The purpose of our survey is to happen a familial nexus between schizophrenic disorder and type-2 diabetes. The thought of this probe comes from the fact that people who suffer from schizophrenic disorder have more hazard of developing type-2 diabetes ( ~9 % ) as compared to the general population ( ~ 4.9 % ) ( Cohen et al 2006 ) . It has besides been noted that there has been high prevalence of approximately 30 % of impaired glucose tolerance in patients holding schizophrenic disorder, subjective to age ( Lindenmayer et al, 2003 ; Subramaniam et Al, 2003 ) . We hypothesize that the nexus between the two conditions is multifactorial that involves familial and environmental, i.e. , antipsychotic drug induced-obesity.
To transport the research forward we performed two familial surveies:
Association survey to see if the two conditions portion common cistrons or familial tract. We chose four campaigner cistrons, AKT1, PPARG, PLA2G4A and PTGS2 in our survey and the made the undermentioned 2 hypotheses:
Functional survey to see the consequence of Clozaril ( untypical antipsychotic drug ) on the cistron look of assorted cistrons identified by genome broad association surveies for type-2 diabetes and fleshiness. The hypothesis we made for this survey is:
The ground of taking PPARG cistron as one of our campaigner cistrons is because it has a function to play as a modulator of insulin sensitiveness, glucose homeostasis and blood force per unit area. GWAS have besides identified PPARG cistron to be associated with type-2 diabetes. Thiazolidinediones, a group of anti-diabetic drugs that improve insulin-resistance, are identified as PPARG-ligands ( Fajas et al. 1997 ) . There is a possibility that PPARG-locus might stand for a pathological nexus between schizophrenic disorder and type-2 diabetes. PPARG is functionally regulated by the desiccation merchandise of prostaglandin D2 ( PGD2 ) , 15-deoxy-i?? prostaglandin ( 15-dPGJ2 ) , which is a major endogenous ligand of PPARG.
PLA2G4A and PTGS2 cistrons are the other two campaigner cistrons studied as they have been associated with schizophrenic disorder in the yesteryear.
In this hypothesis we would prove if PLA2G4A, PTGS2 and PPARG cistrons are associated with schizophrenic disorder singly or in a combined mode. The consequences would assist to find the extent to which a familial nexus explains the association between schizophrenic disorder and type-2 diabetes.
1.1.1. Biological characters of PPARG
Peroxisome Proliferator Activated Gamma ( PPARi?§ ) is a member of the PPAR subfamily of atomic receptors. The atomic receptor superfamily is a group of proteins that act as written text factors to change cistron look when edge to specific ligands. They have a alone belongings of organizing a heterodimer composite with retinoid X receptor ( RXR ) , another member of atomic receptor superfamily. PPARs are involved in a broad assortment of maps such as lipid metamorphosis, embryologic development, and virtually all hormone tracts ( Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear Receptors and Lipid Physiology: Opening the X-Files. Science 294: 1866-1870, 2001 ) . They besides regulate a figure of cellular maps from fatty acerb oxidization to adipocyte distinction. They were foremost identified as proteins that induce peroxisome proliferation in gnawer liver cells in response to exogenic chemicals, many of which are hepatocarcinogens. There are three PPAR isotypes: i?? , I? and i?§ , which have somewhat different look forms and maps but have similar cistron constructions ( Beamer, 1997 ; Fajas, 1997 ) .
2.1.2. Structure of the PPARG protein
Guo & A ; Tabrizchi ( 2006 ) proposed a construction of the PPARi?§ protein which includes 6 structural parts ( A-F ) in 4 functional spheres ( Figure X ) . The inside informations of the location and map of these spheres in given below:
Ligand-independent transactivation ( activation function-1 ) domain- AB part is transcriptionally active in the absence of ligands and can alter the ligand adhering affinity of the receptor when covalently modified by phosphorylation or sumoylation. It is next to the NH2 end point.
Deoxyribonucleic acid adhering sphere – C part, is the most conserved sphere in all atomic receptors. The DNA-binding sphere is formed by two zinc finger-like motives folded in a ball-shaped construction, which can acknowledge a DNA mark composed of 6 bases ( AGGTCA ) . This specific DNA sequence is located within the regulative parts of the mark cistrons and serves as the peroxisome proliferator endocrine response component ( PPRE ) , which allows adhering to this sphere and hence regulates written text.
Hinge sphere – D part, helps in the rotary motion of the DNA-binding sphere and besides connects to the ligand-binding sphere.
Ligand-binding sphere ( activation function-2 ) – EF part, has a big binding pocket that allows the written text receptor to adhere to a assortment of construction different ligands. This lies next to the C end point that is besides portion of the ligand-binding sphere. An agonist locks ligand adhering sphere into an active conformation on adhering whereas an adversary stabilizes the receptor into an inactive province upon binding.
