Pathogenecity Of Diabetes Mellitus And Anti Diabetic Drugs Biology Essay

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Diabetes mellitus describes a metabolic disorder of multiple causes it is characterised by chronic hyperglycaemia with disturbance of carbohydrates, fat and protein metabolism due to lack or reduced insulin secretion. Diabetes can cause long term damage dysfunction and failure of various organs. People with diabetes are at high risk of cardiovascular, peripheral vascular and cerebrovascular disease, several pathogenitic processes are involved in the development of diabetes which includes process which destroys the beta cells of the pancreas with consequent insulin deficiency. The abnormalities in the carbohydrates, fat and protein metabolism are due to deficient action of insulin on target tissues. Diabetes mellitus is very common in the United Kingdom there are 1.4 million people known to have diabetes of whom 20 000 are aged under 20.The British Diabetic Association believes that there may be as many as another 1.4 million people who remain undiagnosed, worldwide there are over 123 million cases. (Day,2002)

There are four main types of anti- diabetic drugs: Sulphonylureas (SU), Biguanide commercially known as metformin, Glitazones and Drugs which delay the absorption of carbohydrate such as acarbose. (Bonnie and Silvio,2005)

Drugs (e.g glyburide,glipzide and glimepiride) improve glucose levels by stimulating insulin secretion by the pancreatic β-cell, patients on SU monotherapy experience a progressive loss of glucose control the same phenomenon is noted in patients taking metformin, a drug that does not increase insulin secretion. Side effects of SUs include weight gain and hypoglycaemia, weight gain is of particular concern given that patients are often obese before therapy initiation. Hypoglycaemia risk becomes a more important issue as patient's overall glucose control approaches the normal range during the past several years the cardiology community has become disquieted because of the potential effects of SUs on myocardial ischemic preconditioning. (Bonnie and Silvio, 2005).


Metaformin a beguanide acts mainly by decreasing hepatic glucose production, primarily gluconeogensis probably through effects on AMP-kinase. Circulating glucose levels are thereby reduced. Metformin is commonly referred to as an "insulin sanitizer" because glucose levels improve without stimulation of insulin secretion. Numerous studies during the past several years have continued to demonstrate a benefit of metformin on these cardiovascular risk markers.In fact metformin is the only antihyperglycemic agent shown to reduce macrovascular events in patients with type 2 diabetes. Metformin is approved for use alone or in combination with all other antidiabetic agents, it is also gaining in popularity as a treatment option for women with polycystic ovary syndrome and has been demonstrated by multiple investigators to improve ovulatory capacity and metabolic parameters in this group of insulin-resistant women. Gastrointestinal side effects of metformin are common but can be minimised by slow dosage titration. Because of the rare risk of lactic acidosis several contraindications limit this drug's use including renal and liver dysfunction, heart failure, dehydration and alcohol abuse. (Bonnie and Silvio, 2005).

Thiazolidinedione TZDs:

The Thiazolidinedione class of drugs or glitazones as they are called (troglitazone, pioglitazone, ciglitazone, englitazone and rosiglitazone) are specific high-affinity lignds for PPARγ. (Mohan et al.,2000). However, TZDs showed to have some side effects including weight gain and edema which precluded their widespread use for patients with heart failure. Recently most concern has arisen regarding the potential effect of TZDs in heart failure patients; it has been advised that the TZDs not to be used for patients with advanced heart failure symptoms. (Gaal and Scheen, 2002)

1.3 Introduction to Peroxisome proliferator receptor:

Peroxisome proliferator receptors (PPARs) are members of the nuclear hormone receptor superfamily of transcription factors and play important role in the regulation of cellular differentiation, development and metabolism. PPAR exist in three isometric forms including PPARα (PPAR alpha) is expressed primarily in brown adipose tissue, liver, kidney, heart, skeletal muscle and plays a major role in lipid catabolisim. PPARσ/β (PPAR beta/delta) is expressed in many tissues but markedly in brain, adipose tissue and skin. (Kiaei, 2002)

PPARγ (PPAR gamma) exists in two isoforms including PPARγ1 and PPARγ2, PPARγ1 is expressed in all tissues including heart, muscle, colon, kidney, pancreas and spleen. Whereas PPARγ2 is mainly expressed in adipose tissue.Some of the known target genes of PPARγ include acyl-CoA oxidase , the adipocyte-specific fatty acid transport protein (aP2), phosphoenolpyruvate carboxiykinase, malic enzyme, leptin, resistin, lipoprotein lipase and adiponectin.(Heikkinen et al,2007).

