mRNA (P < 0.05, n = 4 mice) and Figure 6D shows increased CD36 protein expression in adipose tissue from LDLRAP1−/− mice compared with that from wild-type mice. Thus, despite increased abundance of fatty acid transporters, LDLRAP1 knockout mice remain hyperlipidemic. To determine whether this modified expression is similar to LDLR knockout mice, gene expression in VAT in chow-fed wild-type, LDLR−/−, and LDLRAP1−/− mice was compared. Figure 6E shows that both HIF1α and CD36 mRNA are significantly increased in VAT from LDLRAP1−/− mice compared with both wild-type and LDLR−/− mice, indicating that increased expression of these genes is associated with absence of LDLRAP1. CD36 expression in aorta and BMDM in LDLRAP1−/− and wild-type mice was also examined. Interestingly, and consistent with that observed in VAT, CD36 mRNA was significantly increased in BMDM isolated from LDLRAP1−/− mice compared with controls, but was not significantly increased in aorta in mice fed HFD (Supplemental Figure S1).
      Having established that CD36 abundance is increased and AKT phosphorylation is decreased in LDLRAP1−/− VAT, whether depletion of LDLRAP1 effected the ability of adipose tissue to take up glucose was determined next. Primary adipocytes were isolated from chow-fed male wild-type and LDLRAP1−/− mice, and glucose uptake was determined. Figure 6F shows that adipose tissue from LDLRAP1−/− mice takes up significantly less glucose compared with adipose tissue from wild-type mice (2646.0 ± 50.1 vs 1483.0 ± 162.9 relative fluorescence units, P < 0.01 for wild-type and LDLRAP1−/− mice, respectively), and corroborates the attenuated AKT phosphorylation observed in the LDLRAP1−/− mice. Insulin receptor 1 (IRS1) is important to mediate insulin-dependent regulation of both glucose and lipid metabolism in adipose tissue. Supplemental Figure S2 shows that IRS1 phosphorylation is decreased in VAT in HFD-fed LDLRAP1−/− mice compared with that in wild-type mice.

      Dysregulated Gene Expression in LDLRAP1 Depleted in Adipose Tissue

      VAT is a synthetic organ, and differences in insulin resistance and AKT activation are often reflected in differences in gene expression. To determine whether lack of LDLRAP1 affected VAT gene expression, RNA was isolated and pooled from VAT from three wild-type or LDLRAP1 knockout mice each, fed a HFD for 16 weeks, and gene expression was quantified using a metabolism-biased quantitative RT-PCR–based array (Supplemental Table S1). Figure 7A lists significantly different transcript expression and reflects a dysregulated transcriptional profile characteristic of altered energy metabolism and lipid storage. Expression of a number of these transcripts was validated on VAT isolated and quantitated from individual mice (Figure 7B). mRNA for a number of adipokines are increased in LDLRAP1−/− including Cfd, leptin, and adiponectin, genes involved in adipogenesis such as Bmp7 and Cdkn1a, and PPAgc1a, an important metabolic gene regulatory factor. A decrease in gene expression was noted in transcripts associated with glucose homeostasis, insulin resistance, and metabolism, which together may be reflective of increased hypoxia and inflammation in this tissue.
      Figure thumbnail gr7
      Figure 7Altered gene expression in LDLRAP1 depleted in adipose tissue. A: RNA was isolated and pooled from visceral adipose tissue from three wild-type and three LDLRAP1−/− mice fed HFD, and expression of atherosclerosis-related transcripts was quantitated by biased mouse adipogenesis RT-PCR array. Shown are statistically significant transcripts from the array with P < 0.05. B: Validation of transcripts adiponectin (Adipoq), complement factor D (Cfd), hormone-sensitive lipase (Lipe), and Src proto-oncogene (Src) in individual wild-type or LDLRAP1−/− mice. Values are normalized to wild-type mouse #1, and expression in each LDLRAP1−/− mouse is significantly different from each wild-type control mouse. ∗P < 0.05, ∗∗P < 0.01. WT, wild-type.

      Discussion

      Individuals with ARH are prone to premature atherosclerosis and coronary artery disease, but direct causality of deletion of this gene on atherogenesis has not been reported. In the current study, LDLRAP1−/− mice were shown to have increased atherosclerotic plaque burden compared with controls, with male knockout mice demonstrating the most severe atherosclerotic phenotype compared with female knockout mice. Although it is recognized that sex can exert major effects on the outcome of atherosclerosis studies, there is no consensus linking a particular sex with increased plaque formation, particularly in mice. Many observations are influenced by aortic location, diet, and strain. Initial reports suggest that atherosclerotic lesions in the aortic root were larger in female mice
      • Paigen B.
      • Holmes P.A.
      • Mitchell D.
      • Albee D.
      Comparison of atherosclerotic lesions and HDL-lipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H.
      ; however, this finding is not consistent.
      • Tangirala R.K.
      • Rubin E.M.
      • Palinski W.
      Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.
      ,
      • Daugherty A.
      • Rateri D.L.
      Development of experimental designs for atherosclerosis studies in mice.
      In the current study, male mice clearly weighed more and had increased atherosclerosis and insulin resistance compared with female mice. LDLRAP1 function has been posited to be tissue-specific, because while hepatocytes and lymphoblasts from ARH patients display impaired LDL internalization, lipid uptake is normal in fibroblasts from the same patients.
      • Arca M.
      • Zuliani G.
      • Wilund K.
      • Campagna F.
      • Fellin R.
      • Bertolini S.
      • Calandra S.
      • Ricci G.
      • Glorioso N.
      • Maioli M.
      • Pintus P.
      • Carru C.
      • Cossu F.
      • Cohen J.
      • Hobbs H.H.
      Autosomal recessive hypercholesterolaemia in Sardinia, Italy, and mutations in ARH: a clinical and molecular genetic analysis.
      Disruption of LDLRAP1 has been shown to result in 80% reduction in LDL internalization in cultured hepatocytes.
      • Sirinian M.I.
      • Belleudi F.
      • Campagna F.
      • Ceridono M.
      • Garofalo T.
      • Quagliarini F.
      • Verna R.
      • Calandra S.
      • Bertolini S.
