Metformin Effect on Nontargeted Metabolite Profiles in Patients With Type 2 Diabetes and in Multiple Murine Tissues

Jonathan Adam; Stefan Brandmaier; Jörn Leonhardt; Markus F. Scheerer; Robert P. Mohney; Tao Xu; Jie Bi; Markus Rotter; Martina Troll; Shen Chi; Margit Heier; Christian Herder; Wolfgang Rathmann; Guido Giani; Jerzy Adamski; Thomas Illig; Konstantin Strauch; Yixue Li; Christian Gieger; Annette Peters; Karsten Suhre; Donna Ankerst; Thomas Meitinger; Martin Hrabĕ de Angelis; Michael Roden; Susanne Neschen; Gabi Kastenmüller; Rui Wang-Sattler

doi:10.2337/db16-0512

Genetics/Genomes/Proteomics/Metabolomics

Metformin Effect on Nontargeted Metabolite Profiles in Patients With Type 2 Diabetes and in Multiple Murine Tissues

Jonathan Adam¹,²,
Stefan Brandmaier¹,²,
Jörn Leonhardt³,
Markus F. Scheerer⁴,⁵,
Robert P. Mohney⁶,
Tao Xu¹,²,
Jie Bi⁷,
Markus Rotter¹,²,
Martina Troll¹,²,
Shen Chi¹,²,
Margit Heier²,
Christian Herder⁵,⁸,
Wolfgang Rathmann⁵,⁹,
Guido Giani⁹,
Jerzy Adamski⁴,⁵,¹⁰,¹¹,
Thomas Illig¹²,¹³,
Konstantin Strauch¹⁴,¹⁵,
Yixue Li⁷,
Christian Gieger¹,²,⁵,
Annette Peters¹,²,⁵,¹⁶,
Karsten Suhre³,¹⁷,¹⁸,
Donna Ankerst¹⁹,
Thomas Meitinger²⁰,²¹,
Martin Hrabĕ de Angelis⁴,⁵,¹¹,
Michael Roden⁵,²²,²³,
Susanne Neschen⁴,⁵,
Gabi Kastenmüller³ and
Rui Wang-Sattler¹,²,⁵ ⇑

¹Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany
²Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany
³Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany
⁴Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany
⁵German Center for Diabetes Research (DZD), Neuherberg, Germany
⁶Metabolon, Inc., Durham, NC
⁷Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
⁸Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany
⁹Institute for Biometrics and Epidemiology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany
¹⁰Institute of Experimental Genetics, Genome Analysis Center, Helmholtz Zentrum München, Neuherberg, Germany
¹¹Institute of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Freising, Germany
¹²Hannover Unified Biobank, Hannover Medical School, Hannover, Germany
¹³Institute for Human Genetics, Hannover Medical School, Hannover, Germany
¹⁴Institute of Genetic Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany
¹⁵Genetic Epidemiology, Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universität München, München, Germany
¹⁶Department of Environmental Health, Harvard School of Public Health, Boston, MA
¹⁷Faculty of Biology, Ludwig-Maximilians-Universität, Planegg-Martinsried, Germany
¹⁸Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar (WCMC-Q), Education City–Qatar Foundation, Doha, Qatar
¹⁹Lehrstuhl für Mathematische Modelle Biologischer Systeme, Technische Universität München, Garching, Germany
²⁰Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany
²¹Institute of Human Genetics, Technische Universität München, München, Germany
²²ShanghaiTech University, Shanghai, China
²³Department of Endocrinology and Diabetology, Medical Faculty, Düsseldorf, Düsseldorf, Germany

Corresponding author: Rui Wang-Sattler, rui.wang-sattler{at}helmholtz-muenchen.de.

G.K. and R.W.-S. contributed equally to this study.

Diabetes 2016 Dec; 65(12): 3776-3785. https://doi.org/10.2337/db16-0512

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Figure 1
Differences in relative metabolite concentrations in a human study, in a mouse study, and in organ-specific pathways. A: Mean relative residuals of the concentrations (with SEM) of two metabolites for the NGT, IGT, ndt-T2D, and mt-T2D groups derived in cross-sectional analysis of KORA F4. Residuals were calculated from a linear regression model with full adjustments. B: Mean relative concentrations (with SEM) of two metabolites in four different mouse tissues (plasma, liver, skeletal muscle, and epididymal adipose tissue). C: The connections indicated by liver, muscle, and blood (plasma and serum) show organ specificity between metabolites, pathway-related proteins, metformin targets, and metformin. The metabolites (ellipses) were connected to metformin treatment (straight-sided hexagons), proteins (hexagons), and metformin targets (rectangles). The activation/stimulation is indicated with arrows. For further information, see Tables 3–5 and Supplementary Tables 2 and 3.

