Amalia Gastaldelli,
1,2
Melania Gaggini,
2
and Ralph A. DeFronzo
1
Role of Adipose Tissue Insulin
Resistance in the Natural History
of Type 2 Diabetes: Results From the
San Antonio Metabolism Study
Diabetes 2017;66:815–822 | DOI: 10.2337/db16-1167
In the transition from normal glucose tolerance (NGT)
to type 2 diabetes mellitus (T2DM), the role of b-cell
dysfunction and peripheral insulin resistance (IR) is well
established. However, the impact of dysfunctional adi-
pose tissue has not been fully elucidated. The aim of this
study was to evaluate the role of resistance to the anti-
lipolytic effect of insulin (adipose tissue IR [Adipo-IR]) in
a large group of subjects with NGT, impaired glucose
tolerance (IGT), and T2DM. Three hundred two subjects
with varying glucose tolerance received an oral glucose
tolerance test (OGTT) and euglycemic insulin clamp. We
evaluated Adipo-IR (fasting and mean OGTT plasma free
fatty acid [FFA] 3 insulin concentrations), peripheral IR
(1/[Matsuda index] and (M/I)
21
value), and b-cell func-
tion (calculated as the ratio of the increment in plasma
insulin to glucose [OGTT/IR (DI/DG 4 IR)]). Fasting
Adipo-IR was increased twofold in obese subjects with
NGT and IGT versus lean subjects with NGT (8.0 6 1.1
and 9.2 6 0.7 vs. 4.1 6 0.3, respectively) and threefold in
subjects with T2DM (11.9 6 0.6; P < 0.001). Progressive
decline in DI/DG 4 IR was associated with a progressive
impairment in FFA suppression during OGTT, whereas
the rise in mean plasma glucose concentration only be-
came manifest when subjects became overtly diabetic.
The progressive decline in b-cell function that begins in
individuals with NGT is associated with a progressive in-
crease in FFA and fasting Adipo-IR.
Adipose tissue is an endocrine organ that influences both
glucose and lipid metabolism (1,2) by releasing adipokines,
proinflammatory factors, and free fatty acids (FFAs), which
impair glucose metabolism and muscle ATP synthesis (3),
promote the synthesis of toxic lipid metabolites, and alter
insulin signaling (4,5). Insulin acts on adipose tissue 1)by
stimulating glucose uptake and triglyceride synthesis and
2) by suppressing triglyceride hydrolysis and release of FFA
and glycerol into the circulation (6,7). Adipose tissue in-
sulin resistance (Adipo-IR), that is, the impaired suppres-
sion of lipolysis in the presence of high insulin levels, has
been associated with glucose intolerance, and elevated plasma
FFA levels have been shown to impair muscle insulin signal-
ing, promote hepatic gluconeogenesis, and impair glucose-
stimulated insulin response (7–13). In obese subjects without
diabetes and with type 2 diabetes mellitus (T2DM), subcuta-
neous adipose tissue is resistant to the antilipolytic effect of
insulin. Insulin also is an adipogenic hormone that increases
the uptake of circulating fatty acids and enhances triglyceride
synthesis, thus stimulating the accumulation of subcutaneous
fat as well as ectopic fat in liver, muscle, pancreas, heart, and
other tissues (14–16). Although the role and natural history
of b-cell dysfunction and muscle insulin resistance are well
established in the development of T2DM, the impact of
Adipo-IR in the transition from normal glucose tolerance
(NGT) to T2DM has not been fully elucidated. The in vivo
assessment of Adipo-IR is still controversial because many
different approaches have been used to characterize Adipo-IR.
By using tracers, it is possible to quantitate palmitate turn-
over (17,18) and the ra te of glycerol release (19,20) to pro-
vide an index of lipolysis. Our group was one of the first
to show that in man, the s uppression of lipolysis and
FFA release is related to plasma insulin concentration in a
curvilinear fashion that becomes linear if logarithmically
transformed (18). All studies agree that the relationship be-
tween the circulating plasma insulin concentration and both
1
University of Texas Health Science Center at San Antonio, San Antonio, TX
2
Cardiometabolic Risk Laboratory, Institute of Clinical Physiology, National Re-
search Council, Pisa, Italy
Corresponding author: Amalia Gastaldelli, amalia@ifc.cnr.it.
Received 25 September 2016 and accepted 29 December 2016.
© 2017 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered. More information is available at http://www.diabetesjournals
.org/content/license.