Useful links for PPARG cistron:
OMIM ID: 601487
Entrez Gene ID: 5468
HGNC ID: 9236
1.1.3. The manner of action
PPARG becomes activated upon ligand binding and so forms a heterodimer composite with retinoid X receptor ( RXR ) . The PPARG-RXR composite regulates the transcriptional procedures by adhering to a specific sequence, AGGTCA, i.e. PPREs. This sequence is separated by 1-nucleotide called DR-1 and is located on the booster part of the mark cistron ( IJpenberg et al. 1997 ) . The changes of the cistron written text depend on the nature and type of ligand binding to it. The interactions between the ligand, PPARG-RXR heterodimer, modulator proteins and the written text machinery affect the written text induction and mRNA copiousness of the mark cistron ( Corton, Lapinskas and Gonzalez 2000 ) .
Deoxyribonucleic acid binding sphere
Contains big adhering pocket
Binds to assortment of construction unrelated ligands
C terminal portion of this sphere
Agonist lock the receptor into an active conformation while antagonist stabilise the receptor into an inactive conformation
Transcriptionally active in the absence of ligands
Covalently alteration by phosphorylation & A ; sumuylation changes the ligand-binging affinity of the receptor
Ligand-binding sphere ( AF 2 )
Ligand-independent transactivation sphere ( AF 1 )
Ligand-binding sphere ( AF 2 )
Permits the rotary motion of DNA-binding sphere
Connects AB sphere with EF sphere
Most conserved sphere
PPARs are targeted to a specific sequence ( PPRE ) of bases within the regulative parts of the antiphonal cistrons
Figure Ten: Structure of a PPARG protein proposed by Guo & A ; Tabrizchi ( 2006 )
Modified diagram from: Guo L. , Tabrizchi R. ( 2006 ) Pharmacology & A ; Therapeutics 111: 145-173
1.1.4. Function of the PPARG cistron
The cistron coding for PPARG lies on the short arm of chromosome 3 ( 3p25 ) , spans about 146 kilobit of DNA and contains 7 coding DNAs. The PPARG cistron was ab initio cloned from bone marrow complementary DNA library in 1995 ( Greene et al. 1995 ) . The chief site of look of PPARG is the adipose tissues ( Youssef and Badr 2004 ) although it is besides expressed at the lower degree in many other tissues such as the big bowel, colon, lien, immune cells, endothelial cells and smooth musculus cells ( Moraes, Piqueras and Bishop-Bailey 2006 ) .
The grounds for choosing PPARG as one of our campaigner cistrons is because it is functionally regulated by the desiccation merchandise of prostaglandin D2 ( PGD2 ) , 15-deoxy-i?? prostaglandin ( 15-dPGJ2 ) , which is a major endogenous ligand of PPARG and associated with arachidonate-phospholipid metamorphosis ( Na and Surh 2003 ) . PPARG is besides a modulator of insulin sensitiveness, glucose homeostasis and blood force per unit area. Thiazolidinediones, a group of anti-diabetic drugs that improve insulin-resistance, are the unreal ligands of PPARG ( Fajas et al. 1997 ) . Besides, PPARG is an of import regulator of adipocyte distinction and map.
Several familial fluctuations have been described in the human PPARG cistron. The P115Q transition was foremost identified in 4 morbidly corpulent topics that cause faulty phosphorylation of the protein ensuing in accelerated adipocyte distinction ( Ristow, 1998 ) . Two other mutants ( P467L and V290M ) were found in 3 persons ( in 2 households ) with terrible insulin opposition, dyslipidaemia and high blood pressure. Both of these mutants have been shown to do impaired ligand-dependant transactivation and suppress wild-type PPARG in a dominant negative mode ( Barroso, 1999 ) . Two other more common polymorphisms, a soundless C to T permutation in exon 6 and a C ( proline ) to G ( alanine ) permutation at codon 12, have besides been reported in PPARG ( Vigouroux, 1998 ; Yen, 1997 ) . There is grounds that suggests that the mutant protein produced by the alanine allelomorph at codon 12 has reduced transcriptional and adipogenic, activity in vitro, which could take to lower adipose tissue mass ( Masugi, 2000 ) . In add-on, the alanine allelomorph was found to be associated with lower lipolysis and greater insulin sensitiveness in a group of thin non-diabetic topics ( Stumvoll, 2001 ) .
