Expression and modulation of TUB by insulin and thyroid hormone in primary rat and murine 3T3-L1 adipocytes Clare Stretton a,1 , Gary J. Litherland b,1 , Audrey Moynihan a , Eric Hajduch c,d , Harinder S. Hundal a, * a Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK b Institute of Cellular Medicine, The Medical School, Newcastle University, Newcastle NE2 4HH, UK c INSERM U872, Université Pierre et Marie Curie, Paris 6, France d Université Paris Descartes, Paris F-75006, France article info Article history: Received 26 October 2009 Available online 1 November 2009 Keywords: 3T3-L1 Adipogenesis Zucker rat Insulin Tubby abstract tub encodes a protein of poorly understood function, but one implicated strongly in the control of energy balance and insulin sensitivity. Whilst tub expression is particularly prominent in neurones it is also detectable in extraneuronal tissues. We show here, for the first time, expression of TUB protein in rat adi- pocytes and the murine adipocyte model 3T3-L1 and demonstrate that insulin induces its tyrosine phos- phorylation and association with the insulin receptor. TUB expression is regulated developmentally during adipogenic differentiation of 3T3-L1 cells and in response to cell treatment with thyroid hormone or induction of insulin resistance. TUB was upregulated 5- to 10-fold in adipocytes from obese Zucker rats and 3T3-L1 adipocytes that had been rendered insulin resistant, a response that could be antagonised by rosiglitasone, an insulin-sensitising drug. Our data are consistent with a previously unforeseen role for TUB in insulin signalling and fuel homeostasis in adipocytes. Ó 2009 Elsevier Inc. All rights reserved. Introduction TUB and the tubby-like proteins (TULPs) 1–3 comprise a small gene family whose gene sequences are highly conserved across species [5,11,15,21,23]. This suggests that they may serve a basic function within the cell but, as yet, their exact role remains unclear. The four proteins of the family are highly homologous and have over-lapping expression profiles, but gene-inactivation experiments suggest they serve non-redundant roles. tub was originally identified as the gene involved in the tubby mouse syndrome, characterized by late-onset obesity, insulin resistance and sensory impairments [6,22]. This defect is due to expression of a mutant TUB protein in which the C-terminal 44 amino acid residues are replaced by 24 incorrect residues encoded intronical- ly. Gene knockout studies on TUB result in the same phenotype as the tubby mouse suggesting that the phenotype is caused by a loss of function mutation [22]. In neuronal cell lines TUB can be regulated by a range of stimuli including insulin and thyroid hormone and may regulate several functions including vesicle trafficking, gene transcription and en- ergy homeostasis [12,13,28]. In these latter studies TUB was shown to become tyrosine phosphorylated and associate with the insulin receptor following insulin treatment suggesting that it may act as an adaptor protein in insulin signalling [13]. The ‘‘tubby domain” permits interaction of TUB with phosphatidylinositol-4,5-bisphos- phate (PI(4,5)P 2 ) [27] and, as such, this may facilitate its associa- tion/retention with the inner leaflet of the plasma membrane where it may be placed in close proximity to receptor proteins that alter its function through covalent modification. More recent work has indicated that tubby proteins are also secreted from cells raising the suggestion that they may also possess extracellular functions [4]. Although most abundant in neuronal tissue, tub expression has also been noted previously in insulin-sensitive tis- sues of mice and humans using RT-PCR [10,22] suggesting that, in addition to its role within the CNS, TUB may also regulate fuel and energy balance in peripheral tissues targeted by hormones such as insulin and tri-iodothyronine (T3, i.e. thyroid hormone). We show here that TUB is expressed in primary and cultured adipocytes and that expression of TUB in the latter is regulated in a differentiation- dependent manner. Intriguingly, expression of TUB is enhanced in adipocytes from insulin resistant animals and in 3T3-L1 adipocytes rendered insulin resistant by sustained exposure to a high insulin concentration and in cells incubated with T3. These findings indi- cate that TUB expression is under hormonal control in adipocytes and that TUB may participate in insulin signalling and modulate adipocyte physiology during insulin resistance and altered thyroid status. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.147 * Corresponding author. Fax: +44 1382 385514. E-mail address: h.s.hundal@dundee.ac.uk (H.S. Hundal). 1 These authors have contributed equally to this work. Biochemical and Biophysical Research Communications 390 (2009) 1328–1333 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc