Vanin 1 Gene Role in Modulation of iNOS/MCP-1/TGF-β1 Signaling Pathway in Obese Diabetic Patients

Figures & data

Table 1 Primers Used in Real-Time PCR

Gene	Primer Sequence
Inducible nitric oxide synthase	F: 5′-CGA GGA GGC TGC CCT GCA GAC TGG-3′
	R: 5′-CTG GGA GGA GCT GAT GGA GTA GTA-3′
Vanin-1	F: 5′- -GGAACCCGGTATGTCTTCCC-3
	R: 5′- ACTCCCCAGGTGCGAGC-3
GAPDH (Internal control for qRT-PCR)	F: 5′- CCA TCACCATCTTCCAGG AG-3’
	R: 5′-CCTGCTTCACCACCTTCTTG −3’

Table 2 BMI in Relation to Glucose, Insulin, IRI and HbA1c Among Healthy Persons and Overweight as Well as Class I and II Obese Diabetic Patients

No.	Groups	BMI (kg/m^2)	Glucose (mmol/L)	Insulin (μU/mL)	IRI	HbA1c (%)
(1)	Healthy males (N = 18)	23.25 ± 4.37^a	5.41 ± 0.43^a	5.77 ± 0.76^a	1.39 ± 0.23^a	5.67 ± 0.54^a
(2)	Overweight diabetic patients (N = 16)	30.47± 6.20^b	7.76 ± 0.79^b	7.51 ± 0.72^b	2.59 ± 0.39^b	6.45 ± 1.32^b
(3)	Class I obese diabetic patients (N = 20)	35.20 ± 4.60^c	9.34 ± 0.84^c	8.71 ±1.10^c	3.61 ± 0.57^c	9.88 ± 1.45^c
(4)	Class II obese diabetic patients (N = 13)	38.10 ± 3.37^d	10.63 ± 0.64^d	10.54 ± 1.56^d	4.96 ± 0.73^d	10.31 ± 1.10^d

Notes: Data shown are mean ± standard deviation of number of observations within each treatment. Data followed by different superscript alphabet along the same column are significantly different (p < 0.05). The high significant levels of the parameters were in the order of a < b < c < d. Data with superscript alphabet ^aAre significantly lower than data with superscript alphabet ^bWhile data with superscript ^bAre lower than data with superscript alphabet ^c,dAt p < 0.05. Data followed by the same superscript alphabet are not significantly different at p ≤ 0.05. IRI = insulin (mU/mL) x glucose (mM) / 22.5.

Abbreviations: BMI, Body mass index, BP, Blood pressure, IRI, Insulin resistance index, HbA1c, glycated hemoglobin; N, number of participants in each group.

Table 3 Levels of TC, TG and HDL-C Among Healthy Persons, Overweight as Well as Obese Class I and II Diabetic Patients

No.	Groups	TC (mg/dl)	TG (mg/dl)	HDL-C (mg/dl)
(1)	Healthy males (N = 18)	186.04 ± 25.22^a	122.13 ± 20.08^a	46.04 ± 10.21^b
(2)	Overweight diabetic patients (N = 16)	224.15 ± 13.31^b	167.11 ± 15.12^b	41.00 ± 4.62^b
(3)	Obese Class I obese diabetic patients (N = 20)	247.26 ± 23.79^c	205.92 ± 25.90^c	37.22 ± 4.34^a
(4)	Class II obese diabetic patients (N = 13)	265.94 ± 18.78^d	223.51 ± 25.77^d	34.65 ± 3.71^a

Notes: Data shown are mean ± standard deviation of number of observations within each treatment. Data followed by different superscript alphabet along the same column are significantly different (p < 0.05). The high significant levels of the parameters were in the order of a < b < c < d. Data with superscript alphabet ^aAre significantly lower than data with superscript alphabet ^bWhile data with superscript ^bAre lower than data with superscript alphabet ^c,dAt p < 0.05. Data followed by the same superscript alphabet are not significantly different at p ≤ 0.05.

Abbreviations: TC, Total cholesterol; TG, triacylglycerols; HDL-C, high-density lipoprotein-cholesterol; N, number of participants in each group.

Table 4 Levels of Plasma TNF- α, MCP-1 and TGF-β1 in Healthy Persons and Overweight as Well as Obese Class I and II Diabetic Patients

No.	Groups	TNF-α (pg/mL)	MCP-1 (pg/mL)	TGF-β1 (ng/mL)
(1)	Healthy males (N = 18)	4.04 ± 0.40^a	230.37 ± 18.00^a	35.55 ± 3.901^a
(2)	Overweight diabetic patients (N = 16)	5.03 ± 0.52^b	263.60 ± 19.93^b	41.27 ± 4.98^b
(3)	Class I obese diabetic patients (N = 20)	6.89 ± 0.64^c	307.77 ± 27.58^c	49.25 ± 4.08^c
(4)	Class II obese diabetic patients (N = 13)	8.73 ± 1.28^d	382.23 ± 30.24^d	54.69 ± 3.3^d

Notes: Data shown are mean ± standard deviation of number of observations within each treatment. Data followed by different superscript alphabet along the same column are significantly different (p < 0.05). The high significant levels of the parameters were in the order of a < b < c < d. Data with superscript alphabet ^aAre significantly lower than data with superscript alphabet ^bWhile data with superscript ^bAre lower than data with superscript alphabet ^c,dAt p < 0.05. Data followed by the same superscript alphabet are not significantly different at p ≤ 0.05. Data followed by the same letter are not significantly different at p ≤ 0.05.

Abbreviations: TNF- α, Tumor necrosis factor-α; MCP-1, Monocyte Chemoattractant Protein-1; TGF-β1, Transforming growth factor-beta 1; N, number of participants in each group.

