Conjugated Linoleic Acid Different Isomers Biology Essay

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Conjugated Linoleic Acid (CLA) is a family of different isomers of linoleic acid that are found in the meat and dairy products derived from ruminants (Y.L. Ha et al 1987), that have been shown to have an anti carcinogenic properties and anti inflammatory effects upon human cells in vitro (N S. Kelley et al 2007) but curiously apparent proinflammatory affects in vivo in human trials causing insulin resistance and reduced insulin sensitivity (U Risérus et al 2002). CLA is available as a dietary supplement for its marginal weight management properties, with CLA having a very small impact upon fat mass in humans (S Tricon et al 2004). This study was to assess what, if any impact CLA has upon the expression of AGER on human monocytic cells. From data acquired in the course of this study its apparent that CLA does have an up regulatory affect on AGER gene expression on human monocytic cells in virto.

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The inflammatory process is a controlled response of the body to varying stimuli such as tissue damage, the presence of pathogens or irritants such as tobacco (Cosio 2009). Whilst inflammation benefits wound healing and increases the effectiveness of the body's white blood cells chronic inflammation, irritation and recurrent sites of infection are related to the development of chronic medical conditions such as irritable bowel syndrome, rheumatoid arthritis, arthrosclerosis, cancer and other conditions. The relationship between sites of chronic inflammation and cancer was first hypothised by Virchow in 1863, it is clear that cell proliferation alone does not cause cancer however continued cell proliferation in an area rich in inflammatory cells, growth factors and DNA damaging agents increases and possibly promotes the emergence of a neoplasm (Coussens & Werb 2002). This has also be shown in controlled animal studies where inflammation contributed to carcinogenesis after the administration of a known carcinogenic agent; examples of this include the emergence of tumors in chickens caused by Rous sarcoma virus appearing at sites of injury and inflammation despite systemic viral infection (Weitzman & Gordon 1990).

In response to tissue damage chemical signals at the site of injury initiate an immune response to help heal the affected tissue, this will involve the migration of neutrophils, monocytes and eosinophils from the blood circulation to the site of tissue damage. Chemokines that are released by the damaged tissue allow for chemotaxis of specific leukocytes to the area; neutrophils being the first recruited leucocytes of an acute inflammatory response followed by monocytes which differentiate into macrophages in tissues (Coussens & Werb 2002). When activated macrophages are the main source of growth factors and cytokines which has effects upon the endothelial and epithelial cells in the vicinity with recruited mast cells also being important due to their release of inflammatory mediators such as cytokines and histamines (Coussens & Werb 2002).

With leucocytes able to produce radical oxygen species and nonradical oxygen species such as hydrogen peroxide at the location of tissue damage or infection the release of which will not only damage pathogens but also surrounding tissue possibly leading to further tissue damage and inflammation in the local environment (Weitzman & Gordon 1990). Development of chronic inflammation is dependant upon the cytokines and chemokines that persist at the site of inflammation attracting leucocytes to the vicinity. Whilst inflammation is usually self limiting process a breakdown in the regulation of pro and anti inflammatory cytokines can lead to pathogenesis such as neoplastic progression (Coussens & Werb 2002).

1.2 Conjugated Linoleic Acid

CLA (Conjugated Linoleic Acid) are a family of different isomers of linoleic acid that are found in the meat and dairy products derived from ruminants. It was first isolated from ground beef with its anti-carcinogenic properties becoming apparent after mice that had been administered CLA prior to being treated with 7,12-dimethylbenz[a]anthracene (DMBA) developed half as many papillomas and had a lower tumor incidence than the control mice that were not been administered CLA (Ha et al 1987).

CLA is formed primarily as a result of ruminant gut isomerisation of dietary lioloic acid, with CLA concentrations varying from 2.9 to 8.92 mg CLA/g fat of which CLA isomer 9:11 consists of 73-93% of the total CLA present in the dairy product (G S. Kelly 2001). The level of CLA present within a ruminant product varies considerably; when milk from various herds of cows in New York State was examined, CLA levels ranged from 2.4 to 18 mg CLA/g fat. This large variation of CLA levels in meat and dairy products was attributed to the differences in diet and feed practices between herds with CLA levels being dramatically higher in pasture fed animals (Lin et al 1995). Increasing the proportion of grazed fresh pastoral grass has been shown to increase the CLA content of dairy cows milk five fold when compared to ruminants fed forage and grain (Dhinman et al 1999).

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Figure 1: Isomers of CLA and Linoleic Acid. Structures of the biologically active isomers of conjugated linoleic acid (CLA). Journal of Chemical Education (1996).