      • Sorice M.
      • Torrisi M.R.
      • Arca M.
      Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes.
      ,
      • Jones C.
      • Hammer R.E.
      • Li W.-P.
      • Cohen J.C.
      • Hobbs H.H.
      • Herz J.
      Normal sorting but defective endocytosis of the low density lipoprotein receptor in mice with autosomal recessive hypercholesterolemia.
      ,
      • Jones C.
      • Garuti R.
      • Michaely P.
      • Li W.-P.
      • Maeda N.
      • Cohen J.C.
      • Herz J.
      • Hobbs H.H.
      Disruption of LDL but not VLDL clearance in autosomal recessive hypercholesterolemia.
      As expected, serum cholesterol and triglycerides were also significantly increased in LDLRAP1−/− mice.
      In humans, LDLR polymorphisms have been associated with obesity,
      • Besseling J.
      • Kastelein J.J.P.
      • Defesche J.C.
      • Hutten B.A.
      • Hovingh G.K.
      Association between familial hypercholesterolemia and prevalence of type 2 diabetes mellitus.
      suggesting a link between LDLR function, peripheral lipid deposition, and metabolic dysfunction. Although all mice gain weight as they mature, and particularly so on HFD, surprisingly, the LDLRAP1−/− mice gained significantly more weight compared with control mice. Significant weight gain is often accompanied by loss of insulin sensitivity, and the insulin tolerance tests showed that LDLRAP1−/− mice were indeed insulin insensitive even prior to consumption of the HFD. In support of this observation, serum insulin levels in LDLRAP1−/− mice were significantly higher compared with wild-type mice after 16 weeks HFD, suggesting that the increased insulin level in LDLRAP1−/− is likely compensatory and indicative of insulin resistance. The LDLRAP1 protein plays a critical role in LDLR and LDL internalization. Studies linking the LDLR with type 2 diabetes are complex and often contradictory. In one correlative study, the prevalence of type 2 diabetes among patients with familial hypercholesterolemia was significantly lower than among unaffected relatives.
      • Besseling J.
      • Kastelein J.J.P.
      • Defesche J.C.
      • Hutten B.A.
      • Hovingh G.K.
      Association between familial hypercholesterolemia and prevalence of type 2 diabetes mellitus.
      Several studies have shown that in addition to the amount of lipid in the diet, fatty acid composition affects serum LDL-cholesterol levels both in man and experimental animals.
      • Tyroler H.A.
      Overview of clinical trials of cholesterol lowering in relationship to epidemiologic studies.
      ,
      • Grundy S.M.
      • Denke M.A.
      Dietary influences on serum lipids and lipoproteins.
      One study showed that when fed a HFD, wild-type mice became obese and developed hypercholesterolemia with hyperglycemia, but not hypertriglyceridemia, whereas LDLR−/− mice fed the same diet became more obese than wild-type mice and developed high triglycerides in addition to hyperglycemia and hypercholesterolemia.
      • Schreyer S.A.
      • Vick C.
      • Lystig T.C.
      • Mystkowski P.
      • LeBoeuf R.C.
      LDL receptor but not apolipoprotein E deficiency increases diet-induced obesity and diabetes in mice.
      This study showed that glucose and insulin levels in these mice were only modestly higher, and insulin-to-glucose ratios were not strikingly different from wild-type mice.
      Despite having similar food intake and increased energy expenditure, LDLRAP1−/− mice gained more weight compared with controls. This may be due to the hyperinsulinemia observed in LDLRAP1−/− mice on HFD as mice with improved insulin sensitivity accumulate less adipose tissue even when fed a HFD, and pharmacological reduction of insulin secretion lowers body weight.
      • Mathews S.T.
      • Singh G.P.
      • Ranalletta M.
      • Cintron V.J.
      • Qiang X.
      • Goustin A.S.
      • Jen K.L.
      • Charron M.J.
      • Jahnen-Dechent W.
      • Grunberger G.
      Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene.
      ,
      • He H.
      • Holl K.
      • DeBehnke S.
      • Yeo C.T.
      • Hansen P.
      • Gebre A.K.
      • Leone-Kabler S.
      • Ruas M.
      • Parks J.S.
      • Parrington J.
      • Solberg Woods L.C.
      Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain.
      This implies that the hyperinsulinemia developed by LDLRAP1−/− mice after HFD contributes to increased weight gain due to expanded visceral adipose tissue mass, and may also suggest that insulin levels may predict obesity even in the absence of high-fat feeding and regardless of energy expenditures. Increased activity may be due to absence of LDLRAP1 from neural tissue, which may have unanticipated effects on behavioral activity.
      Dyslipidemia may not only be a consequence of impaired glucose metabolism, but may also cause it. Apolipoprotein A1 (apoA1) knockout in mice results in a phenotype resembling metabolic syndrome, whereas apoA1 overexpression improves glucose metabolism and results in a higher lean body mass and a higher expression of mitochondrial ATP synthase.
      • Lehti M.
      • Donelan E.
      • Abplanalp W.
      • Al-Massadi O.
      • Habegger K.M.
      • Weber J.
      • Ress C.
      • Mansfeld J.
      • Somvanshi S.
      • Trivedi C.
      • Keuper M.
      • Ograjsek T.
      • Striese C.
      • Cucuruz S.
      • Pfluger P.T.
      • Krishna R.
      • Gordon S.M.
      • Silva R.A.
      • Luquet S.
      • Castel J.
      • Martinez S.
      • D'Alessio D.
      • Davidson W.S.
      • Hofmann S.M.
      High-density lipoprotein maintains skeletal muscle function by modulating cellular respiration in mice.
      Interestingly, in the same model, apoA1 overexpression could also prevent diet-induced obesity. Although rodent and human lipoprotein metabolism differs considerably, these findings may also help to explain the interaction of lipoprotein clearance and glucose metabolism. Focusing on adipose tissue, recent studies have shown that LDL metabolism in adipocytes is associated with insulin receptor trafficking and regulation, and glucose metabolism.
      • Hofmann S.M.
      • Zhou L.
      • Perez-Tilve D.
      • Greer T.
      • Grant E.
      • Wancata L.
      • Thomas A.