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Table 1

Characteristics of the KORA F4 cross-sectional study samples (n = 1,604)

Clinical parameters	NGT	IGT	ndt-T2D	mt-T2D
n	1,143	272	115	74
Age, years	58.9 (8.5)	63.8 (8.1)	65.1 (7.1)	66.2 (7.5)
Male, %	45	50	61	58
BMI, kg/m²	27.0 (4.3)	29.8 (4.7)	31.0 (4.8)	32.0 (5.6)
Waist, cm	92 (12.9)	100.2 (14.3)	104.9 (12.1)	106.7 (13.0)
Physical activity, % >1 h per week	63	54	48	36
High alcohol intake, %^*	20	18	25	19
Smoker, %	18	8	12	15
Systolic BP, mmHg	121.6 (17.7)	129.0 (19.0)	134.3 (19.2)	130.7 (18.2)
HDL-C, mg/dL	58.9 (14.8)	54.4 (14.2)	49.6 (11.5)	50.4 (9.6)
LDL-C, mg/dL	140.7 (34.3)	144.5 (36.2)	136.6 (36.0)	124.1 (28.3)
Total cholesterol, mg/dL	223.4 (37.7)	226.4 (41.5)	214.1 (37.0)	203.8 (37.8)
Triglycerides, mg/dL	118.5 (84.6)	150.7 (89.0)	172.5 (128.7)	177.5 (140.5)
HbA_1c, %	5.4 (0.3)	5.6 (0.3)	6.3 (0.9)	6.8 (1.1)
HbA_1c, mmol/mol	35.7 (3.2)	38.1 (3.9)	44.91 (9.9)	51.2 (11.6)
Fasting glucose, mg/dL	93.2 (7.5)	101.2 (10.6)	126.6 (30.5)	142.0 (36.0)
2-h postglucose load, mg/dL	100.8 (20.6)	162.6 (17.5)	216.0 (50.7)^†
Time since diagnosis, years			1.4 (2.6)^‡	7.4 (6.6)
Insulin, µIU/mL	7.1 (26.1)	10.1 (10.9)	14.3 (14.3)	11.6 (11.0)
Leptin, ng/mL	17.2 (19.2)	24.2 (21.3)	26.8 (21.2)	27.8 (25.0)
Statin usage, %	12	15	28	36
β-Blocker usage, %	16	31	43	38
ACE inhibitor usage, %	10	21	32	45
ARB usage, %	8	10	17	14
Insulin therapy, %	0	0	0	0
Metformin usage, %	0	0	0	100
Parental T2D, %	28	30	37	49

KORA F4 study characteristics (including solely subjects with available Metabolon measurements). Percentages of individuals or means (SD) are shown for each variable and each group (NGT, IGT, ndt-T2D, and mt-T2D).
*≥20 g/day for women; ≥40 g/day for men.
†n = 81.
‡For newly diagnosed T2D patients (n = 74), years since T2D diagnosis was defined as 0.

Table 2

Characteristics of the KORA S4 → F4 prospective study samples (n = 683)

Clinical parameters	KORA S4 w/o metformin^§ → KORA F4 w/o metformin^§			KORA S4 w/o metformin^§ → KORA F4 w/ metformin
Clinical parameters	S4	F4	P value	S4	F4	P value
n	646	646		37	37
Age, years	61.4 (4.2)	68.5 (4.2)		63.4 (3.8)	70.4 (3.9)
Male, %	51	51		54	54
BMI, kg/m²	27.9 (3.9)	28.2 (4.2)	0.2	32.8 (4.3)	32 (4.5)	0.49^‖
Waist, cm	93.9 (11.0)	96.8 (11.9)	1.72E-05	106.3 (11.3)	106.7 (12.3)	0.87^‖
Physical activity, % >1 h per week	47	57	5.47E-04	32	43	0.47
High alcohol intake, %^*	20	19	0.65	27	16	0.4
Smoker, %	13	8	9.00E-03	14	8	0.71
Systolic BP, mmHg	132.1 (18.7)	128.2 (19.6)	3.12E-04	144.9 (18.1)	131.6 (18.3)	2.43E-03^‖
HDL-C, mg/dL	59.1 (16.3)	56.7 (14.2)	0.02	53.4 (11.8)	52.4 (7.9)	0.69^‖
LDL-C, mg/dL	154.5 (40.8)	142.5 (36.9)	1.86E-08	143.5 (37.6)	123.5 (23.6)	0.02
Total cholesterol, mg/dL	245.5 (42.0)	225.2 (40.7)	2.2E-16	234.1 (41.0)	202.1 (33.6)	5.20E-04
Triglycerides, mg/dL	129.9 (76.4)	132.7 (83.4)	0.56	170.6 (169)	154.5 (159.2)	0.79
HbA_1c, %	5.6 (0.3)	5.6 (0.5)	0.53	6.4 (0.9)	6.6 (0.7)	0.14
HbA_1c, mmol/mol	37.6 (3.7)	38.0 (5.7)	0.53	46.8 (10.3)	48.3 (7.8)	0.14
Fasting glucose, mg/dL	99.7 (10.9)	100.7 (17.6)	0.53	130.7 (30.1)	129.6 (28.3)	0.9
2-h postglucose load, mg/dL	115.1 (37.1)	126.9 (40.8)	8.34E-09	205.5 (76.8)
Statin usage, %	10	22	8.05E-10	8	29	0.04
β-Blocker usage, %	17	31	5.16E-09	16	29	0.27
ACE inhibitor usage, %	8	21	2.72E-11	16	51	3.18E-03
ARB usage, %	3	12	3.67E-10	3	14	0.2
Insulin therapy, %	0	0		0	0
Metformin usage, %	0	0		0	100
Parental T2D, %	25	25		47	47