PPARγ is known to interact with retinoid X receptor (RXR) bind DNA as a heterodimer and subsequently regulate transcription of PPAR-responsive genes. The PPARγ/RXR heterodimer binds to a direct repeat of a consensus sequence (AGGTCA) by a single nucleotide (DR1).PPARγ has been shown to occupy the 5' half-site of the DR1 element with RXR occupying the 3' half-site. This configuration is in contrast to other nuclear hormone receptors that heterodimerise with RXR (e.g vitamine D receptor, thyroid hormone receptor, and retinoic acid receptor) in which RXR prefers to bind the 5' half-site of the DR element.PPARs were originally identified in xenopus as receptors that induce the proliferation of peroxisomes in cells.(Kintscher and Law,2005)



Figure 1: PPARα and PPARγ pathway. Activated PPARs heterodimerise with retinoid X receptor (RXR), and bind to specific regions on the DNA of target genes. These DNA sequences are termed PPREs (peroxisome proliferator response elements). When the PPAR binds to its ligand, transcription of targets genes are increased or decreased, depending on the gene. The RXR also forms a heterodimer with a number of other receptors: the vitamin D receptor and the thyroid hormone receptor. The ligands for the PPARs are free fatty acids, PPARγ is activated by prostaglandin. Adopted from

1.4 TZDs as ligands for PPARγ:

Identification of TZDs as ligands for PPARγ came from Hrris and Kletzien who showed that pioglitazone increased transcriptional activity from the aP2 enhancer and through the differentiation-linked DNA site. (Spiegelman,1998). PPARγ enhances the expression of a number of genes encoding proteins involved in glucose and lipid metabolism. Adipocytes differentiation responds well to pharmacological PPARγ ligands. Functional PPREs have been identified in several adipocyt- specific genes (phosphoenol pyrvate carboxykinase, ap2, acyl CoA synthase, and fatty acid transport protirn-1 and lipoprotein lipase) and all of them are regulated by PPARγ. This proofs that PPARγ and its target genes have interdependent role in adipocyte differentiation. (Mohan et al., 2000). In some study it was observed that the TZD derivatives enhance the expression of adiponectin mRNA in adipose cells and dramatically increase the plasma adiponectin concentration. TZDs are specific ligands for PPAR{gamma} which induce adipocyte differentiation by activating the expression of adipocyte-specific genes and which is also known as an insulin sensitizer in vivo, presumably by increasing the number of mature adipocytes that can respond to an enhancing effect of insulin on glucose disposal. TZDs might activate the adiponectin promoter by a pathway other than its direct action on PPAR{gamma}. However, it was observed that cotransfection of a dominant-negative form of PPAR{gamma} diminished the action of TZDs on adiponectin promoter. TZDs therefore may enhance adiponectin promoter activity through an unidentified element responsive to PPAR{gamma}. (Maeda et al.,2001). In addition to their role in adipocyte differentiation TZDs also affect lipid metabolism, they increase the lypolysis of triglycerides in very- low-density lipoproteins (VLDL) and thereby reduce triglycerides and increase HDL- cholesterol. Moreover, increased glucose uptake and mRNA expression of the glucose transporter isoforms (GLUT1 and GLUT4) were induced by glitazones through PPARγ cativation. ( Maeda et al.,2001)

1.5 The role of PPARγ agonists in atherosclerosis:

The discovery that PPARγ was expressed at relatively high levels in monocytes and macrophages led to studies showing that PPARγ agonists could promote macrophage differentiation and directly induce the scavenger receptor CD36. These discoveries, coupled with the identification of PPARγ in "foam cell" macrophages within human atherosclerotic lesions, led to fears that TZDs could be promoting atherosclerosis in humans taking these drugs. Endogenous ligands of PPARγ were identified in atherogenic oxidized low density lipoprotein particles in serum and it was shown that these particles could induce expression of PPARγ itself. A pathological cycle was proposed in which these particles induced their own uptake through activation of PPARγ and expression of CD36, leading to foam cell formation. Other evidence, conversely, suggested that PPARγ might be beneficial in atherosclerosis. TZDs, for example, have been shown to reduce blood pressure in several mammalian models. Other atherogenic pathways are also inhibited by TZDs, including proliferation and migration of vascular smooth muscle cells and suppression of proinflammatory signals within macrophages in the vessel wall, such as IL-6, IL-1β, TNF-α, gelatinase, and scavenger receptor A. PPARγ also induces the expression of proteins involved in reverse cholesterol transport, most likely leading to a net reduction of cholesterol in atherosclerotic lesions. Recent genetic studies show that PPARγ is not required for the formation of macrophages from monocytes, although macrophages lacking PPARγ have greatly reduced basal expression of CD36. (Rosen et al., 2001).

Figure 2: PPARγ and Atherosclerosis.Representation of cholesterol trafficking in macrophages. Uptake of oxidized LDL (ox-LDL) by the scavenger receptor CD36, and its intracellular catabolism, leads to the generation of endogenous PPAR ligands (e.g. 9-HODE and 13-HODE), which further activate the receptor, increasing ox-LDL uptake in an 'undesirable' feedforward cycle. This effect is counterbalanced by inhibition of expression of SR-A, a second scavenger receptor, and enhanced cholesterol efflux from cells, via upregulation of the ABCA1 cholesterol transporter. LXR, liver-X-receptor ; SR-A, scavenger receptor-A; ABCA1, ATP-binding cassette transporter A1; HDL, high-density lipoprotein; LDL, low-density lipoprotein; HODE, hydroxyoctadecadienoic acid. Adopted from

1.6 Effect of PPAR-γ on fat cell differentiation:

Some recent studies demonstrated that PPARγ is essential for the development and normal function of white adipose tissue; they have elegantly showed that PPARγ is required to keep up intact insulin sensitivity during caloric intake even gaining importance during high fat diet. By maintaining the intact function of white adipose tissue, PPARγ protects the liver against liver lipid overload therefore ensuring intact hepatic insulin sensitivity. Adipose PPARγ guarantees a balanced and adequate regulation of secretion from adipose tissue of adipocytokines such as adiponectin and leptin which are major contributors to a regular insulin response. Although PPARγ expressed in liver and skeletal muscle contributes importantly to glucose and lipid metabolism, the early studies using tissue specific PPARγ knockout mouse models pinpoint adipose tissue as the major target of TZD mediated improvement of hyperlipidemia and insulinsensitisation (Kintscher and Law ,2005).