      • Pfluger P.T.
      • Basford J.E.
      • Gilham D.
      • Herz J.
      • Tschöp M.H.
      • Hui D.Y.
      Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice.
      Thus, a complex interplay between insulin signaling and adipose LDLRAP1 is likely to exist. Additional studies are needed to determine whether the LDLRAP1-mediated uptake mechanism is altered in the insulin-resistant organism and whether altered LDLRAP1 function further influences glucose metabolism and insulin sensitivity in humans.
      LDLRAP1 effects on dyslipidemia, obesity, metabolism, and insulin sensitivity have not been previously reported, and the next series of experiments aimed to clarify its expression and effects on adipose tissue. Of the three glucometabolic tissues, LDLRAP1 mRNA expression is most abundant in VAT. The role of LDLRAP1 in liver and skeletal muscle insulin sensitivity remains to be determined. Adipose tissue is not homogenous and is composed of adipocytes as well as fibroblasts, vascular cell types, and immune cells, with the latter three collectively referred to as SVF. Notably, LDLRAP1 mRNA was significantly more abundant in adipose cells compared with that in SVF. Adipocyte hypertrophy is indicative of loss of insulin sensitivity both in lean and obese conditions.
      • Cawthorn W.P.
      • Scheller E.L.
      • MacDougald O.A.
      Adipose tissue stem cells meet preadipocyte commitment: going back to the future.
      ,
      • Parlee S.D.
      • Lentz S.I.
      • Mori H.
      • MacDougald O.A.
      Quantifying size and number of adipocytes in adipose tissue.
      Histochemical analysis of VAT revealed that adipocytes were significantly larger in chow- and HFD-fed LDLRAP1−/− compared with wild-type mice. The observation of larger adipocytes in both chow- and HFD-fed LDLRAP1−/− mice compared with wild-type controls is consistent with their altered metabolic profile, obesity, and insulin resistance. VAT from chow-fed LDLRAP1−/− mice also had significantly greater expression of CD36 and HIF1α compared with both wild-type and LDLR−/− mice, suggesting fundamental differences in adipose pathophysiology between the groups. Taken together, LDLRAP1 expression in adipocytes, increased insulin insensitivity and obesity in mice lacking LDLRAP1 indicate a previously unrecognized, but central, function for this protein in VAT, and subsequently in maintenance of a healthy metabolic phenotype.
      Several lines of evidence suggest that loss of LDLRAP1 might decrease glucose uptake in adipocytes. Reduction of insulin-stimulated glucose transport in adipocytes results in whole-body insulin resistance, as demonstrated in adipose tissue–selective GLUT4-null mice.
      • Abel E.D.
      • Peroni O.
      • Kim J.K.
      • Kim Y.B.
      • Boss O.
      • Hadro E.
      • Minnemann T.
      • Shulman G.I.
      • Kahn B.B.
      Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver.
      Further, loss of the LDL receptor function substantially reduces GLUT4 sorting to the plasma membrane, which promotes decreased glucose uptake.
      • Jedrychowski M.P.
      • Gartner C.A.
      • Gygi S.P.
      • Zhou L.
      • Herz J.
      • Kandror K.V.
      • Pilch P.F.
      Proteomic analysis of GLUT4 storage vesicles reveals LRP1 to be an important vesicle component and target of insulin signaling.
      LRP1, a receptor related to LDLR also involved in lipid homeostasis, regulates the intracellular traffic of insulin-responsive vesicles containing the glucose transporter GLUT4 in adipose and muscle cells.
      • Bogan J.S.
      • Kandror K.V.
      Biogenesis and regulation of insulin-responsive vesicles containing GLUT4.
      Blockage of LRP1 exocytosis to the plasma membrane negatively affects insulin-induced intracellular signaling.
      • Actis Dato V.
      • Grosso R.A.
      • Sánchez M.C.
      • Fader C.M.
      • Chiabrando G.A.
      Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller glial cells.
      In the current study, adipocytes isolated from LDLRAP1−/− mice took up significantly less glucose compared with adipocytes isolated from wild-type mice in an ex vivo assay, which can account for the insulin resistance observed in vivo, as well as indicate that LDLRAP1 directly participated in glucose uptake.
      Protein kinase AKT is at the center of many signal transduction pathways and is phosphorylated in insulin-stimulated glucose uptake. VAT from LDLRAP1−/− mice show decreased AKT phosphorylation, which is consistent with insulin resistance demonstrated in vivo and the depressed glucose uptake in isolated adipocytes demonstrated ex vivo. Efficient endocytosis of the LDL–LDLR complex is mediated by LDLRAP1, where, along with endosomal sorting and trafficking machinery, the LDLR is either recycled to the plasma membrane, or in a PCSK9-dependent mechanism, is delivered to lysosomes and degraded.
      • Bartuzi P.
      • Billadeau D.D.
      • Favier R.
      • Rong S.
      • Dekker D.
      • Fedoseienko A.
      • Fieten H.
      • Wijers M.
      • Levels J.H.
      • Huijkman N.
      • Kloosterhuis N.
      • van der Molen H.
      • Brufau G.
      • Groen A.K.
      • Elliott A.M.
      • Kuivenhoven J.A.
      • Plecko B.
      • Grangl G.
      • McGaughran J.
      • Horton J.D.
      • Burstein E.
      • Hofker M.H.
      • van de Sluis B.
      CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.
      The FXNPXY motif in the amino-terminal PTB domain of LDLRAP1 is very similar to the PTB domains of proteins involved in protein trafficking. Signaling from the plasma membrane LDLRAP1 also contains a clathrin binding motif, suggesting that it can interact with other molecules in what is termed a cargo-sorting endocytic web.
      • Garuti R.
      • Jones C.
      • Li W.-P.
      • Michaely P.
      • Herz J.
      • Gerard R.D.
      • Cohen J.C.
      • Hobbs H.H.
      The modular adaptor protein autosomal recessive hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits.
      CD36 is an integral membrane protein and an oxLDL receptor for adipocytes. CD36 plasma membrane translocation involves clathrin coat proteins for endosome formation and transport.
      • Steinbusch L.K.M.
      • Schwenk R.W.
      • Ouwens D.M.