CLA has been shown to reduce inflammation and oxidative stress in animal models (Kelley et al 2007) however Risérus et al found that whilst in vitro CLA has clear anti-oxidative effects in animal studies in human trials consisting of men with metabolic syndrome CLA was pro-oxidative especially when using CLA isomer 10:12 as indicated by a raised C-Reactive Protein (CRP) within the 10:12 trial group. The raised CRP was also seen in the placebo group Risérus et al could not conclude the clinical relevance of the increased CRP, insulin resistance and reduced insulin sensitivity (Risérus et al 2002), however in another study by U Risérus et al this increase in insulin resistance and CRP links oxidative stress already present in patients with metabolic syndrome in contributing to the CLA induced insulin resistance (Risérus et al 2002).

1.3 CLA as a PPAR activator

The ligand activated transcription factor peroxisome proliforator activated receptor (PPAR) modulates lipid metabolisim and has been identified in the regulatory regions of genes coding for the lipid metabolizing enzymes ACO, acyl Co-A synthase, lipoportien and other enzymes. There are three subtypes of PPAR's, PPARα is predominantly expressed in liver, kidney and heart tissue, PPARδ being expressed in almost every human tissue and PPARγ being found in adipose tissue and has been linked to adipocyte differentiation, fatty acid uptake and storage and glucose uptake (Moya-Camarenaa et al 1999). Synthetic PPAR activators are available as novel treatments with Rosiglitazone being one of the most prominent, itself being used to increase insulin sensitivity in diabetics and also being used to manage inflammatory conditions such as ulcerative colitis. The mechanism by which inflammation is decreased by the administration of Rosiglitazone is by Rosiglitazone's binding with PPARγ ligands causing an up regulation of NFκB inhibitor (I-κB) which in turn downregulates the inflammatory pathways (Mohanty et al 2004).

CLA has some structural similarities to peroxisome proliferators, with physiological responses in mice such as reduced body weight and hepatic lipid accumulation being characteristic of PPAR activators. Mohanty et al demonstrated that CLA is a ligand and activator of PPARα and further hypothesized that as CLA induces PPARα response genes it regulates lipid metabolisim through a PPARα mediated pathway (Mohanty et al 2004).

1.4 CLA and immune system modulation

CLA also modulates the immune system by increasing lymphocyte cytotoxicity and macrophage activation. There are numerous dietary oils that are known to modulate macrophage function, dietary fish oil decreases interleukin-1 (IL-1) release by macrophages when compared to corn oil fed controls (Korver & Klasing 1997). Macrophages are the principal source of tumour necrosis factor alpha (TNF-α) with lipopolysaccharide (LPS) which is found in bacterial cell walls being the most potent stimulator of TNF-α production. TNF-α was originally shown to kill tumour cells but it also has a dramatic effect in causing muscle wasting, Mohanty et al showed that the inclusion of CLA into the feed of chicks reduces LPS induced muscle wasting due to the affect of CLA at inhibiting TNF-α release by macrophages when the cells are stimulated with LPS (Mohanty et al 2004).

Immune system T lymphocytes differentiate once activated from naïve T cells (Th0) to T helper (Th1) or T effector (Th2) cells as they secret cytokines and mediate the immune response; Th1 response is cell mediated immunity and inflammation whilst the Th2 cells secret cytokines and mediate the immune response. Once activated naïve T cells differentiate into either Th1 or Th2 cells depending upon the cytokines present; for example in the presence of interleukin-12 (IL-12) Th1 development is preferred whilst in the presence of IL-4 Th0 cells differentiate mostly into Th2 cells. Th1 and Th2 responses by the immune system is interlinked; for example interferon γ (IFN- γ) which is produced by Th1 cells inhibits IL-4 production which is required for Th2 development in turn suppresses Th2 cell development (Mohanty et al 2004).

Dietary CLA increases immunoglobulin (Ig) A, IgG and IgM in rat and lymph node cells whilst reducing IgE implying that CLA promotes Th1 cytokine whilst inhibiting Th2 cytokine production as the Ig class switch required from IgG to IgE wouldn't occur without IL-4 or IL-13 which are both Th2 cytokines (Punnonen et al 1993). It is because macrophages are sensitive to LPS stimulation they are a potential target for CLA in order to decrease an LPS induced response specifically upon macrophage TNF-α production in patients that are having a strong immune response to gram negative bacterial infection such as bacterial meningitis (Mohanty et al 2004).

1.5 Accumulation and creation of AGE's

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Reduced sugars in the body can react non-enzyematically with amino groups of proteins to form Amadori products (Nakamura 2006). The Maillard reaction links protein amino groups with glucose derived carbonyl groups that over months and years create a variety of AGE's within the body (Miyata et al 1998). These glycation products can undergo further reactions including dehydration, rearrangement and condensation to become crosslinked which is mostly irreversible to create AGE's (Nakamura 2006). AGE levels increase normally with age, and AGE accumulation is accelerated in conditions such as diabetes and uremia. Some AGE's can be disposed of via the kidneys as demonstrated by T Miyata et al who demonstrated that renal function directly impacts upon the in vivo half life of the AGE pentosidine in an animal study and AGE's noted to accumulate in animal models with renal failure in animal (Miyata et al 1998).