It has been recently shown that activated PPARγ not only stimulates the differentiation to adipocytes of resident adipose tissue preadipocytes but also promotes the mobilisation of bone marrow-derived circulating progenitor cells to white adipose tissue and their subsequent differentiation in to adipocytes. (Anghel and Wahli,2007). In addition to being involved in the differentiation of adipocytes PPARγ participates in the function of the mature cells. Indeed PPARγ is the major regulator of lipid storage in white adipose tissue, it promotes the release of free fatty acids from circulating lipoproteins by regulating lipoprotein lipase expression and stimulates their uptake by enhancing the expression of the fatty acid translocase CD36 and fatty acid transport protein FATP1. PPARγ also regulates the intracellular retention/transport of free fatty acids by controlling the expression of fatty acid binding proteins, it also promotes the esterficationof free fatty acids in to triglycerides and their storage by regulating the expression of enzymes such as phosphoenol pyruvate carboxykinase, glycerol phosphate dehydrogenase, and diacylglycerol O acyltransferase.PPARγ also participates in the de novo free fatty acids synthesis by regulating directly or indirectly the expression of enzymes such as fatty acid synthase, acetyl CoA synthetase and stearoyl CoA desaturase. (Anghel and Wahli,2007)

Figure3. Physiological effects of PPARγ-activation. PPARγ induces adipocytes differentiation. Plasma derived fatty acids are directed to adipose tissue at the expense of skeletal muscle, which increases glucose uptake and utilization in the muscle. Direct effects of PPARγ activation have also been observed in liver, including decreased glucogensis and storage. PPARγ activation increases cholesterol efflux in macrophages, and increases uptake of proatherogenic oxidised particles via upregulation of CD36. Adopted from

1.7 Role of PPARγ agonists in inflammation:

PPAR γ agonists act by binding to PPAR γ, recent studies shown that TZD causes anti-inflammatory or anti- atherogenic effect. Anti-inflammatory effects of PPAR γ, agonists may be receptor dependent and receptor independent. Receptor dependent actions include inhibition of inflammatory gene expression via inhibition of the NFkB p65 subunit by phosphorylated PPAR γ. Receptor independent actions depend on direct action of TZD on reducing mitochondarial function by binding to Mitoneet (MN), a component of the PDH complex, or a component of complex I. Mitochondrial dysfunction can result in reduced ATP levels, increased ROS levels, and increased RNS, which can activate stress response, AMPK, and glycolysis. (Feinstein et al.,2005)

TZD interfere with the degradation of IkB-B allowing NF-kB to remain in the cytoplasm thus smoothing the transcription of pro-inflammatory cytokines. A study reported a reduction of RAGE expression in human endothelial cells by TZDs, with subsequently reduced endothelial susceptibility toward pro-inflammatory AGE effects. It's known that TNF-{alpha} activates both NF-{kappa}B sites in the RAGE promoter, with only the proximal site being of importance to induce endothelial RAGE expression. This Study demonstrates that TZDs' reduction of TNF-{alpha}-induced RAGE expression is mediated by an inhibition of NF-{kappa}B activation at this respective site. In addition, to that TZDs interaction with this NF-{kappa}B site also explain the reduction in baseline RAGE expression, because baseline promoter activity is also regulated via this NF-{kappa}B site. PPARs have been shown to alter cofactor recruitment as well as cofactor binding to transcription factors like NF-{kappa}B, and such mechanisms may explain the inhibitory effect of TZDs on the proximal NF-{kappa}B site. The reduction of endothelial RAGE expression by TZDs decreases the cells' susceptibility toward proinflammatory AGE effects. (Marx et al., 2004). Another study demonstrated enhanced ubiquitin-proteasome activity in diabetic atherosclerotic lesions, and it provides evidence that the activation of this system by inflammatory cells is associated with an NF-{kappa}B-dependent increase in inflammation, potentially promoting plaque rupture, and also suggest that thiazolodinediones may reduce NF-{kappa}B activation by modulation of ubiquitin-proteasome activity in human atherosclerotic lesions of diabetic patients. (Marfella et al., 2006)

In addition to the cellular anti-inflammatory effects, rosiglitazone caused a fall in plasma concentrations of CRP and MCP-1 in both the non-diabetic obese and the obese diabetic subjects, paralleling the fall in NF{kappa}B. Whereas CRP represents an acute phase response to inflammation; MCP-1 is a specific proinflammatory chemokine, generated by endothelial cells and other inflammatory cells, inducing monocytes, for leukocytes to be chemo-attracted to the inflammatory site. MCP-1 is a key proatherogenic mediator. (Mohanty et al., 2004)