      • Diamant M.
      • Glatz J.F.C.
      • Luiken J.J.F.P.
      Subcellular trafficking of the substrate transporters GLUT4 and CD36 in cardiomyocytes.
      CD36 trafficking is essential for appropriate insulin receptor signal transduction, and its dysregulated expression in adipose is suggested as a link between hypercholesteremia and insulin resistance.
      • Gautam S.
      • Banerjee M.
      The macrophage Ox-LDL receptor, CD36 and its association with type II diabetes mellitus.
      In adipocytes, CD36 can mediate storage and release of fatty acids, and increased CD36 expression is associated with obesity in humans and rodents.
      • Bonen A.
      • Tandon N.N.
      • Glatz J.F.C.
      • Luiken J.J.F.P.
      • Heigenhauser G.J.F.
      The fatty acid transporter FAT/CD36 is upregulated in subcutaneous and visceral adipose tissues in human obesity and type 2 diabetes.
      ,
      • Yang J.
      • Park K.W.
      • Cho S.
      Inhibition of the CD36 receptor reduces visceral fat accumulation and improves insulin resistance in obese mice carrying the BDNF-Val66Met variant.
      Long-term overexposure to circulating lipids as in hypercholesteremic mice has been shown to increase CD36 abundance in vitro and in vivo, which may explain why CD36 abundance is increased in LDLRAP1−/− VAT.
      • Ouwens D.M.
      • Diamant M.
      • Fodor M.
      • Habets D.D.J.
      • Pelsers M.M.A.L.
      • El Hasnaoui M.
      • Dang Z.C.
      • van den Brom C.E.
      • Vlasblom R.
      • Rietdijk A.
      • Boer C.
      • Coort S.L.M.
      • Glatz J.F.C.
      • Luiken J.J.F.P.
      Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification.
      ,
      • Angin Y.
      • Steinbusch L.K.M.
      • Simons P.J.
      • Greulich S.
      • Hoebers N.T.H.
      • Douma K.
      • van Zandvoort M.A.
      • Coumans W.A.
      • Wijnen W.
      • Diamant M.
      • Ouwens D.M.
      • Glatz J.F.
      • Luiken J.J.
      CD36 inhibition prevents lipid accumulation and contractile dysfunction in rat cardiomyocytes.
      Much of CD36 is stored in intracellular endosomes, and its tightly regulated vesicle-mediated trafficking is essential for appropriate insulin receptor signal transduction.
      • Luiken J.J.F.P.
      • Nabben M.
      • Neumann D.
      • Glatz J.F.C.
      Understanding the distinct subcellular trafficking of CD36 and GLUT4 during the development of myocardial insulin resistance.
      In the current study, CD36 expression increased in VAT in LDLRAP1 knockout mice, consistent with the elevated hypercholesteremia observed in LDLRAP1−/− mice. Because CD36 endosomal trafficking involves clathrin coat adaptor proteins such as LDLRAP1, and its appropriate endocytosis trafficking is essential for appropriate insulin receptor signaling, depletion of LDLRAP1 may affect insulin signaling and/or endosome sorting, which is manifested by insulin resistance displayed in the LDLRAP1−/− mice. This is supported by the fact that CD36 is increased in LDLRAP1−/− compared with LDLR−/− mice, which are also hyperlipidemic. Together, deletion of LDLRAP1 in visceral adipose tissue results in impaired insulin signaling, increased CD36 expression, and impaired glucose uptake in adipocytes (Figure 8).
      Figure thumbnail gr8
      Figure 8Schematic representation of proposed role of LDLRAP1 in adipose tissue. Efficient endocytosis of the LDL–LDLR complex is mediated by LDLRAP1. LDLRAP1 contains domains common in adaptor proteins, suggesting that it can interact with other molecules in a cargo-sorting endocytic web. CD36 abundance and trafficking is essential for appropriate insulin receptor signal transduction, and its abundance in adipose is suggested as a link between hypercholesteremia and insulin resistance. Long-term overexposure to circulating lipids increases CD36 abundance. In the absence of LDLRAP1, CD36 abundance is dysregulated, possibly contributing to insulin resistance. LDL, low-density lipoprotein.
      White adipose tissue is a secretory organ, capable of synthesis of many adipokines upon the appropriate stimuli.
      • Grant R.W.
      • Dixit V.D.
      Adipose tissue as an immunological organ.
      Hypertrophic adipocytes demonstrate an altered secretory repertoire which effects local as well as systemic inflammation, obesity, and insulin resistance. Using biased quantitative RT-PCR array, a number of transcripts have been identified that participate in metabolic regulation whose expression was increased, including Bmp7, Src, Cfd, leptin, and PPARγC1a. Cfd is an adipokine that regulates insulin secretion,
      • Gómez-Banoy N.
      • Guseh J.S.
      • Li G.
      • Rubio-Navarro A.
      • Chen T.
      • Poirier B.
      • Putzel G.
      • Rosselot C.
      • Pabón M.A.
      • Camporez J.P.
      • Bhambhani V.
      • Hwang S.J.
      • Yao C.
      • Perry R.J.
      • Mukherjee S.
      • Larson M.G.
      • Levy D.
      • Dow L.E.
      • Shulman G.I.
      • Dephoure N.
      • Garcia-Ocana A.
      • Hao M.
      • Spiegelman B.M.
      • Ho J.E.
      • Lo J.C.
      Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.
      and leptin is an adipokine that regulates insulin secretion and promotes energy expenditure.
      • Allison M.B.
      • Myers Jr., M.G.
      20 years of leptin: connecting leptin signaling to biological function.
      PPARγC1a in particular is implicated in regulation of cellular cholesterol homeostasis and development of obesity.
      • Stefan N.
      • Thamer C.
      • Staiger H.
      • Machicao F.
      • Machann J.
      • Schick F.
      • Venter C.
      • Niess A.
      • Laakso M.
      • Fritsche A.
      • Häring H.U.
      Genetic variations in PPARD and PPARGC1A determine mitochondrial function and change in aerobic physical fitness and insulin sensitivity during lifestyle intervention.