In most tissues AGEs and their precursors are created from the auto oxidation of glucose and fructose, these include deoxyglucosone, methylglyoxal, and glyoxal (S P Baba etal 2009). Methylglyoxal is generated nonenzymatically from intermediates created during glycolysis and enzymatically by peroxidases where other AGE precursors such as deoxyglucosone is generated from the degradation of Amadori compounds. Participation of enzymes in the metabolisim of these intermideates and the creation of AGE precursors remain unclear; however in vitro studies of aldoketo reductase's (AKR) have been shown to catalyze methylglyoxal and are known to convert methylglyoxal to acetol in bacteria though their effectiveness, if any in mammals has not been studied (Baba et al 2009).

Figure 1.2 - (a) Basic scheme for AGEs formation; (b) Non enzymatic glycation products. Bierhaus 1998.

Figure 1.2: Figure 1.2 shows the interaction of protein and sugars required to create AGE products.

1.6 AGER

The receptor for AGE's (AGER) is a multiligand receptor that belongs to the immunoglobulin super family of cell membrane receptors which when bound creates cellular activation and over a sustained period cellular disfunction and tissue destruction. The involvement of AGER in the physiological process has been well demonstrated in murine models using sAGER as a decoy, the introduction of anti-AGER antibodies to destroy the AGER receptor or murine models that do not posses the AGER receptor. AGER contributes to the development of late diabetic complications such as neuropathy, arteriosclerosis and chronic inflammation (Bierhaus et al 2005).

AGER receptors are readily expressed on human tissues, notably that of the lung however expression rapidly becomes measureable at other sites of inflammation on both epithelial cells and inflammatory cells. AGER exists as a membrane bound receptor or as a soluble protein which is increased in areas of stressed and damaged epithelial cells which leads to enhanced survival of the cell (Sparvero et al 2009); as such prolonged or indefinite signaling through the AGER survival pathway in an area of chronic inflammation results in diminished cellular apoptosis which in turn is a setting favorable setting for epithelial malignancies to occur (Sparvero et al 2009).

Figure 1.3 - Structure of RAGE (Amended from Schmidt and Stern 2000).

AGER consists of three immunoglobulin-like regions, one 'V' or Variable immunoglobulin type Ig domain, followed by two 'C' or Constant immunoglobulin Ig domain. The extracelluar region 'V'type particularly is involved in ligand binding and followed by the hydrophobic transmembrane-spanning domain and a cytosolic tail which is responsible for intracellular signalling upon ligand binding. Binding of AGEs to AGER activates NF-B through signal transduction pathways, leading to cellular production of inflammatory mediators. The cytosolic tail is critical for signaling cascades that lead to cellular activation and altered cellular properties.

1.7 Structure of AGER

AGER is found as a full length membrane bound receptor and in a soluble, non-membrane bound form (sAGER) which lacks the transmembrane signalling domain found in AGER. AGER is found in the Class III region of the major histocompatibility complex (MHC). AGER possesses a 'V type domain, two C type domains, a transmembrane domain, and a cytoplasmic tail', sAGER however lacks both the transmembrane domain and the cytoplasmic tail which are both believed to be necessary for intracellular signaling (Sparvero et al 2009).

As AGER receptors are found in the class III region of the MHC, AGER receptors are found on monocytes. AGE binding to monocytic bound AGER induces chemotaxis in the monocyte and allows for infiltration of the monocyte through an intact endothelial monolayer with AGER expressing monocytes being indentified in atherosclerotic plaques in the expanded intima (Aronson & Rayfield, 2002). The binding of AGE to monocytic AGER results in the production of inflammatory mediators such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF- α) both of which have important roles in inflammation and the pathogenesis of diseases such as arteriosclerosis (Aronson & Rayfield, 2002).

AGER transcription is controlled by a number of transcription factors including SP-1, AP-2, NF-κB and IL6, and is readily expressed during embryonic development with its expression being downgraded in adult life, with exceptions being the skin and lungs which continuously express AGER along with monocytes. Endothelial and smooth muscle cells do not express a significant amount of AGER under normal physiological conditions but can be induced where AGER ligands accumulate or transcription factors that regulate AGER expression are activated (Bierhaus et al 2005).

AGE binding to AGER receptors in conditions such as arthrosclerosis on the epithelium induces oxidative stress and the release of NF-κB by the epithelial cells which in turn causes reduced endothelial barrier function with increased permeability of the endothelial cell monolayers. The increased permeability of the endothelium has the potential to allow increased lipid entry into the subendothelium and the increase of adhesive interaction between the endothelium and monocytes allows for migration into the subendothelium which over time allows the accumulation of an atherosclerotic plaque ( Aronson & Rayfield, 2002).

