Abstract 
  
Background
Cardiovascular disease (CVD) remains the leading cause of mortality for type 2 diabetes (T2D) patient, and efficient solutions are yet to be unveiled to solve these twin epidemic of T2D and CVD. Peroxisome proliferator-activated receptors (PPARs) represent the transcriptional master regulator of lipid and glucose metabolism and are thought to be involved in the pathogenic mechanisms of T2D and CVD. Genome-wide association studies (GWAS) have examined over 500,000 single nucleotide polymorphism (SNPs), and seventy-five novel candidate genes for T2D have been identified and replicates. Peroxisome proliferator-activated receptor (PPAR) –γ, Pro12Ala, is one of the prominent SNP found to predispose an individual to T2D and CVD risks significantly. However, the predictive value of SNPs to T2D and CVD is small because lifestyle demonstrated a greater influence on T2D development. T2D is primarily rooted in a dynamic reversible lifestyle factor, such as calorie intake and calorie expenditure; hence meal-replacement therapy (MRT) has become an integral component of the management of T2D for weight loss, glycemic control, and CVD risks.
 
Aim
To uncover the transcriptional regulation underlying the improvement of CVD risks among T2D patients induced by MRT.
 
Method
Obese T2D participants were recruited with sufficient power analysis. These participants underwent well-controlled trial and were randomized to either isocaloric (1200kcal) MRT or standard dietetic regime for 12 weeks. We quantified blood pressure, lipid profile, and inflammatory marker. The peripheral blood’s mononuclear cell (PBMC) was isolated using Ficoll-Paque Plus method during pre and post treatment. DNA and RNA were extracted from PBMC. Allele specific real-time polymerase chain reaction (rt-PCR) was performed to genotype the Pro12Ala SNP. Purified RNA (1ug) was being converted to complementary DNA (cDNA). The cDNA was subsequently undergone transcriptional quantification for PPARs, its’ co-activator and its’ target genes for lipid, mitochondrial oxidation and glucose metabolism with rt-PCR using SYBR green PCR kit. The Shapiro-Wilk data normality tests were conducted to determine the appropriate choice of either parametric or non-parametric statistical analysis is used. Multiple linear regression model was used to inspect the meaningful predictive value of underlying molecular markers and pathway to CVD risk factors.
 
Results
MRT arm had significantly reduced total cholesterol/ high-density lipoprotein cholesterol ratio (TC/HDL-C), a well-known atherogenic index of CVD risks from (4.82 ± 0.43 to 4.43 ± 0.38, P< 0.05). The diastolic and systolic pressures, visceral fat, BMI and C-reactive protein were significantly reduced (P<0.05) in the MRT participants. Participants under standard dietetic regime did not demonstrate significant changes. On univariate logistic regression analysis, Pro12Ala SNP did not show significant association with the outcomes of CVD risks. Clinical phenotypes such as high-sensitivity C-reactive protein (hs-CRP) and visceral fat reduction have demonstrated to predict TC/HDL-C reduction by 42% and 38%, respectively (P< 0.05). Transcriptional PPARγ activation has shown to explain 50.7% of the TC/HDL-C reduction in MRT participants (R2 = 0.507, β =- 0.71 (-0.07- (-0.01), P< 0.05). Fatty acid oxidation, solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 (SLC25A20) activation could potentially explain how visceral fat contributed to reduction of atherogenic index TC/HDL-C by (R2 = 0.650, β =- 0.03 (-0.05 - (-0.01), P< 0.05).
 
Discussion
The obese T2D complied with MRT participants had demonstrated significant improvement of CVD parameters regardless of Pro12Ala SNP genetic predisposition. PPARs analysis has been known to be tissue-specific, probably the key factor that limits human studies due to its invasive nature. However, optimizing the isolation PBMC methodology has assisted current transcriptional quantitation, as PBMC is relatively easy to access and have been used as a surrogate tissue such as adipose tissue, skeletal muscle, breast, lung, and liver. The PPARγ activation has indicated to significantly underline the CVD risks reduction.  The advancement-personalized nutrigenomics such as with MRT has gained popularity. Henceforth to comprehend a clearer mechanistic insight of genes regulating CVD risks, we anticipated embarking on the future study to increase sample size and extend the duration period of trial, as current study additional PPARs target genes fold changed reached marginal significance level. Integrated study synergizing gene-environment interaction such as ongoing research is essential to erase blind spot, finding the niche that could uncover the key early molecular events, facilitating a better diagnostic, treatment and eventually prevention to T2D and reduction of CVD risks.

In search of cardiovascular risks reduction in type 2 diabetes: transcriptional regulation of meal replacement therapy