      It is a potent transcriptional coactivator that regulates many genes involved in energy metabolism by interacting with PPARγ, permitting its interaction with other transcription factors. Because these are considered to be protective, their expression may be increased in a compensatory manner. Abundance of a number of transcripts was decreased, each of which are involved in lipid storage, adipocyte differentiation, and insulin signaling. LMNA is associated with familial partial lipodystrophy of the Dunnigan type (FPLD2), the loss of which is associated with insulin resistance and type 2 diabetes.
      • Wojtanik K.M.
      • Edgemon K.
      • Viswanadha S.
      • Lindsey B.
      • Haluzik M.
      • Chen W.
      • Poy G.
      • Reitman M.
      • Londos C.
      The role of LMNA in adipose: a novel mouse model of lipodystrophy based on the Dunnigan-type familial partial lipodystrophy mutation.
      Sirt1 is expressed in metabolically active tissues and is an important nutrient sensor and regulator of metabolism and energy homeostasis by limiting VAT expansion and promotion of VAT browning.
      • Perrini S.
      • Porro S.
      • Nigro P.
      • Cignarelli A.
      • Caccioppoli C.
      • Genchi V.A.
      • Martines G.
      • De Fazio M.
      • Capuano P.
      • Natalicchio A.
      • Laviola L.
      • Giorgino F.
      Reduced SIRT1 and SIRT2 expression promotes adipogenesis of human visceral adipose stem cells and associates with accumulation of visceral fat in human obesity.
      Expression of each of these can support the observed phenotype of the LDLRAP1−/− mouse. Within the context of this dysregulated gene expression profile, it may not be entirely surprising that LDLRAP1−/− mice are hyperlipidemic, obese, and insulin insensitive.
      In summary, global loss of LDLRAP1 results in dyslipidemia and atherosclerotic plaque formation, which may be expected. However, increased weight gain and insulin insensitivity, and decreased glucose uptake were unexpected findings and indicate a previously uncharacterized role for LDLRAP1 in maintenance of the appropriate metabolic phenotype. Dysregulation of CD36 expression, AKT signaling, and metabolic gene expression was also unexpected and may explain the mechanism(s) driving the observed metabolic phenotype. One limitation to these studies is that LDLRAP1 deletion is global, and future studies are necessary to fully characterize the tissue-specific role of LDLRAP1 in lipid uptake and metabolism. Nevertheless, when taken in its entirety, a picture emerges where LDLRAP1 participates in more than LDLR internalization. LDLRAP1 deletion leads to systemic effects and may act as a molecular link that regulates dyslipidemia, atherosclerosis, insulin resistance, and obesity.

      Supplemental Data

      • Supplemental Figure S1

        CD36 expression in aorta and macrophage. A: RNA was isolated and pooled from three aorta from three wild-type (WT) and LDLRAP1−/− high-fat diet–fed mice, and CD36 expression evaluated by quantitative RT-PCR. B: CD36 mRNA abundance in bone marrow–derived macrophages. RNA was pooled from bone marrow–derived macrophages from three different mice. CD36 mRNA abundance is significantly higher in LDLRAP1−/− compared with wild-type mice. ∗∗∗P < 0.001. ns, not significantly different.

      • Supplemental Figure S2

        Insulin receptor phosphorylation is reduced in VAT from LDLRAP1−/− mice fed high-fat diet compared with wild-type mice. Protein isolated from three wild-type or LDLRAP1−/− mice each was immunoblotted with anti-phospho IRS1 and total IRS1 antibody.

      References

        • Grant R.W.
        • Dixit V.D.
        Adipose tissue as an immunological organ.
        Obesity (Silver Spring). 2015; 23: 512-518
        • Ohman M.K.
        • Shen Y.
        • Obimba C.I.
        • Wright A.P.
        • Warnock M.
        • Lawrence D.A.
        • Eitzman D.T.
        Visceral adipose tissue inflammation accelerates atherosclerosis in apolipoprotein E-deficient mice.
        Circulation. 2008; 117: 798-805
        • van de Sluis B.
        • Wijers M.
        • Herz J.
        News on the molecular regulation and function of hepatic low-density lipoprotein receptor and LDLR-related protein 1.
        Curr Opin Lipidol. 2017; 28: 241-247
        • Au D.T.
        • Strickland D.K.
        • Muratoglu S.C.
        The LDL receptor-related protein 1: at the crossroads of lipoprotein metabolism and insulin signaling.
        J Diabetes Res. 2017; 2017: 8356537
        • Descamps O.
        • Bilheimer D.
        • Herz J.
        Insulin stimulates receptor-mediated uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in adipocytes.
        J Biol Chem. 1993; 268: 974-981
        • Laatsch A.
        • Merkel M.
        • Talmud P.J.
        • Grewal T.
        • Beisiegel U.
        • Heeren J.
        Insulin stimulates hepatic low density lipoprotein receptor-related protein 1 (LRP1) to increase postprandial lipoprotein clearance.
        Atherosclerosis. 2009; 204: 105-111
        • Hurtado-Roca Y.
        • Bueno H.
        • Fernandez-Ortiz A.
        • Ordovas J.M.
        • Ibañez B.
        • Fuster V.
        • Rodriguez-Artalejo F.
        • Laclaustra M.
        Oxidized LDL is associated with metabolic syndrome traits independently of central obesity and insulin resistance.
        Diabetes. 2017; 66: 474-482
        • Liu C.-C.
        • Hu J.
        • Tsai C.-W.
        • Yue M.
        • Melrose H.L.
        • Kanekiyo T.
        • Bu G.
        Neuronal LRP1 regulates glucose metabolism and insulin signaling in the brain.
        J Neurosci. 2015; 35: 5851-5859
        • Ding Y.
        • Xian X.
        • Holland W.L.
        • Tsai S.
        • Herz J.
        Low-density lipoprotein receptor-related protein-1 protects against hepatic insulin resistance and hepatic steatosis.
        EBioMedicine. 2016; 7: 135-145
        • Brown M.S.
        • Goldstein J.L.
        A receptor-mediated pathway for cholesterol homeostasis.
        Science. 1986; 232: 34-47
        • He G.
        • Gupta S.
        • Yi M.
        • Michaely P.
        • Hobbs H.H.