1.1.4. Proposed familial web
As both schizophrenic disorder and type-2 diabetes are extremely heritable diseases, they may portion the same familial web in some instances. Candidate cistron surveies and genome-wide association analyses have revealed tonss of cistrons associated with type-2 diabetes ( E. Zeggini, L.J. Scott, et Al, 2008 ) . Of these associated cistrons, the peroxisome proliferator activated receptor-gamma ( PPARG ) venue might stand for a pathological nexus between schizophrenic disorder and type-2 diabetes as PPARG is functionally regulated by the desiccation merchandise of prostaglandin D2 ( PGD2 ) , 15-deoxy-D prostaglandin ( 15-dPGJ2 ) , which is a major endogenous ligand of PPARi?§ and associated with arachidonate-phospholipid metamorphosis ( H.K. Na and Y.-J. Surh, 2006 ) .
Niacin-induced dermal flower has been used as a non-invasive diagnostic attack to prove for lack in arachidonic acid ( AA ) released from membrane phospholipids and synthesis of AA derived functions such as prostaglandins ( P.E. Ward, J. Sutherland, et Al, 1998 ) . Niacin is a signifier of vitamin B that functionally enhances the synthesis of PGD2, a prostaglandin that can excite blood vass to distend ( J.D. Morrow, J.A. Award et al, 1992 ) . Up to 80 % of patients with schizophrenic disorders have a hapless tegument response to niacin stimulation as compared to 20 % of healthy persons ( P.E. Ward, J. Sutherland, 1998 ; B.K. Puri, T. Easton, 2001 ) . These findings suggest that the lessened niacin flower may stand for a subgroup of patients with schizophrenic disorder who have unnatural phospholipid metamorphosis in their cellular membranes. If this were the instance, such a subgroup might besides hold an increased hazard for type-2 diabetes due to lack of PGD2 and 15-dPGJ2.
Phospholipase A2 ( PLA2 ) is a group of the enzymes modulating the release of polyunsaturated fatty acids ( PUFAs ) , such as AA, from the sn-2 place of phospholipids. This is an of import measure in the coevals of PPARG ligands from arachidonate-phospholipids. The enzymatic activity of assorted members of the PLA2 household has been found to be altered in schizophrenic disorder with some incompatibility ( Gattaz et al 1987 ; Gattaz et Al 1990 ; Gattaz et Al. 1995 ; Albers et Al. 1993 ; Noponen et Al 1995 ; Ross et Al. 1997 ; Ross et Al. 1999 ; Smesny et Al. 2005 ) . Inhibitors of encephalon PLA2 activity have neuropharmacological effects and may turn out to be therapeutically of import in handling neuropsychiatric upsets ( Farooqui et al. 2006 ) . Familial surveies suggest that of the assorted PLA2 members, polymorphisms in the cistron coding for PLA2 group 4 homolog A ( PLA2G4A ) , a cytosolic signifier of PLA2, may lend to the pathogenesis of schizophrenic disorder ( Law et al. 2006 ) . Due to its function in prostaglandin coevals and old association with schizophrenic disorder, PLA2G4A was selected for genotyping in this survey.
Cyclooxygenase 2 ( COX2 or PTGS2 ) is a rate-limiting enzyme responsible for prostaglandin synthesis in response to stimulus including some signaling tracts in the cardinal nervous system ( CNS ) ( Langenbach et al. 1999 ; Samad et Al. 2001 ) . Interestingly the cistrons coding for PTGS2 and PLA2G4A lie near to each other on the long arm of chromosomes 1. While PTGS2 was non associated with schizophrenic disorder in a old survey utilizing a smaller subset of this survey population ( Wei et al. 2004 ) , it is deserving genotyping in this larger population to see if there is a cis-phase interaction between these 2 venue. The present survey hence set out to prove the hypothesis that the PLA2G4A, PTGS2 and PPARi?§ cistrons are associated with schizophrenic disorder singly or in a combined mode.
Hypothesis 1.2. : The function of the AKT1-GSK3 signalling tract
AKT1 is another candidate cistron we chose to analyze as it plays a functional function in intracellular signalling tracts that may be of import for the pathophysiology of schizophrenic disorder. Emamian and colleagues ( 2004 ) were the first to describe on the AKT1 association with schizophrenic disorder in a Northern European population. This initial determination has been replicated in a figure of independent surveies [ Schwab et al. , 2005 ; Bajestan et al. , 2006 ; Ikeda et Al, 2006 ; Norton et al. , 2007 ; Xu et al. , 2007 ; Thiselton et al. , 2008 ] , although some others failed to retroflex the AKT1 association [ Ohtsuki et al. , 2004 ; Ide et al. , 2006 ; Liu et al. , 2006 ; Pinheiro et al. , 2007 ; Turunen et al. , 2007 ; Sanders et al. , 2008 ] . The association of AKT1 and schizophrenic disorder was besides seen in other populations, Nipponese, Irish, Persian and Finnish.
