Fazli Rabbi Awan

Imbalance between Neutrophil Elastase and its Inhibitor α1-Antitrypsin in Obesity Alters Insulin Sensitivity, Inflammation, and Energy Expenditure

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver. NE inhibitor GW311616A reversed insulin resistance and body weight gain in HFD-fed mice. Ela2(-/-) mice also augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK), and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT) and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO, and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance, and liver steatosis.

Investigating the concurrent presence of HCV in serum, oral fluid and urine samples from chronic HCV patients in Faisalabad, Pakistan

Hepatitis C virus (HCV) is normally present in the blood of infected patients; however, it can also be present in some other body fluids. Therefore, in this study, a concurrent presence of HCV-RNA was investigated in oral fluid and urine of 80 Pakistani chronic HCV patients. HCV-RNA was detected in 31 (38.8%) oral fluid and 10 (12.5%) urine samples using RT-PCR in all 80 of the patients whose sera tested positive for HCV-RNA. From this study, it is concluded that, in addition to the blood, HCV RNA can also be found in oral secretions as well as urine of chronic HCV patients.

Role of Leptin Deficiency, Inefficiency, and Leptin Receptors in Obesity

Leptin protein consists of 167 amino acids, which is mainly secreted from the white adipose tissue. This protein acts on the hypothalamic regions of the brain which control eating behavior, thus playing a significant role in maintaining body’s metabolism. Leptin receptors belong to glycoprotein 130 (gp130) family of cytokine receptors and exist in six isoforms (LEPR a–f), and all the isoforms are encoded by LEPR gene; out of these isoforms, the LEPR-b receptor is the ‘longest form,’ and in most of the cases, mutations in this isoform cause severe obesity. Also, mutations in the leptin gene (LEP) or its receptors gene can lead to obesity. Some biochemical pathways affect the bioactivity of leptin and/or its receptors. To date, eleven pathogenic mutations have been reported in the LEP which are p.L72S, p.N103K, p.R105W, p.H118L, p.S141C, p.W121X c.104_106delTCA, c.135del3bp, c.398delG, c.481_482delCT, and c.163C>T. Different mutations in the LEPR have also been reported as c.2396-1 G>T, c.1675 G>A, p.P316T, etc. In some studies, where leptin was deficient, leptin replacement therapy has shown positive impact by preventing weight gain and obesity.

Association of Family History of Type 2 Diabetes with COMT GenePolymorphism (I/D) in Pakistani Population

The Catechol-O-Methyl Transferase (COMT) gene polymorphism (I/D of C nucleotide at base position 900) has been previously associated in the brain disorders and inflammatory reactions but some studies also showed its presence in the development of type 2 diabetes (T2D) and kidney disease. So, the present study aimed to find association of I/D polymorphism with T2D, and its associated factors like family history and nephropathy (End Stage Renal Disease, ESRD) patients in a small group of Pakistani Punjabis. The results identified a significant (p=0.02) correlation of the 900 I/D C polymorphism with family history of diabetes, as it was found that greater number (74%) of patients having I allele had a positive family history of T2D. This has not been previously reported in Pakistani Punjabi population; however, this preliminary finding requires further validation studies.

Leptin Resistance: A Possible Interface Between Obesity and Pulmonary-Related Disorders

Context Under normal physiological conditions, leptin regulates body weight by creating a balance between food intake and energy expenditure. However, in obesity, serum leptin levels increase and become defective to retain energy balance. Evidence Acquisition Elevated serum leptin levels are regarded as an established marker of obesity. It is also reported that obese asthmatic patients have maximum serum leptin levels compared to other groups such as non-obese asthmatics, and normal obese and non obese subjects without asthma. In addition to having an appetite suppressing effect, leptin also regulates certain acute-phase protein expressions including α-1 antitrypsin (A1AT) in the liver. Results A1AT is a protease inhibitor that counterbalances the activity of the neutrophil elastase (NE) enzyme. A1AT reductions in obese-leptin resistant subjects lead to increased NE activity. The overactivity of NE degrades lung tissue proteins, which may lead to pulmonary disorders including asthma. Conclusions On the basis of prior studies, it could be hypothesized that, in obese asthmatic patients, the highest degree of leptin failure/resistance might lead to the creation of an imbalance between NE and its inhibitor A1AT. To ascertain this, large scale prospective studies are warranted to assess the comparative serum leptin and A1AT levels and NE activity in asthmatic non-obese and obese patients, simultaneously. Such studies might help to devise novel interventional therapies for the treatment of pulmonary-related problems including asthma, chronic obstructive pulmonary disorder (COPD), and other lung defects in susceptible obese subjects in the future.