Diabetes Volume 66, April 2017 815
METABOLISM
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the lipolytic rate and the plasma FFA concentration becomes
linear when plotted on a log-log scale (17,21–25). Thus,
the product of the plasma FFA and insulin concentrations
provides an index of Adipo-IR, and this index has been
used to evaluate adipose tissue sensitivity to insulin in a
variety of metabolic conditions (26–30). However, no study
research to our knowledge ha s systematically evaluated
adipose tissue sensitivity to insulin during the transition
from NGT to impaired glucose tolerance (IGT) to T2DM
in a large subject population.
Thegoalofthecurrentstudywastoevaluatetheimpact
of resistance to the antilipolytic effect of insulin during the
natural history of T2DM (i.e., in a large group of subjects
with NGT, IGT, and T2DM). To accomplish this objective,
we have analyzed the data from subjects who participated in
theSanAntonioMetabolism(SAM)studyinwhomwe
measured plasma FFA levels during an oral glucose toler-
ance test (OGTT) and, on a separate day, peripheral insulin
sensitivity by using the euglycemic-hyperinsulinemic clamp.
RESEARCH DESIGN AND METHODS
Subjects
The SAM study cohort comprised 302 subjects (35 lean and
30 obese with NGT, 44 with IGT, and 193 with T2DM) (31).
All subjects were in good general health as judged by medical
history, physical exam inatio n, and blood tests. Body weight
was stable (63 lb) over the preceding 3 months, and no
subject participated in an excessively heavy exercise
program. Subjects with NGT and IGT were not taking
medications known to affect glucose tolerance . Subjects
with T2DM taking sulfonylureas or metformin had their
oral hypoglycemic agent discontinued 3 days before the
study. No subject with d iabetes had received treatment
with a thiazolidinedione, glucagon-like peptide 1 receptor
agonist, dipeptidyl peptidase 4 inhibitor, sodium–glucose
cotransporte r 2 inhibitor, or insulin. None of the sub jects
participated in any regular physical activity program. Obe-
sity was defined as BMI .30 kg/m
2
or body fat .35%
(measured by using tritiated wat er as previously described
[31]). Patients with T2DM were divided into tertiles
according to 2-h plasma glucose values (2h-PG): group I
(2h-PG ,300 mg/dL), group II (2h-PG ,360 mg/dL), and
group III (2h-PG $360 mg/dL). The study protocol was
approved by the institutional review board of the Uni-
versity of Texas Health Science Center at San Antonio
(San Antonio, TX), and informed written consent was
obtained from each subject before participation.
Study Protocol
At 8:00 A.M. after a 10-h overnight fast, all subjects received
a 2-h 75-g OGTT with measurement of plasma glucose, in-
sulin, and FFA concentrations at 2 30, 215,and0minand
then every 30 min after glucose ingestion. On a separate day
after an overnight fast, subjects returned at 7:00 A.M. for a
euglycemic insulin clamp (31). Catheters were placed in an
antecubital vein for the infusion of all test substances and
retrogradely into a vein on the dorsum of the hand for
blood withdrawal. The hand was placed in a heated box at
60°C. Two hours (3 h in T2DM) before the start of the
insulin clamp, 3-
3
H-glucose (DuPont NEN, Boston, MA)
was infused as a primed (40 mCi in subjects without diabe-
tes and [fasting plasma glucose (FPG)/5.6] 3 40 mCi in
subjects with T2DM), continuous infusion (0.4 mCi/min)
throughout the study as previou sly described (31).
Analytical Methods
Plasma glucose concentration was determined by the glucose
oxidase method (Beckman Glucose Analyzer; Beckman Coulter,
Fullerton, CA). Plasma insulin and C-peptide concentrations
were measured by radioimmunoassay with specific kits
(Diagnostic Products Corporation, Los Angeles, CA), and
plasma FFA levels were measured spectrophotometrically
(Wako Chemicals, Neuss, Germany). Plasma 3-
3
H-glucose lev-
els were measured in Somogyi precipitates as previously de-
scribed (31). Plasma adiponectin concentration was measured
by radioimmunoassay (Linco Research, St. Charles, MO).
