Gene Expression Profiling of Apoptotic Proteins in Circulating Peripheral Blood Mononuclear Cells in Type II Diabetes Mellitus and Modulation by Metformin

Figures & data

Table 1 Primer Sequences for All Primers Used in RT-qPCR

Primers	Sense (5ʹ → 3ʹ)	Anti-Sense (5ʹ → 3ʹ)	Accession
Bcl-2	TCCGCATCAGGAAGGCTAGA	AGGACCAGGCCTCCAAGCT	NM_000633.3
Bcl-10	GTGAAGAAGGACGCCTTAGAAA	TCAACAAGGGTGTCCAGACCT	NM_003921.5,
Bid	CCTTGCTCCGTGATGTCTTTC	GTAGGTGCGTAGGTTCTGGT	NM_197966.3
Bad	CCCAGAGTTTGAGCCGAGTG	CCCATCCCTTCGTCGTCCT	NM_004322.3
Bim	ATCCCCGCTTTTCATCTTTA	AGGACTTGGGGTTTGTGTTG	NM_138621.5
Bax	CCCGAGAGGTCTTTTTCCGAG	CCAGCCCATGATGGTTCTGAT	NM_001291428.2
Bak	ATGGTCACCTTACCTCTGCAA	TCATAGCGTCGGTTGATGTCG	NM_001188.4
Apaf1	AAGGTGGAGTACCACAGAGG	TCCATGTATGGTGACCCATCC	NM_013229.3
Caspase-3	AGAACTGGACTGTGGCATTGAG	GCTTGTCGGCATACTGTTTCAG	NM_004346.4
Caspase-7	AGTGACAGGTATGGGCGTTCG	GCATCTATCCCCCCTAAAGTGG	NM_001227.5
Caspase-8	CATCCAGTCACTTTGCCAGA	GCATCTGTTTCCCCATGTTT	NM_001372051.1
Caspase-9	TTCCCAGGTTTTGTTTCCTG	CCTTTCACCGAAACAGCATT	NM_001229.5
Ubiquitin C	ACTACAACATCCAGAAAGAG	CCAGTCAGGGTCTTCACGAAG	NM_021009.
RPL13	AACAAGTTGAAGTACCTGGCTTTC	TGGTTTTGTGGGGCAGCATA	NM_000977.3
Cyclophilin A	CCCACCGTGTTCTTCGACAT	TTTCTGCTGTCTTTGGGACCT	NM_021130.4

Table 2 Demographic Data of the Study Subjects

Group	Gender	Age (Years)	BMI kg/m^2	Glucose (mmol/L)	Insulin µIU/mL	HOMA-IR	HbA1c (%)	LDL (mmol/L)	HDL (mmol/L)	Triglycerides (mmol/L)
Lean	18M, 12F	25.7 ± 1.1	23.0 ± 0.3	5.1 ± 0.1	4.8 ± 0.60	1.1 ± 0.1	5.8 ± 0.10	2.54 ± 0.26	1.35 ± 0.04	0.83 ± 0.09
Obese	11M, 19F	35.1 ± 2.3	39.1 ± 1.7*	5.4 ± 0.1	10.0 ± 1.54*	2.1 ± 0.3*	5.6 ± 0.25	3.04 ± 0.16	1.18 ± 0.05	1.22 ± 0.12
Obese with T2DM	15M, 5F	48.4 ± 3.0*^σ	32.5 ± 1.9*	10.0 ± 1.1*^σ	7.4 ± 2.57*	2.8 ± 0.8*	8.0 ± 0.62*^σ	3.53 ± 0.19*	1.02 ± 0.05*^σ	1.97 ± 0.23*^σ
T2DM on Metformin	12M, 18F	47.1 ± 2.0*^σ	40.5 ± 1.5*	10.0 ± 0.8*^σ	7.8 ± 1.52*	4.1 ± 1.3*	8.7 ± 0.35*^σ	2.60 ± 0.17^δ	0.99 ± 0.04*^σ	1.56 ± 0.16*

Notes: Results are presented as Mean ± S.E.M; *P < 0.05 vs lean subjects; σP < 0.05 vs obese subjects; δP < 0.05 vs T2DM subjects.

Figure 1 mRNA expression of Bcl-2 in PBMC of lean, obese, T2DM, and T2DM on metformin. Results are presented as mean ± S.E.M.

Figure 2 mRNA expression of three members of the Bcl-2 family that promote apoptosis in PBMC of lean, obese, T2DM, and T2DM on metformin: (A) Bak; (B) Bax; and (C) Bcl-10. Results are presented as mean ± S.E.M. *P < 0.05 vs lean subjects; σP < 0.05 vs obese subjects; δP < 0.05 vs T2DM subjects.

Figure 3 mRNA expression of three members of apoptotic BH3 domain-only proteins that bind and regulate the anti-apoptotic Bcl-2 proteins in PBMC of lean, obese, T2DM, and T2DM on metformin: (A) Bad; (B) Bid; and (C) Bim. Results are presented as mean ± S.E.M. *P < 0.05 vs lean subjects; σP < 0.05 vs obese subjects; δP < 0.05 vs T2DM subjects.

Figure 4 mRNA expression of three caspases in PBMC of lean, obese, T2DM and T2DM on metformin: (A) CASP3; (B) CASP7; (C) CASP8; and (D) CASP9. Results are presented as mean ± S.E.M. *P < 0.05 vs lean subjects; σP < 0.05 vs obese subjects; δP < 0.05 vs T2DM subjects.

Figure 5 Apoptotic protease activating factor 1 (Apaf1) mRNA expression in PBMC of lean, obese, T2DM, and T2DM on metformin. Results are presented as mean ± S.E.M.

Figure 6 This figure schematizes steps in the proposed effect of metformin on apoptotic proteins in PBMCs examined in this study. The steps are depicted in a sequence proceeding from right to left. Hyperglycaemia presents in diabetes induces NF-κB pathway by reactive oxygen species (ROS). The low to moderate up-regulation of inflammatory Bcl-10, Bax, Bad, Bid, and the effector caspase-3 coupled with inhibition of caspase-7 in circulating PBMCs of T2DM could be a result of activated NF-κB pathway in T2DM.