Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity

Figures & data

Table 1. Baseline clinical characteristics of the subjects enrolled in this study.

	Group with obesity (n = 7)	Lean group (n = 3)
Age (median, range)	42 (30–55)	49 (35–51)
Sex (F/M)	4/3	2/1
BMI (median, range)	42 (40–49)	22 (21–25)
Hypertension	3	1
Diabetes	1	0
Sleep apnea	1	0
Dyslipidemia	1	0
Arthrosis	1	0
Bariatric surgery	7	0
Colorectal surgery	0	3

Figure 1. Representative Coomassie G250 Blue-stained 2-D-gel pattern of a human liver biopsy homogenate from subject with (A) and without (B) obesity.

Figure 2. Master gel from 2-DE experiments on liver extracts of human subjects with and without obesity. The resulting 12-bit images were automatically analyzed using PDQuest software based on the spots identified across the gels of liver extract from seven patients with obesity and three without obesity. First dimension, IEF pH 3 to 10 NL (left to right); second dimension, SDS-containing 12% T polyacrylamide gel electrophoresis. Spot numbering corresponds to that reported in Table 2. The spots from 1 to 25 indicate a different quantitative representation with respect to control. Spots from 26 to 64 that were common to all compared samples and did not show significant differences.

Table 2. Mass spectrometry-based identification of proteins identified in the liver proteomes of obese (O) and nonobese (NO) subjects.

Spot	Proteins	SWISS PROT entry	NCBI entry	Spot	Proteins	SWISS PROT entry	NCBI entry
1	Calreticulin	CALR_HUMAN	4757900	43	Isocitrate dehydrogenase [NADP] cytoplasmic	IDHC_HUMAN	28178825
2	Catalase	CATA_HUMAN	4557014		4-hydroxyphenylpyruvate dioxygenase	HPPD_HUMAN	4504477
3	Glutamate dehydrogenase 1, mitochondrial	DHE3_HUMAN	4885281	44	Glutamate dehydrogenase 1, mitochondrial	DHE3_HUMAN	4885281
4	S-adenosylmethioninesynthetase isoform type-1	METK1_HUMAN	417297	45	Catalase	CATA_HUMAN	4557014
5	Aminoacylase-1	ACY1_HUMAN	461466		Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial	AL4A1_HUMAN	25777734
6	Aspartate aminotransferase, cytoplasmic	AATC_HUMAN	5902703	46	Catalase	CATA_HUMAN	4557014
	Acetyl-CoA acetyltransferase, mitochondrial	THIL_HUMAN	135755		Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial	AL4A1_HUMAN	25777734
7	Fructose-bisphosphate aldolase B	ALDOB_HUMAN	40354205	47	Aldehyde dehydrogenase, mitochondrial	ALDH2_HUMAN	25777732
8	Short-chain-specific acyl-CoA dehydrogenase, mitochondrial	ACADS_HUMAN	4557233	48	Aldehyde dehydrogenase, mitochondrial	ALDH2_HUMAN	25777732
9	Aldo-keto reductase family 1 member C4	AK1C4_HUMAN	1705823	49	Protein disulfide-isomerase A3	PDIA3_HUMAN	21361657
	Glyoxylate reductase/hydroxypyruvate reductase	GRHPR_HUMAN	47116943	50	Keratin, type II cytoskeletal 8	K2C8_HUMAN	4504919
10	3-mercaptopyruvate sulfurtransferase	THTM_HUMAN	61835204	51	Formimidoyltransferase-cyclodeaminase	FTCD_HUMAN	11140815
11	Phenazine biosynthesis-like domain-containing protein	PBLD_HUMAN	62177133	52	Formimidoyltransferase-cyclodeaminase	FTCD_HUMAN	11140815
12	Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial	ECH1_HUMAN	82654933	53	Keratin, type II cytoskeletal 8	K2C8_HUMAN	4504919
13	Carbonic anhydrase 2	CAH2_HUMAN	4557395	54	Keratin, type I cytoskeletal 18	K1C18_HUMAN	4557888
14	Glutathione S-transferase A1	GSTA1_HUMAN	22091454	55	60 kDa heat shock protein, mitochondrial	CH60_HUMAN	31542947
15	Peroxiredoxin-1	PRDX1_HUMAN	548453	56	60 kDa heat shock protein, mitochondrial	CH60_HUMAN	31542947
16	Protein DJ-1	PARK7_HUMAN	31543380	57	Vimentin	VIME_HUMAN	62414289
17	Apolipoprotein A-I	APOA1_HUMAN	4557321		Tubulin alpha-1B chain	TBA1B_HUMAN	193787715
18	Hemoglobin subunit beta	HBB_HUMAN	4504349	58	ATP synthase subunit beta, mitochondrial	ATPB_HUMAN	32189394
19	Hemoglobin subunit beta	HBB_HUMAN	4504349	59	Tubulin beta chain	TBB5_HUMAN	18088719
20	Hemoglobin subunit beta	HBB_HUMAN	4504349	60	Protein disulfide-isomerase	PDIA1_HUMAN	2507460
21	Hemoglobin subunit beta	HBB_HUMAN	4504349		Alpha-1-antitrypsin	A1AT_HUMAN	1703025
22	Fatty acid-binding protein, liver	FABPL_HUMAN	4557577	61	Calreticulin	CALR_HUMAN	4757900
23	Hemoglobin subunit alpha	HBA_HUMAN	4504345	62	78 kDa glucose-regulated protein	GRP78_HUMAN	16507237
24	Hemoglobin subunit alpha	HBA_HUMAN	4504345	63	Endoplasmin	ENPL_HUMAN	4507677
25	Pterin-4-alpha-carbinolamine dehydratase	PHS_HUMAN	4557831	64	Annexin A6	ANXA6_HUMAN	113962
26	Fatty acid-binding protein, liver	FABPL_HUMAN	4557577		Heat shock cognate 71 kDa protein	HSP7C_HUMAN	123648
27	Acyl-CoA-binding protein	ACBP_HUMAN	118276		Serum albumin	ALBU_HUMAN	4502027
	Hemoglobin subunit beta	HBB_HUMAN	4504349		V-type proton ATPase catalytic subunit A	VATA_HUMAN	22096378
	Cytochrome c oxidase subunit 6B1	CX6B1_HUMAN	117115
	GTP cyclohydrolase 1 feedback regulatory protein	GFRP_HUMAN	2506906
	Fatty acid-binding protein, liver	FABPL_HUMAN	4557577
28	Fatty acid-binding protein, liver	FABPL_HUMAN	4557577
29	Superoxide dismutase [Cu-Zn]	SODC_HUMAN	4507149
30	Eukaryotic translation initiation factor 5A-1	IF5A1_HUMAN	54037409
	Glia maturation factor beta	GMFB_HUMAN	46577593
31	Cytochrome b5	CYB5_HUMAN	41281768
32	Ferritin light chain	FRIL_HUMAN	20149498
33	Peroxiredoxin-2	PRDX2_HUMAN	32189392
	Ferritin light chain	FRIL_HUMAN	20149498
34	Apolipoprotein A-I	APOA1_HUMAN	4557321
35	Ferritin light chain	FRIL_HUMAN	20149498
	Thioredoxin-dependent peroxide reductase, mitochondrial	PRDX3_HUMAN	2507171
36	Enoyl-CoA hydratase, mitochondrial	ECHM_HUMAN	1922287
	Heat shock protein beta-1	HSPB1_HUMAN	4504517
37	Peroxiredoxin-6	PRDX6_HUMAN	4758638
38	Phosphatidylethanolamine-binding protein 1	PEBP1_HUMAN	4505621
39	Glutathione S-transferase Mu 1	GSTM1_HUMAN	23065544
40	Thiosulfate sulfurtransferase	THTR_HUMAN	17402865
41	Betaine–homocysteineS-methyltransferase 1	BHMT1_HUMAN	157266337
42	Betaine–homocysteineS-methyltransferase 1	BHMT1_HUMAN	157266337