1.8 sAGER

sAGER's binding of AGE does not reduce cellular apoptosis as sAGER lacks the transmembrane domain and the cytoplasmic tail which are both believed to be essential for intracellular signaling and thus does not promote cell survival in the same manner as membrane bound AGER does; as such sAGER can be viewed as a competitive inhibitor of AGER activated pathways as sAGER competes for AGE ligands (Schlueter et al 2003). The binding of AGE to membrane bound AGER and the decrease in serum concentrations of sAGER, the subsequent promotion of inflammatory mediators such as TNF- α and IL-1 also play a significant role in the pathogenesis of other medical conditions such as rheumatoid arthritis where decreased serum sAGER has been linked to the promotion of chronic inflammation (Pullerits et al 2005).

The competitive inhibition that occurs between sAGER and AGER in regards to inflammation and the development of chronic and malignant conditions cannot be understated as AGE's which bind to sAGER and AGER ligands once formed are stable and virtually irreversible and accumulate over time. This has been noted in the accelerated development of atherosclerosis in diabetic patients where there is a nonenzymatic reaction between glucose, proteins or lipoproteins in the arterial wall (Aronson & Rayfield, 2002). Glucose forms glycosylation products with reactive amino groups which circulate freely in the blood or become vessel wall proteins, these over time rearrange to become more stable glycosylation products such as HbA1C which is detectable and remain at stable levels whilst blood glucose levels may rise or fall. Glycosylation products that are deposited on longer lived cells such as the collagen of arterial walls will continue to undergo chemical rearrangement to form AGE's and due to the chemical stability of AGE they accumulate continuously whilst binding to AGER receptors on local inflammatory and epithelial cells, leading to a localized shift in favor of oxidative stress which substantially increases local AGE formation which in turn further increases oxidative stress in the local environment (Aronson and Rayfield, 2002).

Excessive AGE accumulation and formation has been linked to the cross linking of collagen, vitronectin and laminin with the binding of AGE's to AGER resulting in the activation of mitogen activated protein kinases, NF-κB and cyclic adenosine monophosphate (cAMP) response element binding (CREB) which stimulate the production of reactive oxygen species and increases local vascular permeability and inflammation (Baba 2009). The significance of AGER mediated events can be seen when AGER is blocked by the competitive inhibition of soluble AGER (sAGER) which was used in a diabetic murine model and restored the diabetic deficit in wound healing (Goova 2001) and that the addition of murine sAGER in diabetic murine models at 7 weeks showed there to be little difference between the diabetic model and the control group in terms of renal nephropathy caused by AGER binding of AGE's and the resulting inflammation; this finding supports the conclusion that sAGER could be used as a novel treatment in preventing the accelerated buildup of AGE's in conditions such as diabetes.

Diabetic murine models with AGER gene deletion also showed renal nephropathy comparable to the control group whilst the diabetic group that wasn't treated with sAGER or had AGER deleted showed accelerated nephropathy in comparison to the control group (Wendt 2003) suggesting that AGE's play a significant role in mediating hyperglycemic injury in diabetic models and by extension humans (Goova 2001).

The gene coding for AGER is found on chromosome 6 in the vicinity of the MHC class III complex in close proximity to the homeobox gene HOX12 and the human counterpart of the mouse mammary tumor gene int-3. AGER is composed of three immunoglobulin like regions which are a V-type domain and two C-type domains, a short transmembrane domain and possesses a cytoplasmic tail (Bierhaus et al 2005). The V-type domain allows the binding of AGE's with the cytoplasmic tail being used for intracellular signaling, with sAGER being an AGER variant that lacks the cytoplasmic tail. AGER is able to bind peptide ligands to its receptor such as amyloid-β peptides which are known to accumulate in Alzheimers disease (Yan et al 1997), amyloid-α which is known to accumulate in systemic amyloidosis (Yan et al 2000) as well as three dimensional peptide structures such as β-sheets and fibrils (Schmidt et al 2001). Further ligands of AGER are S100 (calgranulins) which are a family of closely related calcium binding polypeptides that accumulate at sites of chronic inflammation, the DNA binding protein HMGB1 (amphoterin) which is released by cells and tissues undergoing necrosis (Bierhaus et al 2005). Besides binding these ligands which participate in chronic inflammation and immune responses AGER can also interact with the surface molecules of bacteria (Chapman et al 2002), prions in plaques found in humans infected with Creutzfeldt-Jakob disease (CJD) (Sasaki et al 2002) and leukocytes. With this in mind AGER should be viewed as more than simply a receptor for AGE's but as a pattern recognition receptor (PRR) (Bierhaus et al 2005).

