Lipid abnormalities in alpha/beta2-syntrophin null mice are independent from ABCA1

https://doi.org/10.1016/j.bbalip.2015.01.012Get rights and content

Highlights

  • Mice deficient for alpha and beta 2 syntrophins have normal serum insulin.

  • Hepatic ABCA1 protein is similar to wild type mice.

  • Increased ERK1/2 seems to contribute to low hepatic SR-BI.

  • Sphingomyelin is reduced in the liver and serum of the knock-out mice.

  • Hepatic but not serum cholesterol is lower in the knock-out animals.

Abstract

The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2 −/−) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2 −/− mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2 −/− mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2 −/− mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity.

Introduction

Obesity is a main risk factor for several common diseases including cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD) [1], [2]. Insulin resistance, hyperinsulinemia, and low levels of HDL are characteristic features associated with obesity [3]. HDL metabolism is largely controlled by hepatic ATP binding cassette transporter A1 (ABCA1) which regulates the generation of nascent HDL particles and scavenger receptor-BI (SR-BI) mediating the delivery of HDL cholesterol to the liver [4], [5], [6].

SR-BI and ABCA1 proteins are stabilized by the post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) (PDZ) proteins PDZK1 and syntrophins, respectively [4], [7]. Hepatic SR-BI protein is strongly reduced in PDZK1 deficient mice and abnormally large, cholesterol rich HDL particles circulate in plasma [8]. SR-BI in the adrenal gland is not affected in PDZK1 knock-out mice demonstrating a tissue specific function of this complex [8].

Hepatic ABCA1 deficiency does not only reduce serum HDL but also low density lipoprotein (LDL) [5]. The four carboxy terminal amino acids of ABCA1 bind to the PDZ domains of the syntrophin isoforms alpha (SNTA), beta 1 (SNTB1) and beta 2 (SNTB2). SNTA delays the degradation of ABCA1, and thereby, stimulates apolipoprotein A-I (ApoA-I) mediated release of cholesterol. Association of ABCA1 and SNTB2 has been confirmed by different approaches and the complex is found in the liver, platelets, and macrophages. Knock-down of SNTB2 modestly lowers lipid efflux suggesting a minor role of SNTB2 in ABCA1 biology. SNTB1 stabilizes ABCA1 protein in the macrophages and the liver [4].

Syntrophins are well described components of the skeletal muscle dystrophin associated protein complex (DAPC) and connect various transporters and extracellular proteins with the actin cytoskeleton [9]. SNTA/B2 −/− animals have fewer folds at the neuromuscular junctions, reduced levels of acetylcholine receptors and ran shorter distances on voluntary exercise wheels [10].

Binding of the granule protein islet cell antigen (ICA) 512 to the PDZ domain of SNTB2 anchors insulin transporting vesicles to actin filaments and restrains their mobility. Glucose causes destabilization of this complex and subsequent exocytosis of insulin suggesting a role of SNTB2 in postprandial glucose clearance [9], [11].

Low HDL cholesterol and impaired glucose metabolism are characteristic features of the metabolic syndrome and major risk factors in the pathogenesis of atherosclerotic diseases, type 2 diabetes and NAFLD [1], [2]. Therefore, it is of importance to understand the physiological relevance of SNTA and SNTB2 in ABCA1 function and insulin release which was analyzed herein using mice which lack SNTA and SNTB2 [10].

Section snippets

Cell culture, chemicals and antibodies

Hepa 1–6 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultivated at 37 °C and 5% CO2 in DMEM medium with 10% FBS and 1% penicillin/streptomycin. EndoPorter was ordered from GeneTools (Philomath, OR, USA). Syntrophin isoform specific antibodies have been described elsewhere [12]. Antibodies specific for caveolin-1, Cox-IV, β-actin, AMPK, pAMPK, Akt, ERK1/2, pERK1/2 and GAPDH were from New England Biolabs GmbH (Frankfurt am Main, Germany).

Hepatic ABCA1 and serum cholesterol are normal in SNTA/B2 −/− mice fed a standard diet (SD)

The syntrophins alpha (SNTA), beta 1 (SNTB1) and beta 2 (SNTB2) have been shown to affect ABCA1 activity partly by stabilizing ABCA1 protein [4]. To find out whether deficiency of SNTA and SNTB2 is associated with reduced expression of this transporter, ABCA1 was analyzed by immunoblot in the liver tissues of wild type and SNTA/B2 null mice fed a SD. ABCA1 was similarly expressed in the liver of wild type and double knock-out mice (Fig. 1A). SNTB1 was not changed in the liver of SNTA/B2 null

Discussion

SNTB2 is involved in insulin granule localization and vesicle release in the pancreatic INS-1 cells [11]. Despite this in vitro evidence suggesting an essential role of this adaptor protein in systemic insulin levels no abnormalities have been found in mice lacking SNTA and SNTB2 fed a SD. This excludes a physiologically important function of these adaptor proteins in glucose induced insulin release. Though it is still possible that other family members compensate for the deficiency of SNTB2, a

Funding

The study was supported by a grant from the German Research Foundation (BU 1141/8-1) and the NIH (NS33145).

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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