Plasma and urine metabolic profiles are reflective of altered beta-oxidation in non-diabetic obese subjects and patients with type 2 diabetes mellitus

Abstract

The two primary pathophysiological characteristics of patients with type 2 diabetes mellitus (T2DM) are insulin resistance (IR) and beta cell dysfunction. It has been proposed that the development of IR is secondary to the accumulation of triacylglycerols and fatty acids in the muscle and liver, which is in turn thought to be secondary to an enzymatic defect in mitochondrial beta-oxidation. The purpose of the present study was to analyze the molecules of intermediary metabolism to determine if an alteration in mitochondrial function exists in T2DM patients and, if so, to determine whether this alteration is caused by excess nutrients or an enzymatic defect.
Design and Methods: Seventy-seven subjects were recruited and divided into four groups (21 T2DM patients, 17 non-diabetic overweight/obese subjects, 20 offspring of T2DM patients, and 19 healthy subjects). Anthropometric parameters were determined by air plethysmography, and biochemical and metabolic parameters were measured, including 31 acylcarnitines (ACs) and 13 amino acids quantified by MS/MS and 67 organic acids measured by GC/MS.
Results: Patients with T2DM showed elevation of short-chain ACs (C2, C4), a glycogenic amino acid (valine), a glycogenic and ketogenic amino acid (tyrosine), and a ketogenic amino acid (leucine) as well as altered excretion of dicarboxylic acids. T2DM offspring with abnormal glucose tolerance test GTT showed increased levels of C16. Subjects in the obese group who were dysglycemic also showed altered urinary excretion of dicarboxylic acids and lower levels of a long-chain AC (C14:2).
Conclusions: These results suggest that mitochondrial beta-oxidation is altered in T2DM patients and that the alteration is most likely caused by nutrient overload through a different pathway from that observed in obese subjects.

Full text

Plasma and urine metabolic profiles are reflective of
altered beta-oxidation in non-diabetic obese subjects
and patients with type 2 diabetes mellitus
Jesús Zacarías Villarreal-Pérez1
Email: zacvilla@yahoo.com.mx
Jesús Zacarías Villarreal-Martínez2
Email: chuyzacarias@hotmail.com
Fernando Javier Lavalle-González1
Email: drfernandolavalle@hotmail.com
María del Rosario Torres-Sepúlveda3
Email: qcbrtorres@live.com.mx
Consuelo Ruiz-Herrera3
Email: c_ruiz99@yahoo.com.mx
Ricardo Martín Cerda-Flores4
Email: ricardocerda_mx@yahoo.com.mx
Erick Rubén Castillo-García3
Email: qcberick_gen@hotmail.com
Irám Pablo Rodríguez-Sánchez3
Email: iramrodriguez@gmail.com
Laura Elia Martínez deVillarreal3*
* Corresponding author
Email: laelmar@yahoo.com.mx
1 Universidad Autónoma de Nuevo León, Hospital Universitario, Dr. José
Eleuterio González”, Servicio de Endocrinología, Monterrey, Nuevo León 64460,
México
2 Departamento de Medicina Interna, Universidad Autónoma de Nuevo León,
Hospital Universitario, Dr. José Eleuterio González”, Monterrey, Nuevo León
64460, México
3 Departamento de Genética, Universidad Autónoma de Nuevo León, Hospital
Universitario, Dr. José Eleuterio González”, Av. Gonzalitos s/n, Colonia Mitras
Centro, Monterrey, Nuevo León 64460, México
4 Universidad Autónoma de Nuevo León, Facultad de Enfermería, Avenida
Gonzalitos, 1500 Norte, Col. Mitras Centro, Monterrey, NL, México

Abstract
Objectives
The two primary pathophysiological characteristics of patients with type 2 diabetes mellitus
(T2DM) are insulin resistance (IR) and beta cell dysfunction. It has been proposed that the
development of IR is secondary to the accumulation of triacylglycerols and fatty acids in the
muscle and liver, which is in turn thought to be secondary to an enzymatic defect in
mitochondrial beta-oxidation. The purpose of the present study was to analyze the molecules
of intermediary metabolism to determine if an alteration in mitochondrial function exists in
T2DM patients and, if so, to determine whether this alteration is caused by excess nutrients or
an enzymatic defect
Design and methods
Seventy-seven subjects were recruited and divided into four groups (21 T2DM patients, 17
non-diabetic overweight/obese subjects, 20 offspring of T2DM patients, and 19 healthy
subjects). Anthropometric parameters were determined by air plethysmography, and
biochemical and metabolic parameters were measured, including 31 acylcarnitines (ACs) and
13 amino acids quantified by MS/MS and 67 organic acids measured by GC/MS
Results
Patients with T2DM showed elevation of short-chain ACs (C2, C4), a glycogenic amino acid
(valine), a glycogenic and ketogenic amino acid (tyrosine), and a ketogenic amino acid
(leucine) as well as altered excretion of dicarboxylic acids. T2DM offspring with abnormal
glucose tolerance test GTT showed increased levels of C16. Subjects in the obese group who
were dysglycemic also showed altered urinary excretion of dicarboxylic acids and lower
levels of a long-chain AC (C14:2)
Conclusions
These results suggest that mitochondrial beta-oxidation is altered in T2DM patients and that
the alteration is most likely caused by nutrient overload through a different pathway from that
observed in obese subjects.
Keywords
Acetylcarnitine, Butyrylcarnitine, T2DM, Beta-oxidation defect, Non-Diabetic Obese, Air
plethysmography
Introduction
Type 2 diabetes mellitus (T2DM) and obesity are two deleterious metabolic conditions [1]
whose incidence has increased worldwide in the last decade [2]. Both diet and sedentary
lifestyle are important risk factors for their development [3]. In Mexico, 74% of the adult
population is either overweight or obese, and 14.6% suffer from T2DM [4], which has a high