Data Analysis
Insulin sensitivity was assessed by Matsuda index (32) from
the plasma glucose and insulin concentrations measured dur-
ing the OGTT. Insulin sensitivity also was measured as the
M/I value from the euglycemic-hyperinsulinemic clamp (31)
in 235 subjects (17 of 34 lean subjects with NGT; 21 of 30
obese subjects with NGT; 39 of 44 subjects with IGT; and
53 of 65 subjects with T2DM in group I, 54 of 64 in group II,
and 51 of 64 in group III). Insulin resistance was assessed as
the inverse of insulin sensitivity. During the postabsorptive
state,therateofendogenousglucose production (EGP) equals
R
g
by all tissues in the body and is calculated as the tritiated
glucose infusion (DPM/min) divided by the plasma tritiated
glucose–specific activity (DPM/mg). After the start of insulin
infusion, non–steady-state conditions prevailed, and R
a
was
calculated by using Steele’s equation. Residual EGP was cal-
culated as R
a
minus the exogenous glucose infusion rate. To
calculate total body R
d
, the rate of residual EGP during the
last 30 min of the insulin clamp was added to the exogenous
glucose infusion rate required to maintain euglycemia during
the last 30 min of the insulin clamp. Adipo-IR was calculated
as the product of fasting plasma FFA and fasting plasma
insulin concentrations and as the product of the mean plasma
FFA and insulin concentrations during the OGTT.
b-Cell function (insulin secretion/insulin resistance
index [DI/DG O IR]) was calculated as the ratio of the in-
cremental area under the curve (AUC) in plasma insulin to
the incremental AUC in plasma glucose (DAUC-I/DAUC-G)
during OGTT divided by insulin resistance, measured as the
inverse of the Matsuda index as previously reported (31,33).
Statistical Analysis
Data are mean 6 SEM and presented as mean 6 SE. Non–
normally distributed values were log-transformed before
analysis.
Group values were compared by ANOVA and Bonferroni-
Dunn post hoc analysis. Univariate associations were tested
by Spearman rank correlation. Multivariable analysis tested
816 Adipose Tissue Insulin Resistance in T2DM Diabetes Volume 66, April 2017
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the association between ln(Adipo-IR) (dependent variable)
and age, BMI, sex, presence of diabetes, insulin sensitivity
(ln[Matsuda index]), insulin secreti on (∆AUC-I/∆AUC-G),
and b-cell function (∆AUC-I/∆AUC-G O IR) as independent
variables.
RESULTS
The clinical characteristics of the study subjects are shown
in Table 1. Subjects with diabetes were slightly older than
those without diabetes but had a similar BMI and percent
body fat to obese subjects with NGT and IGT. In subjects
without diabetes (NGT and IGT), the FPG concentrations
were within the normal range and increased progressively
in subjects with T2DM in groups I–III (Table 1). Similarly,
the mean plasma glucose concentrations measured during
the OGTT increased progressively in T2DM groups I–III
(Figs. 1B and 2A).
The fasting plasma FFA concentrations were signifi-
cantly higher in obese NGT and IGT than in lean NGT. The
fasting plasma FFA concentration was the lowest in lean
NGT, increased markedly and linearly from obese NGT to
IGT, and plateaued without further increase in T2DM
(Table 1). During the OGTT, the mean plasma FFA concen-
tration was significantly increased in obese NGT versus
lean NGT and rose progressively from NGT to IGT (Fig.
1E)toT2DMingroupsI–III (Fig. 1F)withnoevidenceof
plateau (Fig. 2B). This pattern closely followed the change
in Matsuda index of insulin sensitivity and insulin secre-
tion, reflecting the progressive declines in insulin sensitiv-
ity (Fig. 2C)andb-cell function (Table 1).
The fasting plasma insulin concentration progressively
increased from NGT to IGT (Fig. 1C) to T2DM (Fig. 1D),
whereas the mean plasma insulin concentration during
OGTT showed the typical inverted U-shaped curve (34),
increasing from NGT to IGT and then decreasing progres-
sively in T2DM in groups I–III (Fig. 2D).
The progressive impairment in FFA suppression during
OGTT was strongly correlated with the progressive decline
in b-cell function (∆AUC-I/∆AUC-G O IR) (r = 20.52; P ,
0.0001) (Fig. 3) and with the progressive decline in insulin
sensitivity measured as the M-value during the insulin clamp
(r = 20.42; P , 0.0001). In marked contrast, the rises in
FPG and mean plasma glucose during the OGTT only be-
came pronounced when subjects became overtly diabetic
(Figs. 1 and 2).
Thus, as lean subjects with NGT became obese (but still
maintained NGT) or IGT or T2DM, both the fasting
plasma and OGTT FFA (as well as glucose) concentrations
increased (Table 1 and Fig. 2A and B), which was
explained by the progressive decline in insulin sensitivity
(Fig. 2C), insulin response during the OGTT (Fig. 2D), and
reduced b-cell function (Table 1 and Fig. 3).