1.9 AGER binding AGE and NF-κB upregulation

Binding of AGE to AGER results in intracellular signaling via the cytoplasmic tail which leads to the activation of proinflammatory transcription factor NF-κB which is activated rapidly as a cellular defense. In resting cells NF-κB is found in the cytoplasam bound in an inactive form to its inhibitor molecule IκBα where upon activation of the cell IκBα is phosphorylated and degraded enabling the release of NF-κB preferentially in the heterodimer p50/p65 form for translocation in the nucleus (Bierhaus et al 2005). Subsequent to nuclear translocation NF-κB binds to DNA sequences and activates the transcription of NF-κB regulated genes such as adhesion molecules, AGER, cytokines, prothrombotic and vasoconstrictive genes. A number of anti-apoptotic genes, including Bcl-XL, Bcl-2 are under the control of NF-κB, which when activated provides a rapid cellular response which in turn promotes cellular survival (Bierhaus et al 2005). AGER mediated NF-κB activation happens over a prolonged length of time which eventually overwhelms the autoregulatory feedback inhibition loops of NF-κB activation. NF-κB's activation is initiated by the degradation of NF-κB inhibitor molecules IκBα and IκBβ followed by the synthesis of NF-κBp65 in the presence of newly synthesized IκBβ with the de novo synthesis of p65 mRNA leading to an expanding pool of excess transcriptionally active NF-κBp65 with an insufficient amount of IκBα to retain NF-κBp65 in the cytoplasm (Bierhaus et al 2005). Newly synthesized IκBβ has been shown to be hyperphsophorylated which is able to dissociate newly formed NF-κB from IκBα (Johnson et al 1996) thus newly synthesized IκBβ is capable of further promoting AGER dependant sustained NF-κB activation (Bierhaus 2001) as AGER expression is induced by NF-κB sustained activation of NF-κB causes an up regulation of AGER which in turn cause further amplification of the signal (Bierhaus et al 2005).

Due to its ability to sustain cellular activation AGER has the potential to be capable of converting a proinflammatory response into sustained cellular dysfunction with the majority of cellular stress coming from the creation of reactive oxygen species and the activation of NF-κB (Yan 1997); this is further compounded by the inflammatory cells directly releasing strong AGE's such as carboxymethyl lysine (CML) which is an AGER ligand found at sites of inflammation (Bierhaus et al 2005).

Figure 1.4 Following release from the inhibitor, the freed NF-κB (p50/p65) translocates itself from the cytoplasm into the nucleus, where it binds to target genes for up-regulation of NF-κB gene expression.

NF-κB is a component that is involved in both the innate and adaptive immune response and the ability of AGER to activate and sustain NF-κB production implicates AGER as being a potential target for the modulation of the immune response in chronic inflammatory disorders as the blocking of AGER by the administration of sAGER may have the affect of down regulating NF-κB production and thus inflammation. The blockade of AGER by the administration of sAGER, anti-AGER antibodies or using models that do not express AGER in their CD4+ T cells have been shown to suppress experimental autoimmune encephalomyelitis in murine models (Yan 2003) along with a significant reduction in the inflammatory response in wild type mice treated with sAGER in a delayed hypersensitivity model (Bierhaus et al 2005). AGER does not play an active role in the adaptive immune response but the deletion of AGER in a murine model did provide protection from the affects of septic shock caused by cecal ligation and puncture which is a model largely based on the innate immune response (Liliensiek et al 2004) and also when multi bacterial peritonitis was induced in AGER-/- mice and in AGER positive control mice the survival rate being up to 80% in the AGER-/- mice but only up to 20% in the AGER positive control group (Liliensiek et al 2004). The treatment of the AGER positive control group with sAGER improved the groups survival, although the protective affect was not as effective as the protection provided the AGER deletion group (Liliensiek et al 2004). With other experiments with wild type and transgenic mice with an over expression of AGER in endotheilial cells demonstrated a strong up regulation of NF-κB with a high associated mortality with AGER-/- mice displaying reduced NF-κB activation and increased survival (Liliensiek 2004).

The blockade of AGER in diabetes and other inflammatory diseases homozygous AGER deficient mice (AGER-/-) and mice with tissue specific AGER expression have been made all of which were viable and displaying normal attributes with no spontaneous disease development noted by 6 months of age. Induction of diabetes in these mice confirmed that AGER contributes to the development of diabetic complications with diabetic neuropathy including renal enlargement, glomerular hypertrophy and albuminuria was significantly increased in the diabetic mice over expressing AGER in their vasculature but reduced in the AGER -/- diabetic mice (Bierhaus et al 2005). Mice that were over expressing AGER had an increase in organ function deficits whilst the AGER -/- mice appeared to be protected from diabetic induced loss of renal function with less intimal expansion seen in the AGER -/- mice than what was seen in the normal control mice when arterial injury was induced (Sakaguchi et al 2003) but interestingly in these models the most protection from diabetic neuropathy and arterial restenosis was seen in the wildtype mice that were treated with sAGER rather than in the AGER-/- mice. In the wildtype mice with induced diabetic neuropathy administration of sAGER restored pain perception where as the AGER-/- mice regained some of their pain perception indicating that sAGER binds to ligands on cellular structures that are different to AGER which also happen to be involved in pain perception (Bierhaus et al 2005).