Development of ARMS-PCR assay for genotyping of Pro12Ala SNP of PPARG gene: a cost effective way for case-control studies of type 2 diabetes in developing countries

Type 2 diabetes (T2D) is a prevalent metabolic disorder across the globe. Research is underway on various aspects including genetics to understand and control the global epidemic of diabetes. Recently, several SNPs in various genes have been associated with T2D. These association studies are mainly carried out in the developed countries through Genome Wide Association Scans, with follow-up replication/validation studies by high-throughput genotyping techniques (e.g. Taqman Technology). Although, similar studies could be conducted in developing countries, however, the limiting factors are the associated cost and expertise. These factors hamper research into the genetic association and replication studies from low-income countries to figure out the role of putatively associated SNPs in diabetes. Although, there are several SNP detection methods (e.g. Taqman assay, Dot-blot, PCR-RFLP, DGGE, SSCP) but these are either expensive or labor intensive or less sensitive. Hence, our aim was to develop a low-cost method for the validation of PPARG (Pro12Ala, CCA>GCA) SNP (rs1801282) for its association with T2D. Here, we developed a cost-effective and rapid amplification refractory mutation specific-PCR (ARMS-PCR) method for this SNP detection. We successfully genotyped PPARG SNPs (Pro12Ala) in human samples and the validity of this method was confirmed by DNA sequencing of a few representative samples for the three different genotypes. Furthermore, ARMS-PCR was applied to T2D patients and control samples for the screening of this SNP.

Aminoacidopathies: Prevalence, Etiology, Screening, and Treatment Options

Inborn errors of metabolism (IEMs) are a group of inherited metabolic disorders which are caused by mutations in the specific genes that lead to impaired proteins or enzymes production. Different metabolic pathways are perturbed due to the deficiency or lack of enzymes. To date, more than 500 IEMs have been reported with most of them being untreatable. However, fortunately 91 such disorders are potentially treatable, if diagnosed at an earlier stage of life. IEMs have been classified into different categories and one class of IEMs, characterized by the physiological disturbances of amino acids is called as aminoacidopathies. Out of 91 treatable IEM, thirteen disorders are amino acid related. Aminoacidopathies can be detected by chromatography and mass spectrometry based analytical techniques (e.g., HPLC, GC–MS, LC–MS/MS) for amino acid level changes, and through genetic assays (e.g., PCR, TaqMan Genotyping, DNA sequencing) at the mutation level in the corresponding genes. Hence, this review is focused to describe thirteen common aminoacidopathies namely: Phenylketonuria (PKU), Maple Syrup Urine Disease (MSUD), Homocystinuria/Methylene Tetrahydrofolate Reductase (MTHFR) deficiency, Tyrosinemia type II, Citrullinemia type I and type II, Argininosuccinic aciduria, Carbamoyl Phosphate Synthetase I (CPS) deficiency, Argininemia (arginase deficiency), Hyperornithinemia–Hyperammonemia–Homocitrullinuria (HHH) syndrome, N-Acetylglutamate Synthase (NAGS) deficiency, Ornithine Transcarbamylase (OTC) deficiency, and Pyruvate Dehydrogenase (PDH) complex deficiency. Furthermore, the etiology, prevalence and commonly used analytical techniques for screening of aminoacidopathies are briefly described. This information would be helpful to researchers and clinicians especially from developing countries to initiate newborn screening programs for aminoacidopathies.