Table 1—Clinical characteristics of the study subjects
NGT T2DM
Lean Obese IGT Group I Group II Group III
n 34 30 44 65 64 64
Sex
Female 14 24 26 36 28 36
Male 20 6 18 29 36 28
Age (years) 40 6 2386 2416 2526 1*§ 53 6 1*§ 51 6 1*§
Weight (kg) 73 6 2796 3* 84 6 3* 86 6 2* 90 6 2*§ 82 6 2*
BMI (kg/m
2
) 25.0 6 0.4 30.5 6 0.8* 31.2 6 0.9* 31.5 6 0.7* 32.3 6 0.7* 30.8 6 0.6*
Percent fat 30 6 1396 1* 38 6 1* 38 6 1* 38 6 1* 38 6 1*
Triglycerides (mg/dL) 144 6 29 97 6 9 153 6 17§ 145 6 10§ 172 6 10§ 157 6 16§
Total cholesterol (mg/dL) 178 6 10 167 6 7 190 6 7§ 180 6 4 176 6 4 179 6 4
LDL (mg/dL) 108 6 8 105 6 6 118 6 6 112 6 4 106 6 4 112 6 4
HDL (mg/dL) 42 6 3426 2416 2396 1376 1406 2
HbA
1c
(%) 5.1 6 0.1 5.3 6 0.2 5.5 6 0.1 7.3 6 0.2*§ 8.3 6 0.2*§ 9.2 6 0.2*§
Fasting glucose (mg/dL) 92 6 1956 1986 1*§ 141 6 4*§ 183 6 4*§ 229 6 5*§
2-h glucose (mg/dL) 100 6 3 112 6 2* 149 6 3*§ 250 6 4*§ 329 6 2*§ 402 6 4*§
Fasting insulin (mU/L) 7.2 6 0.5 11 6 1.2* 12.5 6 0.9* 16.7 6 1.4*§ 20.6 6 1.6*§ 15.1 6 1.1*§
Fasting FFA (mmol/L) 0.51 6 0.03 0.69 6 0.03* 0.77 6 0.03* 0.69 6 0.03* 0.74 6 0.02* 0.78 6 0.03*§
2-h FFA (mmol/L) 0.18 6 0.01 0.17 6 0.01 0.21 6 0.02 0.28 6 0.01*§ 0.37 6 0.02*§ 0.41 6 0.02*§
Clamp FFA (mmol/L) 0.22 6 0.03 0.20 6 0.2 0.24 6 0.03 0.23 6 0.02 0.25 6 0.01 0.22 6 0.01
DAUC-I/DAUC-G 251 6 47 282 6 37 162 6 14*§ 48
6 7*§ 22 6 2*§ 10 6 1*§
DI/DG O IR 1,500 6 281 1,137 6 238* 415 6 25*§ 87 6 7*§ 33 6 2*§ 17 6 1*§
Data are mean 6 SE. Subjects with T2DM were grouped according to tertiles of 2h-PG concentrations (i.e., group I: 2h-PG ,300 mg/dL;
group II: 2h-PG ,360 mg/dL; group III: 2h-PG $360 mg/dL). *P , 0.05 vs. lean subjects with NGT; §P , 0.05 vs. obese subjects
with NGT.
diabetes.diabetesjournals.org Gastaldelli, Gaggini, and DeFronzo 817
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Adipo-IR Index
Fasting Adipo-IR increased twofold in obese NGT and IGT
versus lean NGT (8.0 6 1.1 and 9.2 6 0.7 vs. 4.1 6 0.3; both
P , 0.001) and threefold in T2DM (P , 0.001) (Fig. 2E).
Fasting Adipo-IR was significantly greater in obese T2DM
versus lean T2DM (13.8 6 0.8 vs. 8.0 6 0.7; P , 0.001)
and inversely correlated with b-cell functio n, indicating that
high fasting plasma FFA concentrations and impaired FFA
suppression were mainly a result of insulin secretion defi-
ciency (Fig. 3). This finding was confirmed by analysis of the
euglycemic-hyperinsulinemic clamp data (Table 1) showing
that the plasma FFA concentrations at the end of the hyper-
insulinemic clamp were similar in all study groups.
Although fasting Adipo-IR rose continuously in the
transition from NGT to IGT to T2DM (Fig. 2E), OGTT
Adipo-IR increased from lean NGT to obese NGT to IGT
and then decreased progressively in T2DM groups I–III (Fig.
2F), following the U-shaped insulin response curve during
the OGTT (Fig. 2D). Thus, markedly deficient insulin secre-
tion during the OGTT results in a paradoxical decline in
OGTT Adipo-IR in T2DM (Fig. 2D and F), making the
OGTT Adipo-IR unreliable in subjects with diabetes.