Table 5 Levels of Blood SOD and CAT as Well as Plasma TBARs Among Healthy Persons and Overweight as Well as Class I and II Obese Diabetic Patients

No.	Groups	SOD (U.min/mL)	CAT (μ Moles of H2O2 Consumed min/mL)	TBARs (µ Mole/L)
(1)	Healthy males (N = 18)	5.15 ± 0.74^b	20.92 ± 3.32^a	0.72 ± 0.15^a
(2)	Overweight diabetic patients (N = 16)	4.47 ± 0.60^c	31.60 ± 4.12^b	1.16 ± 0.20^b
(3)	Class I obese diabetic patients (N = 20)	3.90 ± 0.48^b	34.86 ± 2.8^c	1.49 ± 3.38^c
(4)	Class II obese diabetic patients (N = 13)	3.50 ± 0.29^a	38.08 ± 4.47^d	2.06 ± 0.33^d

Notes: Data shown are mean ± standard deviation of number of observations within each treatment. Data followed by different superscript alphabet along the same column are significantly different (p < 0.05). The high significant levels of the parameters were in the order of a < b < c < d. Data with superscript alphabet ^aAre significantly lower than data with superscript alphabet ^bWhile data with superscript ^bAre lower than data with superscript alphabet ^c,dAt p < 0.05. Data followed by the same superscript alphabet are not significantly different at p ≤ 0.05.

Abbreviations: SOD, Superoxide dismutase; CAT, catalase; TBARs, thiobarbituric acid reactive substances; iNOS, inducible NO synthase; N, number of participants in each group.

Figure 1 iNOS gene expression levels among healthy persons and overweight as well as class I and II obese diabetic patients. Data shown are mean ± standard deviation of number of observations within each treatment. Data followed by different superscript alphabet along the same column are significantly different (p < 0.05). The high significant levels of the parameters were in the order of a < b < c < d. Data with superscript alphabet “a” are significantly lower than data with superscript alphabet “b” while data with superscript “b” are lower than data with superscript alphabet “c and d” at p < 0.05. Data followed by the same superscript alphabet are not significantly different at p ≤ 0.05.

Abbreviation: N, number of participants in each group.

Figure 2 Vanin-1 gene expression levels among healthy persons and overweight as well as class I and II obese diabetic patients. Data shown are mean ± standard deviation of number of observations within each treatment. Data followed by different superscript alphabet along the same column are significantly different (p < 0.05). The high significant levels of the parameters were in the order of a < b < c < d. Data with superscript alphabet “a” are significantly lower than data with superscript alphabet “b” while data with superscript “b” are lower than data with superscript alphabet “c and d” at p < 0.05. Data followed by the same superscript alphabet are not significantly different at p ≤ 0.05. N= number of participants in each group.

Table 6 Correlation Between the Expression of Vanin 1 and Glucose Homeostasis Indices, HbA1c, Lipid Profile, Inflammatory Mediators, and Oxidative Stress Biomarkers in Healthy Persons and Overweight as Well as Class I and II Obese Diabetic Patients

Groups	Vanin 1
	Overweight Patients (N = 16)		Class I Obese Diabetic Patients (N = 20)		Class II Obese Diabetic Patients (N = 13)
Parameters	r	p	r	p	r	P
BMI	0.203	0.450*	0.025	0.916*	0.056	0.856*
Glucose	0.561	0.024	0.067	0.780*	0.486	0.092*
Insulin	0.209	0.438	0.039	0.871*	0.421	0.152*
IRI	0.531	0.034	0.002	0.995*	0.620*	0.024*
HbA1c	0.138	0.610	0.596	0.006*	0.415	0.158*
TC	0.335	0.205*	0.233	0.323*	0.047	0.880*
TG	0.013	0.961*	0.066	0.783*	0.327	0.276*
HDL-C	−0.414	0.111*	−0.265	0.259*	−0.031	0.920*
TNF-α	0.234	0.383	0.149	0.529*	0.537	0.059
MCP-1	0.134	0.620*	0.355	0.124*	−0.090	0.770*
TGF-β1	0.342	0.194*	0.361	0.118*	0.151	0.624*
iNOS	0.192	0.475*	0.247	0.293*	0.329	0.272*
SOD	−0.096	0.724*	−0.123	0.605*	−0.613	0.026*
CAT	0.151	0.578*	0.212	0.370*	0.183	0.550*
TBARs	0.114	0.675*	0.176	0.459*	0.337	0.261*

Note: *p significant at 0.05.

Abbreviation: N, number of participants in each group.

Figure 3 Ultrasound (US) imaging of liver in healthy persons and overweight as well as class I and II obese patients. Normal liver contrast with white asterisks demarcating normal hepatic parenchyma for comparison at same depth (A). US of overweight diabetic patients showed the presence of fatty liver with increased liver echogenicity (B). Also, fatty liver with mild hepatic echogenicity obscuring was apparent in class I obese diabetic patients (C). In addition, US liver examination of class II obese diabetic patients showed a marked fatty liver with echogenic liver obscuring the echogenic walls of portal venous branches (D).

Figure 4 CT scan of liver in healthy persons and overweight diabetic as well as class I and II obese patients. Normal appearance of the liver at unenhanced CT (A). CT of overweight diabetic patients showed diffuse fat accumulation in the liver (B). Also, Focal fat accumulation in the liver at CT in class I obese diabetic patients (C). In addition, CT liver examination of class II obese diabetic patients showed multifocal fat accumulation in the liver at CT (D).

Abbreviations: v, Vessel; HU, Hounsfield units; fs, Focal Sparing.

Figure 5 Schematic diagram of the hydrolysis of Pantetheine.