Recent study was done using another type of TZD called troglitazone showing its effect on cellular mediators of inflammation the plasma markers of inflammation. CRP, sICAM-1, MCP-1, and PAI-1 decreased significantly during short treatment period of troglitazone. (Ghanim et al., 2001). These markers are involved in the pathogenesis of atherosclerosis and inflammation. ICAM-1 is an adhesion molecule, which promotes the attachment of leukocytes to the endothelium, whereas MCP-1 is a chemokine attracting monocytes to the site of inflammation. The magnitude of inhibition of intranuclear NF{kappa}B and total cellular NF{kappa}B by troglitazone and the inhibition of ROS generation by MNC suggest that troglitazone may have a potent anti-inflammatory effect. The activation of NF{kappa}B is known to be associated with an increase in TNF{alpha} expression and secretion. It is probable that the TZD moiety of troglitazone is responsible for this effect, because the circulating monocyte has been shown to have PPAR{gamma}. Troglitazone administration to the obese leads to a rapid reduction in intranuclear and total cellular NF{kappa}B, ROS generation by MNC, and p47phox subunit, in association with an increase in I{kappa}B. In addition, troglitazone causes a fall in plasma concentrations of TNF{alpha}, sICAM-1, MCP-1, CRP, and PAI-1. (Ghanim et al., 2001).

1.8 Role of PPAR-γ in endothelial cells:

PPARγ expression in human endothelial cells EC has been demonstrated by reverse transcription polymerase chain reaction (Itoh,1999) and more definitely using western blotting and immunohistochemistry(Marx,1999).Subsequent data suggested that PPARγ activation can influence target genes and processes that are of central relevance to endothelial biology.

The endothelium is now recognised as a biologically active dynamic organ involved in both physiologic and pathologic processes e.g by virtue of their location endothelial cells facilitate metabolic exchange between the circulation and tissues. In obesity and it is associated conditions of insulin resistance and diabetes, endothelial dysfunction is a common feature that may even precede the onset of frank diabetes. Endothelial dysfunction has been previously understood primarily in the context of abnormal vasomotor function. Specific mechanisms, through which the endothelium itself may directly modulate obesity, lipid metabolisim, or insulin sensitivity, have remained largely obscure. The PPARγ is also expressed in endothelial cells in which it regulates target relevant to inflammation and atherosclerosis (Kanda et al., 2009). Endothelial cells contain slightly less but comparable amounts of PPARγ protein relative to preadipocytes and monocytes-derived macrophages (Marx et al.,1999).

Some studies was carried out to obtain a better understanding about the role of PPARγ in endothelial cells it was hypothesised that endothelial PPARγ might be involved in directing metabolic phenotype to prove this animal sample 'mice' was used they were deficient in endothelial PPARγ using floxed PPARγ mice. The mice were investigated under conditions of standard chow and high fat diet both with and without treatment with the PPARγ agonist rosiglitazone as well as before and after bone marrow transplantation to reconstitute hemopoietic PPARγ expression and isolate endothelial PPARγ dependent responses. These studies revealed that mice specifically deficient in endothelial PPARγ apparent a distinct pattern of decreased adiposity, increased insulin action, worsen dyslipidemia, and impaired arterial vasodilation in response to high fat diet challenges as compared with control mice. Inaddition these mice fail to exhibit known metabolic improvements in response to a PPARγagonist. These data found that PPARγ in the endothelium as a previously unrecognised determinant of metabolic status and a potential contributer to metabolic abnormalities found in insulin resistance and diabetes. (Kanda, 2009).

Another study suggested that the PPARγ in endothelial cells not only is an important regulator of hypertension but also mediates the antihypertensive effects of rosiglitazone the data from the study supports a beneficial role for PPARγ-specific ligands in the treatment of hypertension and suggest therapeutic strategies targeting endothelial cells may prove useful (Nicole et al., 2005).

The endothelium has also been suggested to defend against oxidative stress and to maintain appropriate redox status (Kunsch, 1999). In this sense data implicating PPARs in redox responses are also relevant. It was found that both PPARα and PPARγ activators induced expression of the superoxide scavenger enzyme Cu2+, Zn2+ superoxide dismutase.PPARα and PPARγ activators also decreased induction of p22 and p47 phox, two subunits of the superoxide- generating enzyme nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (Inoue,2001).furthermore, the effect of PPARγ ligands on the vascular endothelium was expanded to include enhanced NO release by PPARγ ligands in PAEC, HAEC, and HUVEC suggests that these effects may be generalised to all macrovascular ECs regardless of species they derived from. (Calnek,2002).

