Journal of Neuroendocrinology, 2013, 25, 292–301 ORIGINAL ARTICLE © 2012 British Society for Neuroendocrinology A novel Transcript is Up-Regulated by Fasting in the Hypothalamus and Enhances Insulin Signalling B. Chai, J.-Y. Li, D. Fritze, W. Zhang, Z. Xia and M. W. Mulholland Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, USA. Journal of A transcript of unknown function, regulated by fasting and feeding, was identified by micro- array analysis. The transcript is up-regulated in the fasting state. An 1168-bp cDNA was Neuroendocrinology cloned from rat hypothalamus and sequenced. This sequence is consistent with adipogenesis down-regulating transcript 3 (AGD3) (also known as human OCC-1) mRNA. A protein sequence identical to AGD3 was determined by mass spectrometry. In the rat brain, AGD3 mRNA is distributed in the arcuate nucleus, ventromedial hypothalamus, amygdaloid nuclei, hippocam- pus, and somatic cortex. Double in situ hybridisation showed that AGD3 mRNA is co-localised with pro-opiomelanocortin and neuropeptide Y in arcuate nucleus neurones. AGD3 binds with Correspondence to: insulin receptor substrate 4 and increases insulin-stimulated phospho-Akt and regulates AMP- Michael W. Mulholland, Department activated protein kinase and mammalian target of rapamycin downstream target S6 kinase of Surgery, University of Michigan phosphorylation. Medical School, 2101 Taubman Center, 1500 E. Medical Center Drive, Key words: adipogenesis down-regulating transcript 3, insulin, proopiomelanocortin, neuropeptide Y Ann Arbor, MI 48109-0346, USA (e-mail: [email protected]). doi: 10.1111/j.1365-2826.2012.02378.x Obesity and being overweight represent major public health prob- transcripts (8). When the functional roles have been unclear, these lems. These conditions affect morbidity and mortality as factors have been designated as transcripts of unknown function (TUF). It contributing to diabetes mellitus, cardiovascular disease, pulmonary is widely assumed that the majority of TUFs are noncoding RNAs disease, osteoarthritis and several types of cancers. Research has (9,10). These noncoding RNAs may regulate gene function as established the hypothalamus as a crucial site in controlling body microRNAs, as small nucleolar RNAs or through noncoding repres- weight. Peripherally produced hormones such as insulin, as well as sion of transcription factors (10). We identified a TUF as being nutrients such as glucose and fatty acids, communicate energy sta- strongly up-regulated in the rat ARC by fasting. In the present tus to hypothalamic centres (1–3). The structure of the hypotha- study, this TUF was identified as derived from an mRNA of 1.2 kbp lamic arcuate nucleus (ARC) is consistent with its role as an identical to the previously described overexpressed in colon cancer- integrative centre in energy homeostasis. Neuroanatomical studies 1 (11) and, recently, adipogenesis down-regulating transcript 3 have identified two distinct populations of ARC neurones (4). The (AGD3) (12). The AGD3 mRNA has an open-reading frame that pro- first population expresses the orexigenic neuropeptide Y (NPY) and duces a 63-amino acid peptide. AGD3 is expressed in brain areas agouti-related peptide. The second group of neurones expresses relevant to feeding behaviour and is involved in cellular signalling anorexigenic neuropeptides cocaine- and amphetamine-related pathways that relate to energy metabolism and may be involved in transcript (CART) and pro-opiomelanocortin (POMC) (4,5). controlling feeding behaviour. Gene analysis using microarrays and high-throughput sequencing has been utilised extensively as a platform for biological explora- tion, including conditions associated with obesity. Using this Materials and methods approach, a previous report from our laboratory confirmed the dif- ferential expression of several neuropeptides associated with fasting Antibodies and reagents and obesity (6,7). The study also suggested that intracellular regula- Anti-Myc antibody-conjugated agarose was purchased from Bethyl Labora- tory genes are involved in energy homeostasis and are differentially tories (Montgomery, TX, USA). Anti-Myc monoclonal antibody was pur- regulated during fasting and feeding states. Over the past decade, chased from BD Bioscience (Palo Alto, CA, USA). Protein A SepharoseTM microarray studies of cDNAs have revealed thousands of novel CL-4B was obtained from GE Healthcare (Uppsala, Sweden). Anti-human A novel transcript in hypothalamus enhances insulin signalling 293 insulin receptor substrate 4 (IRS4) antibody (EP907Y) was obtained from pair was 5′GGA ATT CAC GCA ATG GCT GGA TAC CCG AGA G/5′ GGG GTA Abcam (Cambridge, MA, USA). Phospho-Akt (Thr308) antibody, Akt antibody CCG TTG CTT ACA CTG CCG TCAT, covering from 329 to 1168. The PCR prod- phospho-AMP-activated protein kinase (AMPK)a (Thr172) antibody, AMPKa ucts were sequenced. antibody, phospho-S6 (Ser235/236) antibody and S6 ribosomal protein antibody were obtained from Cell Signalling Technologies (Beverly, MA, USA). AGD3 antibody production Polyclonal antibodies against the rat AGD3 sequence were generated and Cell culture purified by affinity chromatography using a custom service from Invitrogen. The specificity of the antibodies was characterised using the myc-AGD3 Human embryonic kidney (HEK293) cells were cultured in Dulbecco’s modi- fusion protein. fied Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS) with 100 U/ml penicillin and 100 lg/ml streptomycin. Human neuroblastoma (SK-N-AS) cells were obtained from ATCC (Manassas, VA, USA) and were cul- Construction of expressing AGD3 vector and its small tured in ATCC-formulated DMEM with 1% non-essential amino acids, 10% interfering (si)RNAs FBS, 100 U/ml penicillin and 100 lg/ml streptomycin. Human colorectal adenocarcinoma (CACO-2) cells were obtained from Sigma-Aldrich (St Louis, AGD3-pcDNA3.1 MO, USA) and were cultured in Eagle’s minimal essential medium. with 2mM glutamine, 1% non-essential amino acids and 10% FBS. Mouse hypo- The sequence 117–529, which was cloned by RT-PCR from the rat hypothal- thalamus (GT1-1) cells were cultured in DMEM with 10% FBS containing amus and has an open-reading frame coding a 63 amino acids protein, was 100 U/ml penicillin and 100 lg/ml streptomycin. subcloned into the pcDNA3.1 using the EcoRI and KpnI sites. The Myc tag was fused to the N-terminus of AGD3. The correct frame of the construct was confirmed by DNA sequencing. Animals and experimental protocol Male Sprague–Dawley rats (Harlan Laboratories, Madison, WI, USA) weigh- AGD3 siRNAs ing 280–300 g were used to study AGD3 expression. Rats were housed in accordance with the University of Michigan Unit of Laboratory Animal Three pairs of double-stranded siRNAs targeting the human overexpression Medicine guidelines. Five rats were assigned to the fed group and five in colon cancer-1 (OCC-1) gene were used. Their sequences were: (i) 5′-CGA rats to the fasted group. Animals in the fed group were allowed free AAG ACA AGU UAA CAA ACU GUC A/5′-UGA CAG UUU GUU AAC UUG UCU access to regular rat chow and water. Animals in the fasted group were UUC GUA; (ii) 5′-AGC AGC CAA AGA UGU AAC AGA AGA A/5′-UUC UUC food-deprived 48 h but were allowed free access to water. At the end of UGU UAC AUC UUU GGC UGC UCC (IDT); and (iii) 5′-CCC UGG GUA CAU 48-h period, animals were sequentially euthanised. Whole brain was AUU GUU GTT/5′-CAA CAA UAU GUA CCC AGG GTT. Lipofectamine RNAiMax removed and frozen. A 2-mm thick section from Bregma À2toÀ4mm was used to transfect siRNAs into cells in accordance with the manufac- was sliced. The ARC was punched out using a Stoelting brain punch set turer’s instructions (Invitrogen). After 72 h of transfection, cells were lysed (Stoelting Co., Wood Dale, IL, USA). Then, total RNA was isolated from the and supernatants were analysed further. samples. Reverse transcription quantitative PCR (RT-QPCR) was used to examine changes in rat AGD3 expression. Reverse transcription was performed using Identification of the AGD3 transcript the SuperScript One-Step RT-PCR System with Platinum Taq DNA Polymer- A Pierce Direct IP Kit (Pierce Biotechnology, Rockford, IL, USA) was used to ase (Invitrogen, Carlsbad, CA, USA). Q-PCR primers were : AGD3: 5′-CCG pull down AGD3 protein from CACO-2 cells. Briefly, AGD3 antibody was ACA GGA GCA GTT AAA GAT G-3/5′-CTT CCA AAT TCA CGA GCA AGC/Probe: linked to agarose beads and incubated with cell lysates. After overnight 5′-56-FAM/AGA TAA CGC AAT GGC TGG ATA CCC G/36-TAMSp/-3′ and GAP incubation at 4 °C, beads were rinsed thoroughly with wash buffer, and DH: 5′-ACC ACA GTC CAT GCC ATC AC/5′-CAG CTC TGG GAT GAC CTT GC/ AGD3 was eluted. Eluates were resolved on 16.5% Tris-Tricine gels (Bio-Rad, 5′-/Probe: 56-FAM/CAC AGC CTT GGC AGC ACC AGT GGA/36-TAMTph/-3′. Hercules, CA, USA). Gels were stained with Bio-Safe Coomassie Blue stain GAPDH primers were used as an internal control. solution. Protein bands recognised by western blotting directed against anti- To analyse AGD3 protein expression in ARC, another set of ten rats, five AGD3 antibody at the 7-kDa position were subjected to mass spectrometry fed and five fasted, were used. Animals were euthanised. The brain was analysis. removed and a 2 9 2 9 2 mm cube was directly dissected from the ARC The protein identification was conducted at the Protein Structure Facility area. The tissue was homogenised in lysis buffer and supernatant was sub- at the University of Michigan (Ann Arbor, MI, USA). Bands of interest were ject to western blotting. excised and the proteins digested in situ with trypsin (Promega, Madison, WI, USA). Digests were analysed by capillary high-performance liquid chro- Cloning of AGD3 cDNA matography-electrospray ionisation tandem mass spectrometry (HPLC- ESI-MS/MS) on a Thermo Fisher LTQ Orbitrap Velos mass spectrometer Our gene microarray study demonstrated that an expressed sequence tag (Thermo Fisher Scientific Inc., Waltham, MA, USA) fitted with a New Objec- (EST) was up-regulated by fasting.