        • Cohen J.C.
        ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2.
        J Biol Chem. 2002; 277: 44044-44049
        • Garuti R.
        • Jones C.
        • Li W.-P.
        • Michaely P.
        • Herz J.
        • Gerard R.D.
        • Cohen J.C.
        • Hobbs H.H.
        The modular adaptor protein autosomal recessive hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits.
        J Biol Chem. 2005; 280: 40996-41004
        • Mishra S.K.
        • Watkins S.C.
        • Traub L.M.
        The autosomal recessive hypercholesterolemia (ARH) protein interfaces directly with the clathrin-coat machinery.
        Proc Natl Acad Sci U S A. 2002; 99: 16099-16104
        • Sirinian M.I.
        • Belleudi F.
        • Campagna F.
        • Ceridono M.
        • Garofalo T.
        • Quagliarini F.
        • Verna R.
        • Calandra S.
        • Bertolini S.
        • Sorice M.
        • Torrisi M.R.
        • Arca M.
        Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes.
        J Biol Chem. 2005; 280: 38416-38423
        • Garcia C.K.
        • Wilund K.
        • Arca M.
        • Zuliani G.
        • Fellin R.
        • Maioli M.
        • Calandra S.
        • Bertolini S.
        • Cossu F.
        • Grishin N.
        • Barnes R.
        • Cohen J.C.
        • Hobbs H.H.
        Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.
        Science. 2001; 292: 1394-1398
        • Zuliani G.
        • Arca M.
        • Signore A.
        • Bader G.
        • Fazio S.
        • Chianelli M.
        • Bellosta S.
        • Campagna F.
        • Montali A.
        • Maioli M.
        • Pacifico A.
        • Ricci G.
        • Fellin R.
        Characterization of a new form of inherited hypercholesterolemia: familial recessive hypercholesterolemia.
        Arterioscler Thromb Vasc Biol. 1999; 19: 802-809
        • Jones C.
        • Hammer R.E.
        • Li W.-P.
        • Cohen J.C.
        • Hobbs H.H.
        • Herz J.
        Normal sorting but defective endocytosis of the low density lipoprotein receptor in mice with autosomal recessive hypercholesterolemia.
        J Biol Chem. 2003; 278: 29024-29030
        • Jones C.
        • Garuti R.
        • Michaely P.
        • Li W.-P.
        • Maeda N.
        • Cohen J.C.
        • Herz J.
        • Hobbs H.H.
        Disruption of LDL but not VLDL clearance in autosomal recessive hypercholesterolemia.
        J Clin Invest. 2007; 117: 165-174
        • Arca M.
        • Zuliani G.
        • Wilund K.
        • Campagna F.
        • Fellin R.
        • Bertolini S.
        • Calandra S.
        • Ricci G.
        • Glorioso N.
        • Maioli M.
        • Pintus P.
        • Carru C.
        • Cossu F.
        • Cohen J.
        • Hobbs H.H.
        Autosomal recessive hypercholesterolaemia in Sardinia, Italy, and mutations in ARH: a clinical and molecular genetic analysis.
        Lancet. 2002; 359: 841-847
        • Ellison S.
        • Gabunia K.
        • Kelemen S.E.
        • England R.N.
        • Scalia R.
        • Richards J.M.
        • Orr A.W.
        • Traylor Jr., J.G.
        • Rogers T.
        • Cornwell W.
        • Berglund L.M.
        • Goncalves I.
        • Gomez M.F.
        • Autieri M.V.
        Attenuation of experimental atherosclerosis by interleukin-19.
        Arterioscler Thromb Vasc Biol. 2013; 33: 2316-2324
        • Gabunia K.
        • Ellison S.
        • Kelemen S.
        • Kako F.
        • Cornwell W.D.
        • Rogers T.J.
        • Datta P.K.
        • Ouimet M.
        • Moore K.J.
        • Autieri M.V.
        IL-19 halts progression of atherosclerotic plaque, polarizes, and increases cholesterol uptake and efflux in macrophages.
        Am J Pathol. 2016; 186: 1361-1374
        • Mina A.I.
        • LeClair R.A.
        • LeClair K.B.
        • Cohen D.E.
        • Lantier L.
        • Banks A.S.
        CalR: a web-based analysis tool for indirect calorimetry experiments.
        Cell Metab. 2018; 28: 656-666.e1
        • Berglund E.D.
        • Li C.Y.
        • Poffenberger G.
        • Ayala J.E.
        • Fueger P.T.
        • Willis S.E.
        • Jewell M.M.
        • Powers A.C.
        • Wasserman D.H.
        Glucose metabolism in vivo in four commonly used inbred mouse strains.
        Diabetes. 2008; 57: 1790-1799
        • Herman A.B.
        • Vrakas C.N.
        • Ray M.
        • Kelemen S.E.
        • Sweredoski M.J.
        • Moradian A.
        • Haines D.S.
        • Autieri M.V.
        FXR1 is an IL-19-responsive RNA-binding protein that destabilizes pro-inflammatory transcripts in vascular smooth muscle cells.
        Cell Rep. 2018; 24: 1176-1189
        • Ray M.
        • Gabunia K.
        • Vrakas C.N.
        • Herman A.B.
        • Kako F.
        • Kelemen S.E.
        • Grisanti L.A.
        • Autieri M.V.
        Genetic deletion of IL-19 (interleukin-19) exacerbates atherogenesis in Il19−/−×Ldlr−/− double knockout mice by dysregulation of mRNA stability protein HuR (Human Antigen R).
        Arterioscler Thromb Vasc Biol. 2018; 38: 1297-1308
        • Kilroy G.
        • Dietrich M.
        • Wu X.
        • Gimble J.M.
        • Floyd Z.E.
        Isolation of murine adipose-derived stromal/stem cells for adipogenic differentiation or flow cytometry-based analysis.
        Methods Mol Biol. 2018; 1773: 137-146
        • Parks B.W.
        • Sallam T.
        • Mehrabian M.
        • Psychogios N.
        • Hui S.T.
        • Norheim F.
        • Castellani L.W.
        • Rau C.D.
        • Pan C.
        • Phun J.
        • Zhou Z.
        • Yang W.P.