Percentages of individuals or means (SD) of participants (with available Metabolon measurements for KORA S4 and F4) are shown for each variable and each group. w/o, without; w/, with.
*>40 g/day in men; >20 g/day in women.
§Includes participants with NGT, isolated IFG, IGT, and ntd-T2D.
‖Normally distributed (every other distribution is not normally distributed).

Table 3

Two human serum metabolites significantly associated with metformin treatment in a cross-sectional analyses (KORA F4)

	Crude linear model mt-T2D (n = 74) vs. ndt-T2D (n = 115)			Full linear model mt-T2D (n = 70)^¶ vs. ndt-T2D (n = 114)^¶
Metabolite	β (95% CI) per SD	P value	FDR	β (95% CI) per SD	P value	FDR
Citrulline	−0.75 (−1.09, −0.41)	2.31E-05	2.83E-04	−0.79 (−1.15, −0.43)	2.54E-05	2.83E-04
X-21365	0.67 (0.38, 0.96)	7.54E-06	1.42E-04	0.65 (0.34, 0.97)	5.20E-05	1.42E-04

Estimates (β) and P values for the comparison of 189 participants (74 mt-T2D and 115 ndt-T2D) were calculated using linear regression analysis with the crude and full adjustments. Because of missing confounding information, the models with full adjustment were based on fewer participants. Significant metabolites are highlighted in boldface type with respect to Bonferroni correction (P < 0.05/353 = 1.42E-04) or the FDR.
¶After exclusion of individuals because of missing confounding information.

Table 4

Citrulline remains significantly associated with metformin treatment in human serum in a longitudinal analysis (KORA S4 → F4)

	Crude GEE model mt-T2D (n = 37) vs. nonmetformin-treated (n = 646) participants^§			Full GEE model mt-T2D (n = 33)^¶ vs. nonmetformin-treated (n = 629)^¶ participants^§
Metabolite	β (95% CI) per SD	P value	FDR	β (95% CI) per SD	P value	FDR
Citrulline	−0.67 (−0.98, −0.36)	2.03E-05	1.76E-03	−0.61 (−0.94, −0.28)	2.96E-04	3.21E-04
X-21365	0.41 (0.12, 0.69)	5.62E-03	0.011	0.14 (−0.17, 0.45)	0.374	0.024

GEE model with crude and full adjustment was used to assess the associations between metformin treatment and metabolite serum values in the longitudinal study of 683 participants with no antidiabetes medical treatment at KORA S4. Of these participants, 37 started metformin treatment after KORA S4. Because of missing confounding information, the models with full adjustment were based on fewer participants. Significant metabolites are highlighted in boldface type with respect to Bonferroni correction (P < 0.05/2 = 0.025) and the FDR.
§Includes participants with NGT, isolated IFG, IGT, and ntd-T2D.
¶After exclusion of individuals because of missing confounding information.

Table 5

Metabolites significantly associated with metformin treatment in mouse models

Metabolite	In plasma		In liver		In skeletal muscle		In adipose tissue
Metabolite	β (95% CI) per SD	P value	β (95% CI) per SD	P value	β (95% CI) per SD	P value	β (95% CI) per SD	P value
Citrulline	−0.39 (−0.49, −0.28)	2.56E-07	−0.02 (−0.06, 0.02)	0.258	−0.35 (−0.41, −0.28)	1.79E-09	−0.26 (−0.33, −0.19)	4.52E-07
X-21365	−0.08 (−0.24, 0.08)	0.311	−0.12 (−0.34, 0.11)	0.295	−0.12 (−0.27, 0.04)	0.126	−0.23 (−0.46, 0.01)	0.063

Estimates (β) and P values for the comparison between metformin-treated (n = 10, in skeletal muscle [n = 9]) and nontreated mice (n = 10) sacrificed at 4 h after the last treatment. Significant metabolites are highlighted in boldface type (P < 0.05).