morbidity and mortality [2]. The primary pathophysiological characteristics in T2DM are
insulin resistance (IR) and beta-cell dysfunction [5,6]. IR is considered to be a state in which
peripheral tissues are rendered unresponsive to the glucose lowering, antilypolytic, and
anabolic properties of insulin, which is a hallmark of obesity and T2DM. IR is also accepted
as an early feature of T2DM because it typically appears one or two decades before the
manifestation of clinically overt diabetes [6].
Several studies suggest that IR occurs secondary to the accumulation of triacylglycerols and
fatty acids in the muscle and liver (lipotoxicity theory) [6]. Although the molecular
pathogenesis of lipotoxicity is not clear, it has been proposed that it occurs secondary to
altered mitochondrial function, resulting from a decline in beta-oxidation, an excess of non-
esterified fatty acids arriving at the mitochondria, or both [7]. Several reports indicate that
there is an increased rate of beta-oxidation in obese individuals as well as in T2DM patients
in a feeding state, whereas the rate of beta-oxidation is reduced during fasting [8]. Although
the mechanisms of this reduction are unknown, it has been proposed that inhibition of
carnitine palmitoyltransferase-1 (CPT1) as a result of increasing levels of malonyl coenzyme
A (malonyl CoA) could be responsible for the decreased beta-oxidation activity [9]. Recent
studies have suggested that lipid accumulation results from a lower oxidative capacity of the
mitochondria [7] or reduced activity of the tricarboxylic acid cycle [6]. Another possible
explanation for this reduction in beta-oxidation is an excessive increase in the delivery of
fatty acids to the mitochondria [10].
Defects in the beta-oxidation of fatty acids can be evaluated based on acylcarnitine (AC)
levels measured by tandem mass spectrometry, which is widely used in neonatal screening
for fatty acid oxidation disorders and organic acidemias [11,12].
This methodology has also been used to analyze mitochondrial function in diabetic patients.
Recent reports indicate that there is an increase in the levels of long-, medium-, and short-
chain ACs in the blood of patients with T2DM and an elevation of long-chain ACs in obese
subjects [13].
We designed this study to quantify intermediate metabolites in plasma and urine to determine
if they reflect a beta-oxidation defect in in patients with T2DM and obese individuals and to
determine whether an excess of nutrients or a blockage of beta-oxidation is the cause of the
alteration. Additionally, we postulated that if there is an alteration in mitochondrial function
in T2DM patients, it should be present before T2DM presents; for this reason, we explored
beta-oxidation in the offspring of T2DM patients.
Materials and methods
Population
We performed a descriptive, comparative, non-blinded study that included 77 subjects (21
T2DM patients, 17 non-diabetic overweight/obese subjects, 20 non-diabetic offspring of
T2DM patients [the latter two groups have a higher risk of developing T2DM], and 19
healthy subjects). Subjects with diabetes were recruited during 2010 from the outpatient
diabetes clinic of the Endocrinology Service of the Dr. José E González University Hospital,
Universidad Autónoma de Nuevo León (UANL), Monterrey, México. Volunteer participants
were recruited from the University Hospital and Medical School population, either as

subjects at risk for developing T2DM (overweight/obese and offspring of T2DM patients) or
as controls. Written informed consent was obtained from all subjects, and the Health
Research Ethics Board of the UANL Medical School approved the study (Approval #: EN-
10-030).
Men and women over 18 years of age were included. Participants were assigned to one of
four groups: 1) patients with T2DM diagnosed according to the criteria established by the
American Diabetes Association; 2) non-diabetic subjects considered to be overweight/obese
with a body mass index (BMI) ≥25 kg/m2 and without a history of T2DM in a first-degree
relative; 3) non-diabetic individuals who had at least one parent diagnosed with T2DM; and
4) healthy individuals with a normal BMI (>20 and <25 kg/m2), a normal oral glucose
tolerance test (OGTT), and no history of T2DM. The group of healthy subjects was
designated as the control group. T2DM patients were required to discontinue medication (oral
hypoglycemic drugs) and should not have received any insulin the night before the study.
Anthropometric, biochemical, and metabolic parameters
Body composition was obtained by air impedance plethysmography (BOD POD). For the
biochemical parameters, 30 mL of venous blood was collected after a 12- to 14-hour fast.
Glucose levels, free fatty acids (FFAs), insulin, transaminases (AST, ALT), uric acid, urea
nitrogen, cholesterol, and alkaline phosphatase as well as a lipid profile were examined in
serum or plasma, depending on the kit used. The Homeostasis Model Assessment (HOMA)
and Matsuda indices were calculated [14].
Blood samples were collected from all subjects to measure the levels of 31 ACs, 13 amino
acids, pyruvate, lactate, and ketone bodies. An OGTT (standardized fasting) was performed
in all groups except for the T2DM group. For AC quantification, blood samples were
collected on filter paper (SS903) and analyzed by tandem mass spectrometry (MS/MS, API
2000, Perkin Elmer Sciex; full-scan, multiple-scan monitoring (MRM)).
A urine sample was collected for the measurement of 67 organic acids with a gas
chromatograph/mass spectrometer (CLARUS 500, Perkin-Elmer Corporation, Norwalk, CT,
USA). The procedure for the extraction of organic acids consisted of calculating the sample
volume, then adjusting the volume according to creatinine excretion, which should be twice
the volume containing 0.06 mg/100 g creatinine. Urinary organic acids were determined by
oximation. Extraction was performed with ethyl acetate. Derivatization was performed by
adding BSTFA-1% TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide with 1%
trimethylchlorosilane) and heating to 60°C in a water bath. Following this, the extract in
solution was injected into the gas chromatograph. Finally, spectral analysis and identification
were performed using the NIST MS Search Program Version 2.0.
Statistical analysis
Each study group was compared with the control group. For quantitative parameters,
Student´s t-test was used, whereas Fisher´s exact test was used for qualitative measurements.
A P <0.05 was defined as significant. IBM SPSS 20 statistical software (IBM Corporation,
Somers, NY) was used for data analysis.