1.9 Role of PPAR-γ in THP cells:

PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. The PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis. (Ricote et al,1998). Recent studies have demonstrated that PPARγ is expressed in monocytes and macrophages and natural and synthetic PPARγ ligands inhibit the production of a number of inflammatory mediators .These observations have raised the possibility that PPARγ may play a physiologic role in modulating the magnitude and duration of inflammatory responses in which macrophages play prominent roles. (Ricote et al,1998). The presence of large numbers of macrophages and a broad spectrum of inflammatory mediators in atherosclerotic lesions raised the question whether PPARγ is expressed in lesion macrophages, whether it is specific to particular stages of lesion development, and whether specific ligands may, in theory, activate this pathway and thus influence the inflammatory process. In the present studies, PPAR-γ was found to be highly expressed in foam cells in atherosclerotic lesions and in thioglycolate-elicited macrophages, whereas very little PPAR-γ was found in bone marrow progenitor cells, resting peritoneal macrophages, or undifferentiated myeloid cell lines. Several factors were found to induce PPAR-γ expression in differentiated macrophages, including M-CSF, GM-CSF, and oxLDL, suggesting that these factors may be of importance in regulating expression of PPARγ in macrophages in vivo. (Ricote et al, 1998).

oxLDL is believed to be generated within the artery wall as the consequence of reaction with pro-oxidant molecules generated by activated macrophages and other vascular cells. Extensive evidence has been provided for the occurrence of lipid peroxidation, and the presence of oxLDL in atherosclerotic lesions of humans and animal models Notably, in early, macrophage-rich lesions, oxidation-specific epitopes were mostly observed within, or in close vicinity to, macrophages. The notable co localization between oxidation-specific epitopes and PPARγ, in particular in the early lesions, suggests that oxLDL, or oxidant stress itself, may be an important regulatory factor of PPARγ expression in lesions. The observation that PPARγ is highly expressed in macrophage foam cells of atherosclerotic lesions underscores the importance of determining the biological role of this transcription factor in the regulation of macrophage gene expression. This issue is especially relevant given the frequency of cardiovascular complications in subjects with type 2 diabetes mellitus, who now have the option to be treated with thiazolidinediones. Although current information would suggest a potential protective role for PPARγ, based on inhibition of inflammatory cytokines, gelatinase B, and the scavenger receptor A gene, the development of atherosclerosis is a complex phenomenon involving many gene products. (Ricote et al,1998).

1.10 Role of housekeeping genes in gene expression studies:

The use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels led to the common use of β- actin housekeeping gene as denominators for comparison of samples. However it is found out that β- actin expression is upregulated with proliferation, activation and differentiation. The use of housekeeping genes in molecular biology assays relies on the postulation that their levels of expression remain the same from cell to cell, sample to sample and treatment to treatment. β-actin a cytokeletal protein and GAPDH an enzyme of glycolisis are the two most widely used housekeeping genes. (Glare et al.,2002) Actin are highly conserved proteins found in all eukaryotic cells and they play a key role in maintaining cytoskeletal structure.β- actin belongs to the actin multigene family it is one isoform of the cytoplasmic actins and a component of cytoskeleton in most eukaryotic cells. It plays an important role in cell motility and structure and in the initiation of transcription. The gene for β- actin has been cloned from diverse species including vertebrates and studies showed that β- actin is expressed in most eukaryotic nonmuscle cells and hence it has been used widely as an internal control gene in the quantification of mRNA levels. (Zhang et al., 2005). The β- actin gene was used as housekeeping gene along with GAPDH for two reasons: initially, the control of RNA quality and cDNA synthesis efficiency without housekeeping gene expression is difficult. Secondly, there has been a substantial lack of validation studies to discredit the methodological concept. ( Glare et al.,2002).


The aim of this project is to determine the PPARγ gene expression in human umblical vein endothelial cells (HUVEC). In addition, to compare the level of PPARγ gene expression in HUVEC and 48 hours dTHP1 cells. The differentiated THP-1 monocytic leukemia cells (dTHP1) were induced by phorbol myristyl acetate (PMA) and its reaction was monitored over a period of 24, 48 and 72 hours. My project aims to observe the PPARγ gene expression in 48 hours dTHP1 and compare it with the PPARγ in HUVEC cells.

Chapter 2

Methods and Materials

3.1 PPARγ expression in human endothelial cells (HUVEC)

and dTHP1.

1 2 3 4 5 6 7



Figure1: Optimisation of RT-PCR reaction. The PPARγ gene expression in HUVEC and dTHP1 cells, PMA was added to the THP1 cells and after 48 hours RT-PCR was performed on both HUVEC and 48 hours dTHP1 cells as they were ran in triplicate. The PCR products were electrophoresed with 0.5X TBE buffer using quantities and conditions described in section 2.7 and 2.8. Lane 1: 100bp DNA ladder (MW). Lanes 2, 3, 4: PPARγ in HUVEC cells at100 bp. Lanes 5, 6, 7: PPARγ in dTHP1 cells at 100 bp.

3.2 β- actin expression in Human endothelial cells (HUVEC) and dTHP1 cells

8 9 10 11 12 13 14

*50 bp

350 bp

100 bp

500 bp

Figure 2: RT-PCR reaction. The β-actin gene expression from HUVEC and 48 hours dTHP1 electrophoresed with 0.5X TBE bbffer using quantities and conditions as described in section 2.7 and 2.8. Lane8: 100bp DNA ladder (MW). Lanes 9, 10, 11: β-actin in HUVEC cells at 350 bp. Lanes 12, 13, 14: β-actin in dTHP1 at 350 bp. β-actin used as a control gene its expression reveals that RT-PCR has run properly and the cells were proprly activated.

* Primer dimer was formed and appeared at 50 bp, because primers are present at high concentrations, weak interactions can occur between them.