V-akt murine thymoma viral transforming gene homologues ( AKTs ) are a subfamily of the phosphoinositide-dependent serine-threonine protein kinases and play a critical function in cellular procedures such as glucose metamorphosis, cell proliferation, programmed cell death, written text and cell migration ( Brazil and Hemmings 2001 ) . AKTs besides interact with neural dopaminergic signalling ( Tan et al. 2008 ) . There are three isoforms of AKT enzymes identified in mammalian cells, viz. AKT1, AKT2 and AKT3, which are encoded by distinguishable cistrons located on chromosomes 14q32 ( AKT1 ) , 19q13 ( AKT2 ) and 1q43 ( AKT3 ) in worlds. All three cistrons have greater than 85 % indistinguishable DNA sequences and their protein merchandises portion the same structural organisation ( Kandel and Hay 1999 ) . All 3 AKT isoforms are ubiquitously expressed in mammals although the degrees of look vary among tissues ( Bellacosa et al. 1993 ) . The most common isoforms prsent in most tissues is AKT1, which is extremely expressed in renewing nerve cells ( Owada et al. 1997 ) . The highest look of AKT2 is observed in insulin-responsive tissues, such as skeletal musculus, bosom, liver and kidney ( Altomare et al. 1995 ) , proposing that this isoform is of import for the insulin signaling tract. Unlike AKT1 and AKT2, the AKT3 isoform shows a more restricted form of look and higher degrees are found in the testicles and encephalon than in the grownup pancreas, bosom and kidney ( Brodbeck, Cron and Hemmings 199 ) .
1.2.2 Structure of AKT1
Figure Ten: Structure of the AKT molecule. ( Walker et al. 1998 ) Walker et Al in 1998 proposed that all three AKT-isoforms portion similar construction. The elaborate construction of AKT1 is shown in Figure V-1 and contains the undermentioned functional spheres: ( 1 ) pleckstrin homology ( PH ) domain that binds phospholipids, ( 2 ) a catalytic sphere, ( 3 ) a regulative sphere and ( 4 ) short glycine-rich part ( Bridgess to the catalytic part ) . All three isoforms besides possesse a conserved threonine and serine residue ( threonine 308 and serine 473 in AKT1 ) and the phosphorylation of those sites is critical for full activation of AKT. The distance between the two phosphorylation sites/residues is around 160 – 170 amino acids ( Kandel and Hay, 1999 ) .
1.2.3 The proposed tract
AKT molecule is activated inside the cell. Briefly, coevals of D3-phosphorylated phosphoinositides by phosphoinositide 3-kinases ( PI3K ) induce the translocation of AKT on the plasma membrane via its PH sphere where it is activated. In the plasma membrane, AKT co-localizes with the constitutively active phosphoinositide-dependent kinase-1 ( PDK-1 ) , which activates AKT by phosphorylation on the different residues, for illustration, Thr308 and Ser473 in the AKT1 isoform ( Kandel and Hay, 1999 ) . After activation, AKT can phosphorylate a figure of substrates, one of which is glycogen synthase kinase 3 ( GSK-3 ) ( Norton et al. 2007 ) . On phosphorylation, AKT1 inhibits GSK-3 that upregulates the animal starch synthase activity, which is portion of the insulin signal transduction tract ( Ali, Hoeflich and Woodgett 2001 ) . Impaired AKT1-GSK3 beta signaling has been reported in schizophrenic disorder ( Emamian et Al. 2004 ) . Furthermore, GSK3 has besides been implicated in the multifactorial etiology of skeletal musculus insulin opposition in carnal theoretical accounts and in patients with type 2 diabetes ( Dokken, Sloniger and Henriksen 2005 ) . The disfunction of the AKT1-GSK3 signal tract may be involved in both type-2 diabetes and schizophrenic disorder. Several surveies have shown the ATK1 association with schizophrenic disorder ( Emamian et Al. 2004 ; Neuton et al. , 2007 ) .
The present survey therefore was to retroflex the initial findings in the British samples and besides to research if the AKT1 cistron was involved in development of major tranquilizers induced type-2 diabetes.
Effectss of Clozaril on look of the cistrons associated with type-2 diabetes and fleshiness
The patients who suffer from schizophrenic disorder have to be on antipsychotic medicine. The major side consequence of these drugs is enormous weight addition, hence fleshiness. The major hazard factor of type-2 diabetes is fleshiness. Therefore we tested the consequence of Clozaril, an untypical antipsychotic drug, on the look of cistrons associated with fleshiness and type-2 diabetes identified by genome-wide association surveies through tissue civilization techniques. These surveies have late identified more than 20 cistrons for fleshiness and type-2 diabetes ( Table X ) .We besides tested the campaigner cistrons selected in the above mentioned hypothesis and besides the cytokine cistrons.