Results
In total, 111 biochemical and metabolic parameters, including ACs, amino acids, and organic
acids, were measured in all groups. Comparison of the study groups with the control group
showed that in the three groups of cases, the average BMI, anthropometric parameters
obtained by BOD POD, and ages were higher.
Tables 1, 2, and 3 provide the biochemical parameters and metabolites that displayed
significant differences between the case groups and the control group. Regarding biochemical
parameters, subjects with T2DM had basal glucose, insulin levels, and ketone bodies levels in
blood and a HOMA index that were significantly higher than those of healthy controls.
Triacylglycerol, acetylcarnitine (C2), butyrylcarnitine (C4), alanine, tyrosine, and the
branched chain amino acids leucine and valine (Table 1) were also significantly elevated in
this group.
Table 1 Comparison of anthropometric and biochemical measurements in T2DM
patients and the control group
T2DM patients
Control (n =19) (X + SD)
P value
(n =21) (X + SD)
Age
52 ± 11.1
24.3 ± 3.7
<0.005
BMI
32.4 ± 6.0
22.8 ± 1.4
<0.005
Waist cm
103.3 ± 13.8
70.0 ± 7.7
<0.005
Hip cm
109.0 ± 13.3
95.0 ± 7.7
<0.005
% Fat
42.0 ± 8.9
25.6 ± 9.8
<0.005
% Lean mass
58.0 ± 9.0
74.3 ± 9.9
<0.005
Total weight
84.2 ± 17.1
63.1 ± 9.2
<0.005
Glucose 0´
148.2 ± 51.4
83.9 ± 12.5
<0.005
Glucose 30´
ND
ND
ND
Glucose 60´
ND
ND
ND
Glucose 90´
ND
ND
ND
Glucose 120´
ND
ND
ND
Insulin 0´
15.6 ± 7.9
7.3 ± 2.0
<0.005
HOMA IR
5.4 ± 2.7
1.5 ± 0.44
<0.005
Free fatty acids
0.6 ± 0.2
0.5 ± 0.3
>0.05
Beta-hydroxybutyrate
0.2 ± 0.1
0.2 ± 0.04
>0.05
ALP
63 ± 17
62.10 ± 16.9
>0.05
Uric Acid
5.8 ± 1.5
5.2 ± 1.0
>0.05
Serum creatinine
0.7 ± 0.17
0.8 ± 0.15
>0.05
Cholesterol
202.5 ± 50.7
200.6 ± 39.6
>0.05
Total bilirubin
0.2 ± 0.10
0.3 ± 0.2
>0.05
Direct bilirubin
.05 ± .02
0.06 ± 0.03
>0.05
Indirect Bilirubin
0.15 ± 0.1
0.2 ± 0.1
>0.05
Total protein
8.1 ± 0 .7
8.5 ± 0.9
>0.05
Albumin
4.7 ± 0.5
5.13 ± 0.5
<0.05
Globulin
3.4 ± 0.4
3.4 ± 0.5
>0.05
AST
17 ± 11.5
10.4 ± 2.0
<0.05

ALT
14.5 ± 10.1
8.0 ± 2.6
<0.005
C4
0.24 ± 0.10
0.18 + 0.08
<0.05
C2
10.1 ± 2.2
8.7 ± 1.6
<0.005
Leu
117.7 ± 22.3
96.1 ± 23.7
<0.05
Tyr
51.3 ± 10.4
41.5 ± 11.0
<0.005
Val
165.2 ± 19.0
135.0 ± 31.0
<0.005
Gly
242.0 ± 36.2
226.5 ± 47.0
>0.05
Arg
27.5 ± 9.8
27.4 ± 7.8
>0.05
Cit
18.4 ± 4.7
17.5 ± 4.2
>0.05
Met
21.7 ± 5.1
22.4 ± 5.2
>0.05
Orn
83.7 ± 13.8
76.6 ± 9.6
>0.05
Phe
41.2 ± 6.2
39.3 ± 10.8
>0.05
Ala
288.8 ± 63.0
238.0 ± 44.2
<0.005
Table 2 Comparison of anthropometric and biochemical measurements in the
overweight/obese patient group and the control group
Overweight/obesity
Control (n =19) (X + SD)
P value
(n =17) (X + SD)
Age
39.2 ± 14.0
24.3 ± 3.7
<0.005
BMI
30.2 ± 7.4
22.8 ± 1.4
<0.005
Waist cm
99.1 ± 17.6
70.0 ± 7.7
<0.005
Hip cm
111.3 ± 11.8
95.0 ± 7.7
<0.005
% Fat
37.3 ± 11.4
25.6 ± 9.8
<0.005
% Lean mass
62.7 ± 11.4
74.3 ± 9.9
<0.005
30.6 ± 12.1
18.1 ± 11.7
<0.005
Total weight
84.1 ± 17.3
63.1 ± 9.2
<0.005
Glucose O´
90.5 ± 11.9
83.9 ± 12.5
>0.05
Glucose 30´
144.2 ± 34.6
131 ± 30.9
>0.05
Glucose 60´
126.5 ± 53.5
113.9 ± 25.3
>0.05
Glucose 90´
120.5 ± 48.8
109.27 ± 33.33
>0.05
Glucose 120´
107.6 ± 46.4
94.9 ± 21.7
>0.05
Insulin 0´
10.7 ± 7.0
7.3 ± 2.0
>0.05
Insulin 30´
81.4 ± 51.5
56.7 ± 24.6
>0.05
Insulin 60´
89.9 ± 92.4
57.9 ± 36.2
>0.05
Insulin 90´
72.9 ± 63.3
54.8 ± 35.8
>0.05
Insulin 120´
76.3 ± 69.67
41.4 ± 37.0
>0.05
Matsuda index
4.9 ± 2.5
6.2 ± 1.8
>0.05
HOMA IR
2.3 ± 1.3
1.5 ± 0.44
<0.05
Free Fatty Acids
0.5 ± 0.20
0.5 ± 0.3
>0.05
Beta-hydroxybutyrate
0.2 ± 0.05
0.2 ± 0.3
>0.05
ALP
57.9 ± 13.1
62.16 + 16.9
>0.05
Uric acid
6.3 ± 1.4
5.2 ± 1.0
<0.05
Serum creatinine
0.9 ± 0.3
0.8 ± 0.15
>0.05
Cholesterol
214.0 ± 66.8
200.6 ± 39.6
>0.05
Total bilirubin
0.4 ± 0.3
0.3 ± 0.2
>0.05