In a multivariable regression analysis (with age, BMI,
sex, presence of diabetes, insulin sensitivity [ln(Matsuda
index)], insulin secretion [∆AUC-I/∆AUC-G], and b-cell
function [∆AUC-I/∆AUC-G O IR] as independent variables),
ln(Adipo-IR) (dependent variable) was found to be indepen-
dently correlated (total r =0.81;P , 0.0001) with BMI (P ,
0.0001), insulin sensitivity (P , 0.0001), and male sex (P ,
0.0001). By performing the same analysis separately in
males and females, we found that in both females (total r =
0.82; P , 0.0001) and males (total r = 0.80;
P , 0.0001),
the association holds with BMI (P , 0.0001) and insulin
sensitivity (P , 0.0001). In a subgroup of 54 subjects for
whom plasma was available, adiponectin correlated nega-
tively with ln(Adipo-IR) independently of age, sex, and
BMI (r = 20.49; P = 0.009).
DISCUSSION
We previously have shown that during the transition from
NGT to IGT to T2DM, b-cell function progressively
declines and peripheral insulin resistance progressively
increases (31,35). Adipo-IR also is increased in patients
with T2DM, but the natural history of its development
as individuals progress from NGT to IGT to T2DM has
been poorly studied. As recently reviewed (21), a number
of indices of adipocyte insulin resistance have been pro-
posed that are based on tracer turnover (i.e., labeled pal-
mitate or glycerol) or FFA suppression during insulin
infusion (euglycemic-hyperinsulinemic clamp) or OGTT. In
the current study, we used the product of fasting plasma
FFA and fasting plasma insulin concentrations as the index
Figure 1—Changes in plasma glucose (A), insulin (C), and FFA (E ) concentrations during the OGTT in subjects without diabetes. Changes
in plasma glucose (B), insulin (D), and FFA (F) concentrations during the OGTT in subjects with T2DM. D, diabetic.
818 Adipose Tissue Insulin Resistance in T2DM Diabetes Volume 66, April 2017
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of Adipo-IR. Because the circulating plasma FFA concentra-
tion closely reflects the rate of peripheral lipolysis, Adipo-IR
represents an index for adipose tissue resistance to the
antilipolytic effect of insulin. The hyperbolic relationship
between plasma insulin and FFA concentrations initially was
demonstrated by Groop et al. (18) who examined the relation-
ship between insulin and the inhibition of plasma FFA con-
centration and rate of lipolysis (measured by
14
C-palmitate)
during a five-step euglycemic-hyperinsulinemic clamp. Sim-
ilar results were reported by Bugianesi et al. (26) who mea-
sured the plasma FFA concentration and rate of lipolysis
(by
2
H-glycerol turnover) during a two-step hyperinsulinemic-
euglycemic clamp. However, the number of subjects in these
previous studies was small, and the changes in Adipo-IR
in NGT to IGT to T2DM was not evaluated.
In this cross-sectional study, we evaluated changes in
plasma FFA concentration during the fasting state and
OGTT in subjects across a wide range of glucose tolerance
and insulin resistance. Subjects with insulin resistance
(ranging from obese NGT to IGT and T2DM) were compared
with lean subjects with NGT. The fasting plasma FFA
concentration increased markedly and linearly as subjects
progressed from lean NGT to obese NGT and IGT and
plateaued without further increase in T2DM, reflecting the
progressive decline in insulin sensitivity. The increase in
plasma FFA concentration during the OGTT tracks with
worsening whole-body insulin resistance and worsening
Adipo-IR during the OGTT over the range of NGT to IGT.
With progression of IGT to T2DM, the plasma FFA
concentration during the OGTT continues to rise, whereas
whole-body insulin resistance plateaus and OGTT Adipo-IR
declines. Thus, as we previously have shown, patients with
IGT are maximally/near maximally insulin resistant with
respect to glucose metabolism, and the rise in fasting plasma
FFA concentrations closely follows the increase in insulin
resistance. Further progression from NGT to IGT is associated
with parallel increases in fasting plasma FFA and worsening
whole-body insulin resistance and Adipo-IR. As IGT prog-
resses to T2DM, no further increase in fasting plasma FFA
occurs, even though Adipo-IR continues to worsen. A likely
explanation is the observation that the fasting plasma insulin
concentration increases sufficiently to offset the worsening of
Figure 2—Mean plasma glucose (A), FFA (B), and insulin (D) concentrations during OGTT; Matsuda index of insulin sensitivity (C); and
fasting (E) and OGTT (F ) Adipo-IR index. The figure demonstrates that as lean subjects with NGT become obese (but still maintain NGT) or
IGT or T2DM, not only glucose but also FFA concentrations increased during fasting and OGTT, which is explained by the progressive
decline in the insulin secretion/insulin resistance index. G, group; Ins, insulin.
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