2.0 AGER splice variants

The AGER gene is located on chromosome 6 in the MHC class III region and consists of a 5' flanking region that regulates the transcription of the AGER gene, 11 exons and a 3' untranslated region with the resulting mRNA is translated into a protein of 404 amino acids. AGER is composed of a number of protein domains, an extracellular region and signal peptide which is followed by three immunoglobulin like domains (Hudson et al 2008). The domains include a Ig like V domain which itself contains a ligand binding site, and two Ig like C2 domains and a single transmembrane domain with a short cytoplasmic tail. Like other members of the immunoglobulin family of receptors splice variants exist for AGER which results in changes to the amino acid sequence which in turn affects the ligand binding domains of AGER with the removal of the transmembrane region that leads to the production of secreted nonmembrane bound forms of the receptor (Hudson et al 2008).

There are 17 AGER splice variants currently known with the presence and quantity of the splice variant varying from tissue to tissue. It is believed that the AGER v1 is the primary secreted splice variant of AGER and that it is the primary splice mechanisim for the production of sAGER (B. Hudson etal 2008).

Aims

The aim of this experiment is to ascertain whether AGER gene expression is affected by the administration of CLA to human monocytic cells and using CLA isomers 9:11 and 10:12 at varying concentrations to observe their affect, if any upon AGER gene expression. It is expected that CLA will up regulate AGER expression due to CLA's structural similarities to peroxisome proliferators with results from murine studies showing CLA to have strong similarities to a PPAR activators; with CLA also known to be a ligand and activator of PPARα; however we wish to identify if CLA causes an up regulation of sAGER as opposed to AGER's membrane bound form.

2.Methods

2.0 Methodology

This study was performed, after full ethical approval was granted by the School of Health Sciences Ethical Committee at University of Wales Institute, Cardiff, UK, using the human monoclonal monocytic cell line Monomac 6 treated with either 20µl/ml or 100µl/ml of CLA isomers 9:11 or 10:12.

2.1.1 Cell culture of human monocytic cells

Cultured human monoclonal monocytic cells (monomac 6) were obtained from Lowri Mainwairing, PhD student of University Institue of Wales Cardiff. For this study Monomac 6 (MM6) cells were treated by one of two different isomers of CLA and cDNA was obtained from the cells with which we were able to monitor the affect of CLA if any; upon the gene expression of the AGER. Two CLA isomers were chosen as both isomers (9:11, 10:12) have demonstrable anti inflammatory and anti carcinogenic effects in animal models with some limited data available of human trials (N S. Kelley et al 2007). MM6 cells were treated with CLA isomers 9:11 and 10:12 at two different concentrations (20µg/ml and 100µg/ml) and over four different time frames (4 hours, 24 hours, 48 hours and 72 hours) and cDNA obtained from the MM6 cells.

Primer selection

All primers for the real time-PCR were designed using the Applied Biosystems Primer Express ® software V 2.0 (Warrington, UK). The following guidelines were considered and adhered to during the design of primer sequences. Both primer sequences should have similar if not identical percentage of guanine-cytosine (GC) content and should have comparable melting temperatures (Tm), within the range of 55 - 60°C. In order to minimise the risk of primer dimer formation, sequences of identical nucleotides. Finally all potential primer sequences were screened using a Basic Local Alignment Search Tool (BLAST) search on Pub Med to ensure that they were specific only to the region of the AGER gene. The primer sequences were obtained from Sigma (Poole, Dorset, UK).

TaqMan primers were used with the following sequences:

AGER

5'-ATGGAAACTGAACACAGGCCG-3'; Forward

5'-AAATCCCCTCATCCTGGATCC - 3' Reverse

GAPDH

5'-CATTGACCTCAACTACATG-3'; Forward

5'-TCTCCATGGTGGTGAAGAC-3' Reverse

2.1.4 Primer and cDNA storage

Forward and reverse primers were stored at -20­oC at a concentration of 10µl, cDNA was stored at -20­oC at a concentration of 15ng/µl. Each primer was diluted 1:2 in distilled H2O and each cDNA sample was diluted 1:3 with distilled H2O. A master mix series was made and used to determine primer optimization using a series of varying dilutions.