Direct bilirubin
0.05 ± 0.03
0.06 ± 0.03
>0.05
Indirect bilirubin
0.3 ± 0.26
0.2 ± 0.18
>0.05
Total protein
8.2 ± 1.07
8.5 ± 0.9
>0.05
Albumin
4.9 ± 0.4
5.1 ± 0.5
>0.05
Globulin
3.3 ± 0.62
3.4 ± 0.5
>0.05
AST
16.1 ± 9.3
10.4 ± 2.0
<0.05
ALT
10.4 ± 3.3
8.2 ± 2.6
<0.05
C4
0.19 ± 0.11
0.18 ± 0.08
>0.05
C2
8.5 ± 2.3
8.7 ± 1.6
>0.05
Leu
95.3 ± 28.0
103.8 ± 21.5
>0.05
Tyr
43.4 ± 10.8
48.2 ± 10.5
>0.05
Val
126.1 ± 41.7
138.8 ± 34.2
>0.05
Gly
210.8 ± 54.1
220.6 ± 38.4
>0.05
Arg
24.3 ± 10.5
24.1 ± 6.25
>0.05
Cit
17.7 ± 4.1
17.9 ± 6.25
>0.05
Met
21.26 ± 5.7
23.5 ± 6.25
>0.05
Orn
85.5 ± 23.4
80.2 ± 15.0
>0.05
Phe
38.2 ± 8.5
41.1 ± 8.3
>0.05
Ala
209.9 ± 52.4
238. ± 45.2
>0.05
Table 3 Comparison of anthropometric and biochemical measurements in the T2DM
patient offspring group and the control group
T2DM patient’s offspring
Control (n =19) (X + SD)
P value
(n =20) (X + SD)
Age
37.15 ± 13.4
24.3 ± 3.7
<0.005
BMI
29.3 ± 6.0
22.8 ± 1.4
<0.001
Waist cm
95.3 ± 17.1
70.0 ± 7.7
<0.0005
Hip cm
106.0 + 17.7
95.0 ± 7.7
<0.005
% Fat
32.5 ± 11.2
25.6 ± 9.8
<0.05
% Lean Mass
67.5 ± 11.2
74.3 ± 9.9
<0.05
Total Weight
82.54 ± 21.67
63.1 ± 9.2
<0.0005
Glucose O
90.25 ± 11.47
83.9 ± 12.5
>0.05
Glucose 30´
140.7 ± 32.76
131 ± 30.9
>0.05
Glucose 60´
141.2 ± 40.0
113.9 ± 25.3
<0.05
Glucose 90´
128.5 ± 46.46
109.27 ± 33.33
>0.05
Glucose 120
120.05 ± 33.21
94.9 ± 21.7
<0.005
Insulin 0´
11.1 ± 4.9
7.3 ± 2.0
<0.0005
Insulin 30´
80.7 ± 49.8
56.7 ± 24.6
<0.05
Insulin 60´
87.7 ± 58.3
57.9 ± 36.2
<0.05
Insulin 90´
79.4 ± 57.2
54.8 ± 35.8
<0.05
Insulin 120´
73.2 ± 49.6
41.4 ± 37.0
<0.0005
Free fatty acids
0.62 ± 0.24
0.5 ± 0.3
>0.05
Beta-hydroxybutyrate
0.18 ± 0.10
0.2 ± 0.3
>0.05
ALP
58.5 + 20.17
58.6 ± 20.2
>0.05
Uric acid
6.21 ± 1.33
5.2 ± 1.0
<0.05

Serum creatinine
0.91 ± 0.17
0.8 + 0.15
>0.05
Cholesterol
198.65 ± 28.4
200.6 ± 39.6
>0.05
Total bilirubin
0.34 ± 0.21
0.3 ± 0.2
>0.05
Direct bilirubin
0.05 ± 0.04
0.06 ± 0.03
>0.05
Indirect bilirubin
0.28 ± 0.21
0.2 ± 0.18
>0.05
Total protein
8.38 ± 0.80
8.4 ± 0.9
>0.05
Albumin
4.98 ± 0.39
5.11 ± 0.5
>0.05
Globulin
3.39 ± 0.55
3.4 ± 0.5
>0.05
AST
14.85 ± 8.64
10.4 ± 2.0
<0.05
ALT
13.7 ± 8.11
8.2 ± 2.6
<0.0005
Triacylglycerol
154.2 ± 114.9
110.6 ± 71.2
>0.05
C4
0.17 ± 0.08
0.18 ± 0.08
>0.05
C2
9.14 ± 1.44
8.7 ± 1.6
>0.05
Leu
103.8 ± 21.52
96.1 ± 23.7
>0.05
Tyr
48.2 ± 10.5
41.5 ± 11.0
>0.05
Val
138.8 ± 34.2
135.0 ± 31.0
>0.05
Gly
220.6 ± 38.4
226.5 ± 47
>0.05
Arg
24.1 ± 7.3
27.4 ± 7.84
>0.05
Cit
17.9 ± 3.09
17.5 ± 4.2
>0.05
Met
23.5 ± 6.25
22.4 ± 5.2
>0.05
Orn
80.2 ± 15.0
76.6 ± 9.5
>0.05
Phe
41.1 ± 8.3
39.3 ± 10.8
>0.05
Ala
231.0 ± 45.23
238.0 ± 44.2
>0.05
In addition to differences in anthropometric measurements, non-diabetic overweight/obese
subjects only had an increased HOMA index; we did not observe elevations of
triacylglycerols, amino acids, or ACs (Table 2). In this group, six (35.3%) patients with pre-
diabetes (basal glucose =101 - 125 mg/dl and/or OGTT 120 min =141 - 199 mg/dl) showed a
decrease in the level of C14:2 (tetradecenoyl carnitine) in addition to glucose elevations at
30, 60, 90, and 120 min during the OGTT and elevation of insulin levels at 120 min
compared with the control group (Table 4).
Table 4 Comparison of anthropometric and biochemical measurements in the
dysglycemic patient group and the control group
Dysglycemic/obese
(n = 6 ) (X ± SD)
Dysglycemic/ offspring
Control
(n = 5 )
(n =19)
(X ± SD)
(X ± SD)
Age
53.0 ± 7.66 *
46.6 ± 12.0*
24.3 ± 3.7
BMI
33.2 ± 8.28*
33.8 ± 6.7*
22.8 ± 1.4
Waist cm
99.9 ± 14.7*
109.0 ± 20.9*
70 ± 7.7
Hip cm
107.2 ± 8.7*
116.2 ± 24.5
95 ± 7.7
% Lean mass
66.8 ± 7.9
62.8 ± 15.1
74.3 ± 9.9
Total weight
87.2 ± 21.8*
95.8 ± 26.0*
63.1 ± 9.2
Glucose O´
97.2 ± 16.5
100.6 ± 8.8*
83.9 ± 12.5
Glucose 30´
169.7 ± 35.3*
177 ± 39.91
131 ± 30.9