        • Neuhaus I.
        • Gargalovic P.S.
        • Kirchgessner T.G.
        • Graham M.
        • Lee R.
        • Tontonoz P.
        • Gerszten R.E.
        • Hevener A.L.
        • Lusis A.
        Genetic architecture of insulin resistance in the mouse.
        Cell Metab. 2015; 21: 334-347
        • Schefer V.
        • Talan M.I.
        Oxygen consumption in adult and AGED C57BL/6J mice during acute treadmill exercise of different intensity.
        Exp Gerontol. 1996; 31: 387-392
        • Goralski K.B.
        • Sinal C.J.
        Type 2 diabetes and cardiovascular disease: getting to the fat of the matter.
        Can J Physiol Pharmacol. 2007; 85: 113-132
        • Hosogai N.
        • Fukuhara A.
        • Oshima K.
        • Miyata Y.
        • Tanaka S.
        • Segawa K.
        • et al.
        Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation.
        Diabetes. 2007; 56: 901-911
        • Sharma M.
        • Dey C.S.
        AKT ISOFORMS-AS160-GLUT4: the defining axis of insulin resistance.
        Rev Endocr Metab Disord. 2021; 22: 973-986
        • Karlsson H.K.R.
        • Zierath J.R.
        • Kane S.
        • Krook A.
        • Lienhard G.E.
        • Wallberg-Henriksson H.
        Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects.
        Diabetes. 2005; 54: 1692-1697
        • Liu Y.
        • Neumann D.
        • Glatz J.F.C.
        • Luiken J.J.F.P.
        Molecular mechanism of lipid-induced cardiac insulin resistance and contractile dysfunction.
        Prostaglandins Leukot Essent Fatty Acids. 2018; 136: 131-141
        • Kashyap S.R.
        • Ioachimescu A.G.
        • Gornik H.L.
        • Gopan T.
        • Davidson M.B.
        • Makdissi A.
        • Major J.
        • Febbraio M.
        • Silverstein R.L.
        Lipid-induced insulin resistance is associated with increased monocyte expression of scavenger receptor CD36 and internalization of oxidized LDL.
        Obesity (Silver Spring). 2009; 17: 2142-2148
        • Paigen B.
        • Holmes P.A.
        • Mitchell D.
        • Albee D.
        Comparison of atherosclerotic lesions and HDL-lipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H.
        Atherosclerosis. 1987; 64: 215-221
        • Tangirala R.K.
        • Rubin E.M.
        • Palinski W.
        Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.
        J Lipid Res. 1995; 36: 2320-2328
        • Daugherty A.
        • Rateri D.L.
        Development of experimental designs for atherosclerosis studies in mice.
        Methods. 2005; 36: 129-138
        • Besseling J.
        • Kastelein J.J.P.
        • Defesche J.C.
        • Hutten B.A.
        • Hovingh G.K.
        Association between familial hypercholesterolemia and prevalence of type 2 diabetes mellitus.
        JAMA. 2015; 313: 1029-1036
        • Tyroler H.A.
        Overview of clinical trials of cholesterol lowering in relationship to epidemiologic studies.
        Am J Med. 1989; 87: 14S-19S
        • Grundy S.M.
        • Denke M.A.
        Dietary influences on serum lipids and lipoproteins.
        J Lipid Res. 1990; 31: 1149-1172
        • Schreyer S.A.
        • Vick C.
        • Lystig T.C.
        • Mystkowski P.
        • LeBoeuf R.C.
        LDL receptor but not apolipoprotein E deficiency increases diet-induced obesity and diabetes in mice.
        Am J Physiol Endocrinol Metab. 2002; 282: E207-E214
        • Mathews S.T.
        • Singh G.P.
        • Ranalletta M.
        • Cintron V.J.
        • Qiang X.
        • Goustin A.S.
        • Jen K.L.
        • Charron M.J.
        • Jahnen-Dechent W.
        • Grunberger G.
        Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene.
        Diabetes. 2002; 51: 2450-2458
        • He H.
        • Holl K.
        • DeBehnke S.
        • Yeo C.T.
        • Hansen P.
        • Gebre A.K.
        • Leone-Kabler S.
        • Ruas M.
        • Parks J.S.
        • Parrington J.
        • Solberg Woods L.C.
        Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain.
        Physiol Genomics. 2018; 50: 605-614
        • Lehti M.
        • Donelan E.
        • Abplanalp W.
        • Al-Massadi O.
        • Habegger K.M.
        • Weber J.
        • Ress C.
        • Mansfeld J.
        • Somvanshi S.
        • Trivedi C.
        • Keuper M.
        • Ograjsek T.
        • Striese C.
        • Cucuruz S.
        • Pfluger P.T.
        • Krishna R.
        • Gordon S.M.
        • Silva R.A.
        • Luquet S.
        • Castel J.
        • Martinez S.
        • D'Alessio D.
        • Davidson W.S.
        • Hofmann S.M.
        High-density lipoprotein maintains skeletal muscle function by modulating cellular respiration in mice.
        Circulation. 2013; 128: 2364-2371
        • Hofmann S.M.
        • Zhou L.
        • Perez-Tilve D.
        • Greer T.
        • Grant E.
        • Wancata L.
        • Thomas A.
        • Pfluger P.T.
        • Basford J.E.
        • Gilham D.
        • Herz J.
        • Tschöp M.H.
        • Hui D.Y.
        Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice.
        J Clin Invest. 2007; 117: 3271-3282
        • Cawthorn W.P.
        • Scheller E.L.
        • MacDougald O.A.
        Adipose tissue stem cells meet preadipocyte commitment: going back to the future.
        J Lipid Res. 2012; 53: 227-246
        • Parlee S.D.
        • Lentz S.I.
        • Mori H.
        • MacDougald O.A.
        Quantifying size and number of adipocytes in adipose tissue.
        Methods Enzymol. 2014; 537: 93-122
        • Abel E.D.
        • Peroni O.
        • Kim J.K.
        • Kim Y.B.
        • Boss O.
        • Hadro E.
        • Minnemann T.
        • Shulman G.I.
        • Kahn B.B.
        Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver.
        Nature. 2001; 409: 729-733
        • Jedrychowski M.P.