3.3 PPARγ Expression in HUVEC and dTHP1:

Figure 3: PPARγ expression in HUVEC and dTHP1 cells. The expression of PPARγ in HUVEC cells is 3% less than it in *dTHP1. However, dTHP1 cells were treated with PMA and after 48 hours the PPARγ expression was investigated. The cells were run in triplicate the mean and the S.D value was calculated. The expression of PPARγ noticed to be higher in dTHP1 cells. However, it was an insignificant as the p-value= 0.432 (p> 0.05).

* Data for differentiated THP-1 was kindly provided by Abbas Fayyad (BSc

BMS 2009).

3.4 β- actin expression in HUVEC and THP1 cells:

Figure 4: β-actin expression in dTHP1 and HUVEC. β-actin expression in dTHP1 is 4% higher than it in HUVEC cells since the dTHP1 cells were induced by PMA. The samples ran in triplicate the mean and the S.D values were calculated. β- actin is a housekeeping gene, in RT-PCR the use of housekeeping gene is essential as denominators for comparison of samples, expression of β-actin gene remains constant.

T-Test report for PPAR-γ expression in HUVEC and dTHP-1 cells in relation to β-actin gene. This test was done by using Minitab 15.

Two- sample T for HUVEC VS dTHP1s

N Mean St Dev SE Mean

HUVEC 3 1.0667 0.0321 0.019

dTHP1 3 1.0867 0.0208 0.012

Difference = mu (HUVEC) - mu (dTHP1)

Estimate for difference: -0.0200

95% CI for difference: (-0.0904, 0.0504)

T-Test of difference= 0 (VS not =): T-Value= -0.90 P-Value= 0.432 DF= 3

3.5 Mathematical results of PPARγ gene in HUVEC and dTHP1 cells in relation to β- actin:



INT /mm2




Mean ratio PPARγ/


S.D Ratio






PPARγ/ βactin-Huvac


PPARγ/ βactin -Huvac























β-actin -HUVAC





β-actin -HUVAC



β-actin -HUVAC




-d THP1






-d THP1




-d THP1



Table 1: PPARγ and β-actin intensity results as expressed per mm.2 Based on the above data a bar chart was plotted in order to compare the PPARγ gene expression in HUVEC and dTHP1 cells by using the mean ratio of PPARγ/ β-actin. Intensity readings for PPAR-γ and β-actin gene bands were obtained from gel by using UV illumination.

Chapter 4


4.1 Discussion:

Peroxisome proliferator- activated receptor- gamma (PPARγ) found to play a role in the pathological processes of diabetes, obesity, atherosclerosis and cancer. Peroxisome proliferator-activated receptor-gamma (PPARγ) belongs to a nuclear receptor superfamily of transcription factors. It is mainly known to regulate adipocyte differentiation and fatty-acid uptake and storage (Knouff and Auwrx, 2004). The transcriptional activity of PPARγ is controlled by the loose binding of small lipophilic ligands into the ligand-binding pocket. Although a natural compound exhibiting specific, high-affinity binding characteristics remains unidentified, endogenous polyunsaturated fatty acids and eicosanoids, derived from nutrition or metabolic pathways, have been known as ligands for PPARγ. (Krey et al.,1997). In addition, many synthetic compounds, most particularly the thiazolidinediones (TZDs), are potent PPARγ agonists. (Picard and Auwerx,2002).

This study has focused on the determination of PPARγ gene expression in human umbilical vein endothelial cells (HUVEC) and it was found that the PPARγ gene is there in HUVEC (see figure 1 section 3.1). According to a study by (Kanda et al 2008) PPARγ is also expressed in ECs where it regulates target relevant to inflammation and atherosclerosis. This study has also compared the PPARγ gene expression in endothelial cells to that in dTHP1cells. PPARγ expression was induced in dTHP1 cells by phorbol myristyl acetate (PMA) the results showed that the expression of PPARγ in dTHP1 is 3% higher than in ECs, the P-value was >0.05 (p=0.432) which indicate an insignificant difference in the expression of PPARγ between the two cells (see figure 3 section 3.3). Similarly, (Marx et al., 1999) have stated that the ECs contain slightly less but comparable amounts of PPARγ protein relative to preadipocytes and monocytes derived macrophages. Reports from previous studies stated that the PPARγ expression increases in THP1 cells (human monocyte cell line) when they are treated with PMA. (Rios et al.,2008).

In addition, the β- actin gene was used in this study as a housekeeping gene; in RT-PCR the use of housekeeping gene is essential as denominators for comparison of samples, expression of β-actin gene remains constant. (Glare et al.,2002). The expression of PPARγ gene was calculated in relation to β- actin gene.

Difficulties encountered during the investigation include: centrifuging pellet where the cells were not exposed satisfactorily to ensure a firm pellet, hence loss of cells in the supernatant occurred, in combating this problem the centrifugation speed was increased to 12000 RCF for duration of 10 minutes. Another difficulty experienced was presence of particles in the gel hindering an accurate observation, possible causative factors for this occurrence include dirty ethedium bromide (stain) used, inaccurate TBE buffer concentration and under washing of the gel after electrophoresis. In overcoming this problem fresh ethedium bromide was used, accurately concentrated buffer was prepared before performing the gel electrophoresis and sufficient washing of the gel was carried out. During the gel electrophoresis primer dimer (PD) formation was noticed (see section 3.2 figure 2). There are several reasons of PD formation e.g because primers are present at high concentrations, weak interactions can occur between them. Complementarity of just one nucleotide between amplimer 3'-ends can give rise to PD, or the cold-start conditions, every possible combination of two different primers of a multiplex ARMS reaction would give rise to PDs, (Brownie et al., 1997). PD formation can be reduced by careful primer design, the application of strict conditions, the use of optimum temperature or `hot-start', touch-down PCR and/or enzyme formulations such as AmpliTaq Goldtm . (Brownie et al., 1997).