Glucose 60´
182.8 ± 55.0*
196.2 ± 23.8*
113.9 ± 25.3
Glucose 90´
171.0 ± 42.8*
198.0 ± 38.5*
109.27 ± 33.33
Glucose 120´
150.0 ± 45.9*
165.0 ± 20.92*
94.9 ± 21.7
Insulin 0´
16.0 ± 9.6
12.6 ± 5.6
7.3 ± 2.0
Insulin 30´
87.4 ± 42.6
98.7 ± 54.3
56.7 ± 24.6
Insulin 60´
153.2 ± 135.6
126.2 ± 56.4*
57.9 ± 36.2
Insulin 90´
130.3 ± 77.3
131.7 ± 59.0*
54.8 ± 35.8
Insulin 120´
132.2 ± 84.1*
109.1 ± 46.0*
41.4 ± 37.0
Matsuda Index
2.9 ± 2.1*
2.4 ± 1.0*
6.2 ± 1.8
HOMA IR
3.5 ± 1.5*
3.2 ± 1.5
1.5 ± 0.4
Free Fatty Acids
0.5 ± 0.3
0.7 ± 0.2
0.5 ± 0.3
Uric Acid
6.4 ± 1.4
7.12 ± 0.6*
5.2 ± 1.0
Serum Creatinine
0.9 ± 0.3
0.9 ± 0.2
0.8 ± 0.15
Cholesterol
234.5 ± 22.7*
180.8 ± 21.5
200.6 ± 39.6
AST
16.5 ± 7.6
18.2 ± 10.8
10.4 ± 2.0
ALT
12.0 ± 3.2*
19.4 ± 9.9
8.2 ± 2.6
Triacylglycerol
204.8 ± 80.0*
283.6 ± 177.3
110.6 ± 71.2
C4
0.18 ± 0.08
0.25 ± 0.13
0.18 ± 0.08
C2
8.4 ± 1.6
9.8 ± 1.05
8.7 ± 1.6
C16
0.74 ± 0.17
0.85 ± 0.14*
0.67 ± 0.17
C 14: 2
0.03 ± 0.02*
0.05 ± 0.03
0.06 ± 0.3
* P value: <0.05.
The non-diabetic offspring of T2DM patients showed higher blood glucose levels during the
OGTT at 60 and 120 min as well as increased insulin levels at 0, 60, 90, and 120 min relative
to the control group (Table 3). Additionally, the Matsuda and HOMA indices were lower and
higher, respectively, as compared to control group (Table 3). In this group, 14 (66%) patients
were overweight/obese, and 5 (25%) were pre-diabetic. The pre-diabetic offspring exhibited a
significant elevation of palmitoylcarnitine (C16) and decreased glycine as well as significant
differences in anthropometric measurements, insulin at time 0, and the HOMA index relative
to the control group (Table 4).
All case groups demonstrated a significant elevation of transaminases (AST and ALT),
although uric acid was only increased in the overweight/obese and offspring groups.
Analysis of the organic acids in urine showed the presence of intermediate metabolites of
glycolysis and the Krebs cycle including lactic, 3OH-butyric, succinic, adipic, palmitic, citric,
and phenyl acetic acids in all subjects in all four groups. Some metabolites were detected in a
limited number of study participants. A significantly higher proportion of subjects with
T2DM excreted 2OH-butyric acid relative to the control group (90% vs. 20%, P <0.05), and
none exhibited sebacic acid excretion, compared with 40% in the control group (P <0.05). A
lower number of obese subjects excreted suberic acid relative to the control group (36% vs.
89%, P <0.05). There were no differences in the excretion of organic acids between the
offspring of T2DM patients and the control group.