Tube No

Plate No

Mastermix (μl)

5 μM Forward

Primer (μl)

5 μM Reverse Primer (μl)

Template (μl) (cDNA 5ng/μl)

Distilled H2O

Total

1

1

15

0.6

0.6

3

10.8

30

2

2

15

0.6

1.5

3

9.9

30

3

3

15

0.6

3

3

8.4

30

4

4

15

0.6

4.5

3

6.9

30

5

5

15

0.6

6

3

5.4

30

6

6

15

1.5

0.6

3

9.9

30

7

7

15

1.5

1.5

3

9

30

8

8

15

1.5

3

3

7.5

30

9

9

15

1.5

4.5

3

6

30

10

10

15

1.5

6

3

4.5

30

11

11

15

3

0.6

3

8.4

30

12

12

15

3

1.5

3

7.5

30

13

13

15

3

3

3

6

30

14

14

15

3

4.5

3

4.5

30

15

15

15

3

6

3

3

30

16

16

15

4.5

0.6

3

6.9

30

17

17

15

4.5

1.5

3

6

30

18

18

15

4.5

3

3

4.5

30

19

19

15

4.5

4.5

3

3

30

20

20

15

4.5

6

3

1.5

30

21

21

15

6

0.6

3

5.4

30

22

22

15

6

1.5

3

4.5

30

23

23

15

6

3

3

3

30

24

24

15

6

4.5

3

1.5

30

25

25

15

6

6

3

0

30

Figure 2.0 Master mix table for primer optimization: Aliquots of these dilutions were pipette into an RT-PCR plate, covered to ensure the mix did not evaporate and centrifuged prior to being run on an Applied BioSystems 7500 Fast Real Time PCR system with the following thermal cycle:

Figure 2.1 PCR cycle and temperature ramping used for AGER gene

DNA Amplification

Amplification and dissociation curves obtained in the real time-PCR analysis.

Amplification curves are the plot of the fractional cycle number at which the fluorescence passes the fixed threshold (CT) versus the cycle number (Rn) were linear with no evidence of non-specific amplification. Figures 3.0 and 3.1 are examples of dissociation and amplification curves obtained from the study, with linear amplification and single dissociation peak reassuring the quality of the real time-PCR products obtained.

2.3 Statistical Analysis

All statistical analysis was performed on Minitab® version 17. Only multiple comparisons were undertaken using one-way ANOVA and results were deemed significant when p<0.05

3. Results

Results

Figure 3.0: This is the plot of fluorescence signal (y-axis) versus the number of cycles (x-axis). It shows a typical linear amplification for AGER mRNA with no evidence of non-specific product amplification. The appearance of this amplification plot is typical for that seen for AGER and GAPDH genes studied.

Figure 3.1: This curve is an example of the dissociation obtained showing the rate of change of the relative fluorescence units (derivative, y-axis) and temperature (x-axis). A single peak (melting temperature) suggests the absence of any substantial primer-dimer products and further indicating that the optimum primer concentrations were used in this RT-PCR analysis.

Figure 3.2: The relative gene expression of human monocytic cells that have been treated with CLA isomer 9:11 and at 20µg/ml and 100µg/ml concentration. The control used during this experiment was the house keeping gene GAPDH which showed no increase in expression in the monocytic cells used when treated with CLA isomer 9:11. The expression of AGER mRNA was significantly increased in 10:12 treated cells at 24 and 48 hours and with 9:11 at 72 hours as compared to control cells (p<0.05 ANOVA)

As can be seen in figure 3.2, the affect of CLA isomer 9:11 upon AGER gene expression in human monocytic cells is variable depending on the concentration used and the duration post dosage.

Figure 3.3: Shows the relative gene expression of human monocytic cells that have been treated with CLA isomer 9:11 and 10:12 at 20µg/ml concentration. The control used during this experiment was the house keeping gene GAPDH which showed no increase in expression in the monocytic cells used when treated with CLA isomer 9:11 and 10:12.

As can be seen in figure 3.3, the affect of CLA isomer 9:11 and 10:12 upon AGER gene expression in human monocytic cells peaks at 24 hours post exposure with relative AGER gene expression diminishing as time progresses.

Figure 3.4 :The relative gene expression of human monocytic cells that have been treated with CLA isomer 9:11 and 10:12 at 100µg/ml concentration. The control used during this experiment was the house keeping gene GAPDH which showed no increase in expression in the monocytic cells used when treated with CLA isomer 9:11 and 10:12 at 100µg/ml concentration.

As can be seen in figure 3.4, the effect of CLA isomer 9:11 upon AGER gene expression in human monocytic cells is variable; however unlike in Figure 3.1 that uses CLA 9:11 and 10:12 at 20µg/ml concentration AGER gene expression peaking at 24 hours there is a clear increase in AGER gene expression after 24 hours and continuing to increase up until 72 hours post exposure in Figure 3.2.

Figure 3.5: The relative gene expression of human monocytic cells that have been treated with CLA isomer 10:12 at 20µg/ml and 100µg/ml concentration. The control used during this experiment was the house keeping gene GAPDH which showed no increase in expression in the monocytic cells used when treated with CLA isomer 10:12 at 20µg/ml and 100µg/ml concentration. The expression of AGER mRNA was significantly increased in 10:12 treated cells at 24 and 48 hours and with 9:11 at 72 hours as compared to control cells (p<0.05 ANOVA)

As can be seen in figure 3.3, the affect of CLA isomer 10:12 upon AGER gene expression in human monocytic cells is variable; however monocytic cells treated with the 20µg/ml concentration of CLA 10:12 showed elevation in AGER gene expression 24 hours after treatment whereas CLA 10:12 at 100 ug/ml concentration caused a decrease in AGER gene expression for 48 hours post treatment with AGER gene expression increasing at 72 hours whilst the 20 µg/ml concentration AGER gene expression peaked at 24 hours and decreased afterwards.