Endothelial cells are an important source of plasminogen activator inhibitor type-1 (PAI-1) plasma activity, considerable evidence links PAI-1 to myocardial infarction and deep venous thrombosis endothelial production of PAI-1 likely influences these events. The regulation of PAI-1 expression in endothelial cells has received focused attention. Cytokines such as transforming growth factor-β and tumour necrosis factor-α increase PAI-1 expression, circulating lipids, some lipid- lowering therapies, and the clinical condition of obesity itself all effect PAI-1 expression. This response to lipids as well as the evidence that adipocytes themselves can express PAI-1 raises the possibility that transcriptional mediators important in adipogensis and adipocyte signalling may play similar roles in endothelial cells. (Marx et al.,1999).

Although PPARγ has been extensively studied in adipocytes, monocytes/macrophages, and vascular smooth muscle cells, essentially not much is known about PPARγ in EC biology and gene expression. In a study carried out by (Marx et al.,1999) who investigated the PPARγ expression in human EC and the regulation of PAI-1 by PPARγ, their study has focused on the role of PPARγ signalling in ECs, such conclusions offer a novel molecular link between obesity, coagulation status and vascular events. Their study revealed that the human endothelial cells express PPARγ protein though it has slightly less but comparable amounts of PPARγ protein comparing to preadipocytes and monocyte- derived macrophages. They also found out that the PPARγ positively controls gene expression of PAI-1 in ECs thus potentially promoting thrombosis. It was also concluded that PPARγ inhibits macrophage activation thereby reducing cytokine production and macrophage gene expression. PPARγ as a highly regulated central transcriptional pathway present in various cell types might have varying effects on a complex pathological process like atherosclerosis. (Marx et al.,1999). The PPARγ expression is detected in the nucleus of many cells but only adipose tissue, large intestine and haematopoietic cells express the highest level of PPARγ mRNA and protein. Whereas, human muscles tissue expresses lower or trace amount of PPARγ under basal conditions. In addition, PPARγ mRNA has been identified in skeletal muscle and is found to be increased in obese subjects with insulin resistance. The expression of PPARγ in adipose tissue changes under the influence of a number of metabolic and hormonal variables, while temporarily change in food intake do not affect the expression of human PPARγ, hypocaloric diets for a longer period result in its down regulation. (Mohan et al.,2000). PPARγ plays an important role in regulating lipid metabolism in mature adipocytes,although much more of PPARγ role was known after the discovery of thiazolidinedione(TZD) which is an antidiabetic drug and are a high affinity agonist ligands for PPARγ. TZDs appear to co-ordinately activate gene expression leading to an increase in net lipid partitioning into adipocytes. Target genes directly regulated by PPARγ that are involved e.g lipoprotein lipase, fatty-acid transport protein and oxidised LDL receptor 1. Although increased fat storage would be expected to boost the size of adipocytes, TZD treatment actually leads to smaller adipocytes. This is partly due to increased adipocyte differentiation leading to new smaller cells. In addition, TZDs induce the coactivator PPARγ -coactivator which promotes mitochondrial biogensis leading to an increase in fatty-acid oxidation that further protects against adipocyte hypertrophy. (Lehrke and Lazar, 2005).

PPARγ is expressed in white blood cells and differentiated macrophages and has been implicated in the process of atherosclerosis. Initially, PPARγ activation was proposed to be proatherogenic by stimulating uptake and storage of oxidised lipids in macrophages via upregulation of the scavenger receptor/ fatty acid transporter CD36. This process leads to foam cell development and is a key event in the development of atherosclerosis. In contrast, treatment with thiazolidinediones has been shown to reduce the development of atherscloresis in mouse models suggesting that PPARγ is antiatherogenic. The inhibitory effect on atherosclerosis may be mediated by upregulating expression of the ABCA1 transporter in macrophages thereby promoting cholesterol efflux. Furthermore, PPARγ activation strongly reduces inflammatory gene expression in macrophages including MCP-1, VCAM-1, ICAM-1, IFNγ and TNFα. (Stienstra et al., 2007).

Patients with type 2 diabetes suffer from endothelial dysfunction. Endothelial damage results in a lower production of nitric oxide which inhibits thrombosis, inflammation and vascular smooth muscle (VSMC) growth and migration to prevent the vascular injury response. (Gujra,1999). Endothelial dysfunction occurs when vasoconstrictive effects place over vasodilator effects as a result of reduced nitric oxide (NO) bioavailability and consequent loss of its vascular protective action. Endothelial dysfunction is regarded an early step in the atherosclerotic process. Several mechanisms have been described to explain the improvement in endothelial function following treatment with PPARγ agonists. The decline in insulin resistance may increase NO delivery, since physiologically insulin increases endothelial nitric oxide synthase (eNOS) expression. Recent studies showed that PPARγ agonists directly stimulate eNOS expression in vitro. The reduction of circulating free fatty acid afforded by TZDs also improves endothelial function. The anti-inflammatory effects of TZDs have already been well demonstrated in animals evidenced by decreased NFKB activation, cytokines and vascular adhesion molecules release. (Bahia et al., 2006).