Effects of Reg3 Delta Bioactive Peptide on Blood Glucose Levels and Pancreatic Gene Expression in an Alloxan-Induced Mouse Model of Diabetes.

OBJECTIVE The endocrine regeneration of the pancreas holds great potential for stable diabetes therapy. The Regeneration (Reg) family of proteins has been associated with pancreas regeneration. Hence, the Reg3 delta bioactive peptide from a mouse was evaluated to see whether it can reverse hyperglycemia in a mouse model of diabetes with any effects on pancreatic gene expression. METHODS In this study, we administrated the synthetic Reg3 delta bioactive peptide to healthy mice and to alloxan-induced mouse models of diabetes for 30 days, with weekly measurements of body weight and blood glucose levels. After 1 month, pancreatic gene profiling of these mice was performed for the Ngn-3, Pdx-1, MapK8, IGF-1, IGF2bp2, Reg3 beta and Reg3 delta genes. RESULTS The glycemic levels in mice with diabetes were decreased significantly, restored almost to normal. Furthermore, the gene expression levels measured by quantitative polymerase chain reaction (qPCR) showed that messenger RNA (mRNA) levels of 2 important transcription factors (Ngn-3 and Pdx-1) were increased during the Reg3 delta peptide treatment. CONCLUSIONS This study shows that Reg3 delta has the potential to reverse hyperglycemia by modulating gene expression in pancreatic endocrine precursor markers Pdx-1 and Ngn-3, which require further investigation at the protein and immunohistology levels.

Identification of hepatitis B virus core mutants by PCR-RFLP in chronic hepatitis B patients from Punjab, Pakistan.

SummaryChronic hepatitis B virus (HBV) infection remains a major health issue worldwide. Several factors including core gene variation are responsible for the development of chronicity of HBV infection. The present study was designed to identify the variations in the core region of the HBV genome in a local population of chronic hepatitis B patients (n = 57) using a PCR-based restriction fragment length polymorphism (PCR-RFLP) method. Fifty subjects were found to be positive for the presence of HBV DNA. For the core region genotyping, the Ava II and Msp I restriction enzymes were used. Mutations at nucleotide (nt) 2147 and nt 2362 in the HBV genome in the core region for Ava II (A4 type, 74%) and nt 2331 for Msp I (M1 type, 66%) were observed as the most common pattern. These results are different from those of previously reported studies on other populations and thus appear to be unique to the Pakistani population. This type of characterization of core mutants may be useful for the design of vaccines based on viral epitopes that are effective for the Pakistani population. Moreover, these unique genotypic patterns for the HBV core gene might be some of the main factors responsible for understanding the underlying mechanism by which HBV chronicity is developed in the Pakistani population.

A case control association study of ACE gene polymorphism (I/D) with hypertension in Punjabi population from Faisalabad, Pakistan

ABSTRACT Angiotensin converting enzyme (ACE) is a key component of renin angiotensin aldosterone system. It converts angiotensin I to angiotensin II. Insertion/deletion (I/D) polymorphism of ACE gene is found associated with several complications. However, its association with hypertension and related metabolic diseases is still controversial. So, the aim of the present study was to check this association for Punjabi population from Faisalabad, Pakistan. For this purpose, blood samples (patients = 100, controls = 48) were collected and several biochemical parameters were measured. Genotyping for ACE (I/D) polymorphism was performed by polymerase chain reaction (PCR) assay. ID genotype is found prevalent in the studied population as 41% in control subjects and 61% in patients. Furthermore, chi-square analysis showed significant (p = 0.005) difference for genotypic frequencies between both groups. One-way ANOVA for association of II, ID, and DD genotypes with anthropometric, clinical, and biochemical parameters showed that in patient group, DD genotype is significantly (p = 0.041) associated with systolic blood pressure (SBP). Moreover, ID genotype is found associated with the presence of cardiovascular diseases. This study concludes that DD genotype is strongly associated with higher SBP in hypertensive patients.