The first 25 spots reports proteins identified as differentially represented in the O subjects.

Table 3. Proteins identified as over- and down-represented in the liver of O subjects, compared to control (NO subjects).

Spot	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25
Protein	Calreticulin	Catalase	Glutamate dehydrogenase 1	S-adenosylmethionine synthetase isoform type 1	Aminoacylase-1	Acetyl-CoA acetyltransferase, mitochondrial; aspartate aminotransferase, cytoplasmic	Fructose- bisphosphate aldolase B	Short-chain specific acyl-CoA dehydrogenase, mitochondrial	Aldo-keto reductase family 1 member C4; Glyoxylate reductase/ hydroxypyruvate	3-Mercaptopyruvate sulfurtransferase	Phenazine biosynthesis-like domain- containing protein	Δ(3,5)- Δ(2,4)-dienoyl-CoA isomerase, mitochondrial	Carbonic anhydrase 2	Glutathione S-transferase A1	Peroxiredoxin-1	Protein DJ-1	Apolipoprotein A-I	Hemoglobin subunit beta	Hemoglobin subunit beta	Hemoglobin subunit beta	Hemoglobin subunit beta	Fatty acid-binding protein	Hemoglobin subunit alpha	Hemoglobin subunit alpha	Pterin-4-alpha-carbinolamine dehydratase
Sample
1	–	++	–	–	+++	+	++	++	++		+++	+++	+	–	+	+	–	–	–	–	–	–	–	++	++
2	+	++	–	–	+	+	+	+	–	+++	++	++	++	–	–	++	–	+	++++	+++	++	++++	+	+++++	+
3	++++	+++	–	–	+	+	+++	–	++		–	–	–	–	+++	+++	–	+	+++	++	++	+	+	+	++
4	–	+	–	–	++	–	–	++	+++	+	+	++	+	–	+	–	–	–	++++	+	++	+	++	++	–
5	+	–	–	–	–	+++++	–	–	++	+	+	–	++	–	++	+	–	+	–	++	+++		–	+	++
6	+	+++	–	–	++	–	+	+	–	+	–	+	–	–	+	++	–	++	–	++	++	++	+++++	++	++
7	+++++	–	–	+	+	+	++++	+++	++	+	++	+	+	+++	+	+	–	+	–	–	+	+	+	++	–
+	>0 < 5		–	>0 < −5
++	>5 < 10		–	>−5 < −10
+++	>10 < 15		–	>−10 < −15
++++	>15 < 20		–	>−15 < −20
+++++	>20		–	>−20

The protein value was calculated by subtracting the average volume value of the NO subjects (assumed as reference data) to that of each measured spot. The protein in the liver biopsies was judged to be positive or negative according to the higher or lower value than 0 score in comparison to the reference samples. Volume measurements (expressed as percentage) were indicated with + or – signs, depending on the degree of protein expression.

Figure 3. Gene Ontology (GO) annotation of identified proteins. The graphs show the percentages of corresponding GO terms to the 25 differentially represented components. The identified proteins were clustered according to three different criteria: cellular component, biological process, and molecular function.

Figure 4. Enzymes involved in fatty acid β-oxidation, ketogenesis, pyruvate metabolism and gluconeogenesis and acetyl-CoA generation/consumption deregulated process in human subjects with obesity. Over-represented proteins and promoted biological processes were underlined; down-represented proteins and reduced biological processes were in italics.