Figure 3.4: Typical PCR gel of AGER gene products as compared to DNA ladder. This gel confirms the presence of various splice variants of AGER in CLA treated MM6 cells.

4. Discussion

Discussion

From the results obtained it is clear that AGER gene expression is increased by the addition of both CLA isomers 9:11 and 10:12 with both isomers causing a peak of AGER expression in human monocytic cells approximately 24 hours post exposure with the control gene GAPDH showing very little to no up regulation by the addition of CLA.

CLA isomer 9:11 had an affect on the up regulation of AGER, increasing the mean expression by 14% 24 hours after exposure (20µg/ml concentration) and surprisingly by only 5% in the 9:11 100µg/ml concentration) (Figure 3.2). This is demonstrated again with CLA isomer 10:12 where the 20µg/ml concentration increases the mean expression of AGER by 60% 24 hours post exposure whilst 10:12 at 100µg/ml concentration increases the mean AGER gene expression by less than 10% at its peak 72 hours post exposure (Figure 3.5).

The combination of CLA isomers 9:11 and 10:12 at 20µg/ml caused AGER gene expression to be upregulated almost 40% at its peak expression 24 hours after exposure (Figure 3.3), however this is a lower increase of AGER expression when compared to CLA isomer 10:12 at 20µg/ml concentration which achieved 60% up regulation of AGER gene expression (Figure 3.5) suggesting that the addition of isomer 9:11 retards the action of isomer 10:12 by causing a decrease of approximately 20% in AGER up regulation compared to the increase in AGER gene expression achieved by CLA isomer 10:12 at 20µg/ml concentration having been administered on its own (Figure 3.5).

The higher concentration of 100µg/ml of CLA isomers 9:11 and 10:12 appear to have a comparable affect to the expression of AGER in that both isomers upregulated AGER expression 50% (Figure 3.4) which is comparable to the increase in expression seen in Figure 3.5 where isomer 10:12 at 20µg/ml upregulated expression 60% and in Figure 3.2 where 9:11 at 20µg/ml achieved a 14% increase in AGER gene expression. What is clear from Figures 3.2, 3.4 and 3.5 is that the affect of isomers at 100µg/ml concentration upon human monocytic cells show an increase in AGER gene expression at 48 hours, continuing to increase at 72 hours but due to the timescales used whether AGER expression continues to be upregulated or the affects of the CLA decreases after this time is a limitation of this study.

The increase in AGER gene expression achieved by administering 20µg/ml of CLA isomers 9:11 and 10:12 in comparison to the administration of 100µg/ml of those isomers clearly shows (Figure 3.2, 3.5) that the administration of 20µg/ml caused a larger increase in AGER gene expression than that caused by administration of 100µg/ml of the same CLA isomer. This could be explained by optimal dosage, in that 20µg/ml is closer to the optimal dosage required for the upregulation of AGER gene expression whilst 100µg/ml is further from the optimal dosage required and thus has a smaller impact upon the upregulation of AGER gene expression than that of the 20µg/ml concentration used to treat the cultured human monocytic cells.

There is no information currently available about the optimal dosage of CLA in regards to its affects upon AGER gene expression however there is data available in regards to the affect of CLA upon weight loss in human trials (L D Whigham et al 2007) where an equal mixture of CLA isomers 9:11 and 10:12 at a dose of 3.4g per day resulted in an average weight loss of 0.14kg per week and the larger dose of 6.8g per day resulted in an average weight loss of 0.11kg per week (L D Whigham et al 2007).

This data indicates that the affects of CLA upon human weight loss is subject to optimal dosage of CLA in the diet, which also supports the data gathered from this study in that lower doses (20µg/ml) administrated of CLA have a larger affect upon AGER gene expression whilst larger administered doses (100µg/ml) of CLA have sufficiently less impact upon AGER gene expression than would be expected; leading to the conclusion that as with CLA's ability to cause weight loss and its affect upon AGER gene expression relies upon an optimal dosage of CLA.

Optimal dosage of dietary supplemented CLA is highly variable within human trials, compounded by the short duration of most trial data (<12 weeks) (Whigham et al 2007); thus it can be expected that the data collected in this study of CLA's affect upon AGER gene expression in vitro using a monoclonal cell line (MM6 cells) would vary considerably if this study used monocytic cells derived from a variety of test subjects due to natural variation in the genetics of each cell; which would be expected to provide better data in regards to the affect of CLA if given to the general population than that provided by a monoclonal cell line.

Conclusion