Discussion
The present study was conducted to obtain more information regarding biochemical and
metabolic parameters in T2DM patients and to compare these parameters with healthy
subjects to determine whether altered mitochondrial beta-oxidation, secondary to either an
overload of nutrients or an enzymatic defect, exists in these patients. Additionally, the study
examined whether subjects at risk for developing T2DM present metabolic alterations prior to
developing the disease.
In the present study all the anthropometric parameters (i.e., BMI, waist, %fat) were
significantly higher in the individuals of the case groups when comparing against the control
group. However, only the offspring of T2DM patients showed altered plasma glucose and
insulin levels during the OGTT; obese subjects did not.
In the biochemical measurements, transaminases (AST, ALT) were significantly higher in all
case groups than in controls. It has been reported that individuals with T2DM have a higher
incidence of liver function test abnormalities than individuals who do not have diabetes.
Additionally, mild chronic elevations of transaminases often reflect underlying insulin
resistance [15].
The elevation of transaminases in diabetics, overweight/obese individuals, and offspring of
diabetic patients found in the present study may reflect fatty acid accumulation in the liver
[16], although FFAs were not significantly elevated. It has been previously reported that
elevated ALT in non-diabetic Swedish men is a risk factor for T2DM, independent of obesity,
body fat distribution, plasma glucose, lipid, AST, bilirubin concentration, and family history
of diabetes. In another study, non-diabetic Pima Indians were followed for an average of 6.9
years to determine whether hepatic enzyme elevations could be linked to the development of
T2DM. At baseline, ALT, AST, and the OGTT were related to percent body fat. After
adjusting for age, sex, body fat, whole body insulin sensitivity, and acute insulin response,
only elevated ALT at baseline was associated with an increase in hepatic glucose output.
Prospectively, increasing ALT concentrations were associated with a decline in hepatic
insulin sensitivity and risk of T2DM. The authors concluded that higher ALT is a risk factor
for T2DM and indicates a potential role of increased hepatic gluconeogenesis and/or
inflammation in its pathogenesis [15]. Our results are in agreement with the aforementioned
studies because only subjects with dysglycemia showed increased ALT in the subgroup
analysis of the overweight/obese and offspring groups. As expected T2DM patients, showed
high plasma triacylglycerol concentration, as well as disglycemic subjects from the obese and
offspring groups..
It has been reported that an overload of mitochondrial lipid oxidation results in the
accumulation of β-oxidation intermediates (ACs) and the depletion of Krebs cycle
intermediates, leading to mitochondrial stress and activating currently unknown signaling
pathways that interfere with insulin action [6]. Reports regarding the elevation of β-oxidation
intermediates in diabetics are controversial. Shure et al. reported elevation of long-chain ACs
and decreased levels of short-chain ACs [17], whereas Adams et al. and Mihalik et al.
reported elevation of short-, medium- and long-chain ACs in diabetic patients [13,18]. In a
study with streptozotocin-induced diabetic rats, short-chain ACs (C2 and C4) was
significantly elevated In a recent study, considerably higher levels of short-chain ACs and

lower levels of some long-chain ACs were detected in T2DM patients and patients with
metabolic syndrome [19].
In the present study, T2DM patients showed elevated levels of short-chain ACs (C2 and C4)
in the blood as compared to healthy controls. Disglycemic offspring showed elevation of a
long-chain AC (C:16). Notably, in contrast to these groups, dysglycemic obese patients had
lower levels of a specific long-chain AC (C14:2), which suggests the involvement of the
same metabolic systems in a different manner, as postulated by Bene et al. 2013 [19].
Acetylcarnitine (C2) is the final metabolite of the beta-oxidation pathway, which produces
acetyl CoA as a substrate for the Krebs cycle, whereas butyrylcarnitine (C4) is a marker of
ketogenesis and mitochondrial beta-oxidation. C4 levels reflect the concentration of tissue
butyryl CoA, which is a metabolite of glutamate and alpha-ketoglutarate, both of which are
intermediate metabolites of the Krebs cycle [20]. These findings exclusively in the T2DM
group would indicate an increase in mitochondrial beta-oxidation [21].
We believe that the differences in the levels of ACs found in the aforementioned studies, may
be the result of the conditions or characteristics of the patients at the time of the study. In a
previous study (unpublished results), we observed elevations of short-, medium-, and long-
chain ACs in individuals with diabetes; however, in that study, patients had overt diabetes
and were naive to treatment, which is in contrast to the patients in the present study, who
were under treatment, and the differences among the reported studies could be related to the
time of diagnosis, drug use, or BMI.
The AC profile is used in neonatal screening as an early marker of fatty acid disorders such
as beta-oxidation enzymatic defects. The results of the present study do not support the
hypothesis that there is an enzymatic defect in beta-oxidation that leads to fatty acid
accumulation and precludes IR because the earlier manifestations of beta-oxidation overload
were observed in the offspring of T2DM patients, who were already pre-diabetic. The pre-
diabetic offspring showed significant elevation of long-chain fatty acid (C16) concentrations,
whereas euglycemic offspring only showed a higher HOMA index relative to the control
group, which suggests that some insulin resistance was already present. However, the
elevation of C16 could be an early marker for the risk of developing diabetes, as previously
reported by Zhao et al. [22].
T2DM encompasses not only changes in glucose metabolism but also alterations in fatty acid
and protein levels with subsequent metabolic alterations in the pathways involved [23]. The
branched-chain amino acids (BCAAs) leucine and valine are glycogenic and modulators of
insulin secretion. According to Wang et al. [17], hyperaminoacidemia can promote diabetes
via hyperinsulinemia.
In a previous report, Vannini P et al. reported that the BCAAs leucine and valine were
increased in diabetic patients, indicating impaired short-term metabolic control [24].
BCAAs contribute to glucose recycling via the glucose-alanine cycle. Under normal
conditions, alanine arising from BCAA nitrogen likely accounts for 25% of gluconeogenesis
from amino acids [25,26].
Elevation of BCAAs in IR adults independent of BMI in conjunction with increases in
plasma ACs derived from amino acid oxidation suggests an increase in amino acid flux [27].