The endothelium has been also suggested to defend against oxidative stress and to maintain appropriate redox status (Kunsch,1999). The effect of PPARγ ligands on the vascular endothelium was expanded to include enhanced NO release. However, similar stimulation of NO release by PPARγ ligands in PAEC (Porcine Aortic Endothelial Cells), HAEC (Human Arterial Endothelial Cells) and HUVEC suggests that these effects may be generalised to all macrovascular ECs, regardless of species or vascular bed from which they are derived. (Calnek,2002).

The role of PPARγ has been investigated widely in different cells, and types of ligands that regulate PPARγ activity in vivo were discovered. Natural ligands such as fatty acids and 15-deoxy-Δ12,14-prostaglandin J2 (15ΔPGJ2) and the synthetic ligands such as rosiglitazone, troglitazone have been identified as PPARγ activators. PPARγ function is accelerated in presence of these ligands in the cell. (Marx et al,2004). The PPARγ plays a role in lipid metabolism, regulating genes that take part in the release, transport and storage of fatty acids. PPARγ-activating thiazolidinediones (TZDs) in use as anti-diabetic agents improve insulin sensitivity through their transcriptional effects. The effects of pharmacological PPARγ activation have been attributed to decrease free fatty acid level and increase lipid storage in adipose tissue in which it is most highly expressed thus reducing lipotoxicity in muscle and liver. (Rico et al,1998). The PPARγ is also expressed in ECs in which it regulates target relevant to inflammation and atherscloresis. However it is suggested to have more experimental trials on roziglitazone effect in ECs.

4.2 Future outlook:

RT-PCR technique was used to determine the PPARγ gene expression but there are other techniques that can measure the activity of the protein rather than its expression such as:

Reporter gene assay: Reporter genes have become an invaluable tool in studies of gene expression. They are widely used in biomedical and pharmaceutical research and also in molecular biology and biochemistry. A gene consists of two functional parts: One is a DNA-sequence that gives the information about the protein that is produced (coding region). The other part is a specific DNA-sequence linked to the coding region; it regulates the transcription of the gene (promoter). The promoter is either activating or suppressing the expression of the gene. The purpose of the reporter gene assay is to measure the regulatory potential of an unknown DNA-sequence. This can be done by linking a promoter sequence to an easily detectable reporter gene such as that encoding for the firefly luciferase. Common reporter genes are β-galactosidase, β-glucuronidase and luciferase. Various detection methods are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence. [berthold on line]

Detection of CD36 gene: CD36 is an 88 kDa glycoprotein originally identified as a platelet receptor and also known as fatty acid translocase, which is expressed in numerous cell types including monocytes/macrophages, platelets, endothelial cells, and adipocytes. CD36 is a multiligand receptor that is recognized by fatty acids, anionic phospholipids, thrombospondin, and oxidized lipoproteins. It is this latter property of scavenging (e.g., clearing) oxLDL which implicates CD36 in the initial steps of atherogenesis.(Demers et al.,2008).

Expression of CD36 has been shown to be regulated through the peroxisome proliferator activated receptor gamma (PPARγ) pathway. Its expression is dramatically increased on monocytes upon their interaction with activated endothelium. In addition it has been revealed that a variety of specific PPARγ ligands can by themselves up regulate CD36 indicating that the activation of PPARγ is sufficient to induce it expression.(Han and Sidell, 2002). A study carried by Rios et al, (2008) investigated the effect of modified LDLs on the expression of CD36 in human monocytic cell line (THP1). The CD36 expression in monocytes/macrophages is directly related to the increased uptake of the oxidised LDL particles. Several stimuli or downstream signals have been described to be involved in the over expression of CD36 in monocytes/macrophages including PPARγ activation.

Transcription factor assay: It is an enzyme linked immunosorbeant assay (ELISA) which is used to detect the specific transcription factor DNA binding activity in nuclear extracts and whole cell lysates. A specific double strand DNA (dsDNA) sequence containing the PPAR response element is immobilised on to the bottom of wells of a 96 well plate. PPARs contained in a nuclear extract, bind specifically to the PPAR response element. PPARalpha, delta, or gammas are detected by addition of specific primary antibodies directed against the individual PPARs. A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm. Cayman's PPARalpha, delta, gamma Complete Transcription Factor Assay detects PPARalpha, delta, gamma. [Cayman Chemical on line]

Chapter 5


5.0 Conclusion:

My in vitro results demonstrated that the PPARγ is expressed in endothelial cells. The PPARγ expression was observed in THP1 cells after 48 hours of treatment with PMA (phorbol myristate acetate) and the results were compared to PPARγ expression in HUVEC. The result illustrated that the expression of PPARγ in dTHP1 cells is 3% higher than in HUVEC cells. However, the difference was insignificant as the p value was > 0.05 (p=0.0432).