        • Gartner C.A.
        • Gygi S.P.
        • Zhou L.
        • Herz J.
        • Kandror K.V.
        • Pilch P.F.
        Proteomic analysis of GLUT4 storage vesicles reveals LRP1 to be an important vesicle component and target of insulin signaling.
        J Biol Chem. 2010; 285: 104-114
        • Bogan J.S.
        • Kandror K.V.
        Biogenesis and regulation of insulin-responsive vesicles containing GLUT4.
        Curr Opin Cell Biol. 2010; 22: 506-512
        • Actis Dato V.
        • Grosso R.A.
        • Sánchez M.C.
        • Fader C.M.
        • Chiabrando G.A.
        Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller glial cells.
        Biochem J. 2018; 475: 1669-1685
        • Bartuzi P.
        • Billadeau D.D.
        • Favier R.
        • Rong S.
        • Dekker D.
        • Fedoseienko A.
        • Fieten H.
        • Wijers M.
        • Levels J.H.
        • Huijkman N.
        • Kloosterhuis N.
        • van der Molen H.
        • Brufau G.
        • Groen A.K.
        • Elliott A.M.
        • Kuivenhoven J.A.
        • Plecko B.
        • Grangl G.
        • McGaughran J.
        • Horton J.D.
        • Burstein E.
        • Hofker M.H.
        • van de Sluis B.
        CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.
        Nat Commun. 2016; 7: 10961
        • Steinbusch L.K.M.
        • Schwenk R.W.
        • Ouwens D.M.
        • Diamant M.
        • Glatz J.F.C.
        • Luiken J.J.F.P.
        Subcellular trafficking of the substrate transporters GLUT4 and CD36 in cardiomyocytes.
        Cell Mol Life Sci. 2011; 68: 2525-2538
        • Gautam S.
        • Banerjee M.
        The macrophage Ox-LDL receptor, CD36 and its association with type II diabetes mellitus.
        Mol Genet Metab. 2011; 102: 389-398
        • Bonen A.
        • Tandon N.N.
        • Glatz J.F.C.
        • Luiken J.J.F.P.
        • Heigenhauser G.J.F.
        The fatty acid transporter FAT/CD36 is upregulated in subcutaneous and visceral adipose tissues in human obesity and type 2 diabetes.
        Int J Obes (Lond). 2006; 30: 877-883
        • Yang J.
        • Park K.W.
        • Cho S.
        Inhibition of the CD36 receptor reduces visceral fat accumulation and improves insulin resistance in obese mice carrying the BDNF-Val66Met variant.
        J Biol Chem. 2018; 293: 13338-13348
        • Ouwens D.M.
        • Diamant M.
        • Fodor M.
        • Habets D.D.J.
        • Pelsers M.M.A.L.
        • El Hasnaoui M.
        • Dang Z.C.
        • van den Brom C.E.
        • Vlasblom R.
        • Rietdijk A.
        • Boer C.
        • Coort S.L.M.
        • Glatz J.F.C.
        • Luiken J.J.F.P.
        Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification.
        Diabetologia. 2007; 50: 1938-1948
        • Angin Y.
        • Steinbusch L.K.M.
        • Simons P.J.
        • Greulich S.
        • Hoebers N.T.H.
        • Douma K.
        • van Zandvoort M.A.
        • Coumans W.A.
        • Wijnen W.
        • Diamant M.
        • Ouwens D.M.
        • Glatz J.F.
        • Luiken J.J.
        CD36 inhibition prevents lipid accumulation and contractile dysfunction in rat cardiomyocytes.
        Biochem J. 2012; 448: 43-53
        • Luiken J.J.F.P.
        • Nabben M.
        • Neumann D.
        • Glatz J.F.C.
        Understanding the distinct subcellular trafficking of CD36 and GLUT4 during the development of myocardial insulin resistance.
        Biochim Biophys Acta Mol Basis Dis. 2020; 1866: 165775
        • Gómez-Banoy N.
        • Guseh J.S.
        • Li G.
        • Rubio-Navarro A.
        • Chen T.
        • Poirier B.
        • Putzel G.
        • Rosselot C.
        • Pabón M.A.
        • Camporez J.P.
        • Bhambhani V.
        • Hwang S.J.
        • Yao C.
        • Perry R.J.
        • Mukherjee S.
        • Larson M.G.
        • Levy D.
        • Dow L.E.
        • Shulman G.I.
        • Dephoure N.
        • Garcia-Ocana A.
        • Hao M.
        • Spiegelman B.M.
        • Ho J.E.
        • Lo J.C.
        Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.
        Nat Med. 2019; 25: 1739-1747
        • Allison M.B.
        • Myers Jr., M.G.
        20 years of leptin: connecting leptin signaling to biological function.
        J Endocrinol. 2014; 223: T25-T35
        • Stefan N.
        • Thamer C.
        • Staiger H.
        • Machicao F.
        • Machann J.
        • Schick F.
        • Venter C.
        • Niess A.
        • Laakso M.
        • Fritsche A.
        • Häring H.U.
        Genetic variations in PPARD and PPARGC1A determine mitochondrial function and change in aerobic physical fitness and insulin sensitivity during lifestyle intervention.
        J Clin Endocrinol Metab. 2007; 92: 1827-1833
        • Wojtanik K.M.
        • Edgemon K.
        • Viswanadha S.
        • Lindsey B.
        • Haluzik M.
        • Chen W.
        • Poy G.
        • Reitman M.
        • Londos C.
        The role of LMNA in adipose: a novel mouse model of lipodystrophy based on the Dunnigan-type familial partial lipodystrophy mutation.
        J Lipid Res. 2009; 50: 1068-1079
        • Perrini S.
        • Porro S.
        • Nigro P.
        • Cignarelli A.
        • Caccioppoli C.
        • Genchi V.A.
        • Martines G.
        • De Fazio M.
        • Capuano P.
        • Natalicchio A.
        • Laviola L.
        • Giorgino F.
        Reduced SIRT1 and SIRT2 expression promotes adipogenesis of human visceral adipose stem cells and associates with accumulation of visceral fat in human obesity.
        Int J Obes (Lond). 2020; 44: 307-319