In a recent report, leucine, valine, tyrosine, and phenylalanine were found to be significantly
associated with the incidence of diabetes [28]. In the present study, we observed a significant
elevation of leucine, valine, tyrosine, and alanine in diabetic patients, although in contrast to
the study of Wang, we did not find any association of the amino acid concentration pattern
with the prediction of diabetes. Subjects in our study who were at risk for developing diabetes
(overweight/obese and the offspring of T2DM subjects) did not show any increase in amino
acid levels. In a study with diabetic db-/db- mice, clear evidence of increased
gluconeogenesis was found, as demonstrated by strongly decreased concentrations of the
gluconeogenic amino acids alanine, glycine, and serine [15]. Although in the present study
we observed increased alanine in T2DM patients, dysglycemic obese subjects had lower
levels of glycine, perhaps as a sign of altered glucose metabolism.
Obesity and a family history of T2DM are highly associated with development of the disease.
The underlying mechanisms that trigger and exacerbate obesity-associated insulin resistance
and the transition to T2DM remain unclear [29]. It has been suggested that a chronic positive
energy balance and increased storage of energy as fat are linked to impaired glucose
homeostasis and the development of diabetes [29]. Obesity is caused by the excessive
accumulation of triacylglycerols, and its effects on the use and storage of various fuels
(glucose, fatty acids, and amino acids) result in abnormalities in metabolism [30]. In the
present study, several analytes such as transaminases, glucose, and insulin were elevated in
both the obese group and the offspring group, which suggests that these metabolic alterations
are already present in both groups. When we analyzed the pre-diabetic subjects, these
metabolic abnormalities were present, and markers of altered mitochondrial beta-oxidation
were also detected. We found a lower level of unsaturated long-chain fatty acids (C14:2) in
the obese group, which could indicate a reduction in long-chain fatty acid metabolism.
Increases in short-, medium-, and long-chain AC levels in diabetic individuals suggest a
different and more complex defect compared with that of obese subjects.
Organic acids exist as intermediate compounds in many biochemical pathways, (glycolysis,
glyconeogenesis, lipolysis). The Krebs cycle is the central metabolic pathway for energy
molecules, and deficiencies in any of the Krebs cycle enzymes can cause inefficient cycling
of organic acid intermediates, which consequently increase their concentration in the urine of
the affected individual. In our study, differential urinary excretion of dicarboxylic acids was
observed, whereas the excretion of adipic acid and 3OH-butyrate, which are indicators of
ketogenesis, was similar in all groups. Ketosis is secondary to the fasting state and is a
metabolic marker of fatty acid metabolism that is accompanied by excessive urinary
excretion of adipic (C6) and suberic acids (C8) [31]. In the present study, a significantly
lower proportion of obese subjects excreted suberic acid (C8) relative to the control group
(36% vs. 89%), and none of the T2DM patients excreted sebacic acid (C10). This result could
be related to a decrease in medium-chain fatty acids in T2DM patients, which has been
reported previously [17] .
The lack of sebacic acid (C10) excretion in diabetic subjects would indicate active lipid
metabolism, as this has been reported in association with starvation, fat feeding, or
experimental diabetes [32], as well as an elevated rate of beta-oxidation to form C6-C8 [33],
contrary to what we found in dysglycemic obese patients.
The increased urinary excretion of hydroxyisobutyric acid in T2DM patients, which reflects
an increase in serum C4 levels (derived from fatty acid oxidation or amino acid catabolism),
indicates an increased metabolism of fats or proteins.

Specific patterns were observed for each analyzed group, and patients with T2DM had an
abnormal AC pattern that suggests an increase in the substrate for mitochondrial beta-
oxidation. This alteration could be present at earlier stages of the disease because pre-diabetic
T2DM offspring showed increased levels of C16. The group of obese subjects also showed
altered urinary excretion of dicarboxylic acids and lower levels of the long-chain AC C14:2
when pre-diabetic, suggesting altered mitochondrial beta-oxidation. Our results are in
agreement with those reported by Koves, et al. (6), who suggested that there is a
mitochondrial substrate overload in T2DM patients.
Conclusion
Patients with T2DM exhibit defective beta-oxidation that suggests an overload of nutrients,
as shown by higher levels of TGL and elevation of acetylcarnitine and butyrylcarnitine (both
of which are derived from the final products of beta-oxidation) as well as augmented urinary
excretion of intermediate metabolites. This beta-oxidation defect could be present at earlier
stages of the disease because the pre-diabetic offspring of T2DM patients showed increased
levels of C16. Moreover, obese subjects also showed altered mitochondrial beta-oxidation as
well as altered urinary excretion of dicarboxylic acids and reduced levels of C14:2 only when
disglycemic, indicating differential involvement of the metabolic pathways.
Acknowledgements
The authors acknowledge the help of Michelle de J. Zamudio-Osuna, M.S., Brenda
Navarrete, M.D., and César Antonio Garza-Osorio, M.D. The authors also gratefully
acknowledge Sergio Lozano-Rodriguez, M.D., for his critical reading of the manuscript.
Funding
The work was performed using resources provided by each of the participating departments.
References
1. Galgani J, Diaz E: Obesity and fatty acids in the etiology of insulin resistance. Rev Med
Chil 2000, 128:1354–1360.
2. Aguilar-Salinas CA, Mehta R, Rojas R, Gomez-Perez FJ, Olaiz G, Rull JA: Management
of the metabolic syndrome as a strategy for preventing the macrovascular complications
of type 2 diabetes: controversial issues. Curr Diabetes Rev 2005, 1:145–158.
3. Hu FB: Sedentary lifestyle and risk of obesity and type 2 diabetes. Lipids 2003,
38:103–108.
4. Encuesta Nacional de Salud y Nutrición: Encuesta Nacional de Salud y Nutrición. In
2006. http://www.insp.mx/ensanut/ensanut2006.pdf.
5. Maassen JA, Romijn JA, Heine RJ: Fatty acid-induced mitochondrial uncoupling in
adipocytes as a key protective factor against insulin resistance and beta cell dysfunction:

a new concept in the pathogenesis of obesity-associated type 2 diabetes mellitus.
Diabetologia 2007, 50:2036–2041.
6. Koves TR, Ussher JR, Noland RC, Slentz D, Mosedale M, Ilkayeva O, Bain J, Stevens R,
Dyck JR, Newgard CB, et al: Mitochondrial overload and incomplete fatty acid oxidation
contribute to skeletal muscle insulin resistance. Cell Metab 2008, 7:45–56.
7. Lieber CS, Savolainen M: Ethanol and lipids. Alcohol Clin Exp Res 1984, 8:409–423.
8. Brands M, Verhoeven AJ, Serlie MJ: Role of mitochondrial function in insulin
resistance. Adv Exp Med Biol 2012, 942:215–234.
9. Hesselink MK C, Mensink M, Schrauwen P: Lipotoxicity and mitochondrial
dysfunction in type 2 diabetes. Immunol Endoc Metab Agents Med Chem 2007, 7:3–17.
10. Goodpaster BH, Wolf D: Skeletal muscle lipid accumulation in obesity, insulin
resistance, and type 2 diabetes. Pediatr Diabetes 2004, 5:219–226.
11. Zytkovicz TH, Fitzgerald EF, Marsden D, Larson CA, Shih VE, Johnson DM, Strauss
AW, Comeau AM, Eaton RB, Grady GF: Tandem mass spectrometric analysis for amino,
organic, and fatty acid disorders in newborn dried blood spots: a two-year summary
from the New England Newborn Screening Program. Clin Chem 2001, 47:1945–1955.
12. Spiekerkoetter U: Mitochondrial fatty acid oxidation disorders: clinical presentation
of long-chain fatty acid oxidation defects before and after newborn screening. J Inherit
Metab Dis 2010, 33:527–532.
13. Adams SH, Hoppel CL, Lok KH, Zhao L, Wong SW, Minkler PE, Hwang DH, Newman
JW, Garvey WT: Plasma acylcarnitine profiles suggest incomplete long-chain fatty acid
beta-oxidation and altered tricarboxylic acid cycle activity in type 2 diabetic African-
American women. J Nutr 2009, 139:1073–1081.
14. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC:
Homeostasis model assessment: insulin resistance and beta-cell function from fasting
plasma glucose and insulin concentrations in man. Diabetologia 1985, 28:412–419.
15. Harris EH: Elevated liver function tests in type 2 diabetes. Clin Diabetes 2005,
23:115–119.
16. Begriche K, Massart J, Robin MA, Bonnet F, Fromenty B: Mitochondrial adaptations
and dysfunctions in nonalcoholic fatty liver disease. Hepatology 2013, 58:1497–1507.
17. Suhre K, Meisinger C, Doring A, Altmaier E, Belcredi P, Gieger C, Chang D, Milburn
MV, Gall WE, Weinberger KM, et al: Metabolic footprint of diabetes: a multiplatform
metabolomics study in an epidemiological setting. PLoS One 2010, 5:e13953.
18. Mihalik SJ, Goodpaster BH, Kelley DE, Chace DH, Vockley J, Toledo FG, DeLany JP:
Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and
identification of a marker of glucolipotoxicity. Obesity (Silver Spring) 2010, 18:1695–
1700.

19. Bene J, Marton M, Mohas M, Bagosi Z, Bujtor Z, Oroszlan T, Gasztonyi B, Wittmann I,
Melegh B: Similarities in serum acylcarnitine patterns in type 1 and type 2 diabetes
mellitus and in metabolic syndrome. Ann Nutr Metab 2013, 62:80–85.
20. Fiehn O, Garvey WT, Newman JW, Lok KH, Hoppel CL, Adams SH: Plasma
metabolomic profiles reflective of glucose homeostasis in non-diabetic and type 2
diabetic obese African-American women. PLoS One 2010, 5:e15234.
21. Schooneman MG, Vaz FM, Houten SM, Soeters MR: Acylcarnitines: reflecting or
inflicting insulin resistance? Diabetes 2013, 62:1–8.
22. Zhao X, Fritsche J, Wang J, Chen J, Rittig K, Schmitt-Kopplin P, Fritsche A, Haring HU,
Schleicher ED, Xu G, Lehmann R: Metabonomic fingerprints of fasting plasma and spot
urine reveal human pre-diabetic metabolic traits. Metab 2010, 6:362–374.
23. Wurtz P, Makinen VP, Soininen P, Kangas AJ, Tukiainen T, Kettunen J, Savolainen MJ,
Tammelin T, Viikari JS, Ronnemaa T, et al: Metabolic signatures of insulin resistance in
7,098 young adults. Diabetes 2012, 61:1372–1380.
24. Vannini P, Marchesini G, Forlani G, Angiolini A, Ciavarella A, Zoli M, Pisi E:
Branched-chain amino acids and alanine as indices of the metabolic control in type 1
(insulin-dependent) and type 2 (non-insulin-dependent) diabetic patients. Diabetologia
1982, 22:217–219.
25. Ruderman NB: Muscle amino acid metabolism and gluconeogenesis. Annu Rev Med
1975, 26:245–258.
26. Layman DK, Walker DA: Potential importance of leucine in treatment of obesity and
the metabolic syndrome. J Nutr 2006, 136:319S–323S.
27. Huffman KM, Shah SH, Stevens RD, Bain JR, Muehlbauer M, Slentz CA, Tanner CJ,
Kuchibhatla M, Houmard JA, Newgard CB, Kraus WE: Relationships between circulating
metabolic intermediates and insulin action in overweight to obese, inactive men and
women. Diabetes Care 2009, 32:1678–1683.
28. Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, McCabe E, Lewis GD, Fox CS,
Jacques PF, Fernandez C, et al: Metabolite profiles and the risk of developing diabetes.
Nat Med 2011, 17:448–453.
29. Adams SH: Emerging perspectives on essential amino acid metabolism in obesity and
the insulin-resistant state. Adv Nutr 2011, 2:445–456.
30. Quiroga AD, Lehner R: Liver triacylglycerol lipases. Biochim Biophys Acta 1821,
2012:762–769.
31. Gregersen N, Kolvraa S, Mortensen PB, Rasmussen K: C6-C10-dicarboxylic aciduria:
biochemical considerations in relation to diagnosis of beta-oxidation defects. Scand J
Clin Lab Invest Suppl 1982, 161:15–27.

32. Mortensen PB: C6–C10-dicarboxylic aciduria in starved, fat-fed and diabetic rats
receiving decanoic acid or medium-chain triacylglycerol. An in vivo measure of the rate
of beta-oxidation of fatty acids. Biochim Biophys Acta 1981, 664:349–355.
33. Mortensen PB, Gregersen N: The biological origin of ketotic dicarboxylic aciduria. II.
In vivo and in vitro investigations of the beta-oxidation of C8-C16-dicarboxylic acids in
unstarved, starved and diabetic rats. Biochim Biophys Acta 1982, 710:477–484.