Clinical Science (2008) 115, 197–202 (Printed in Great Britain) doi:10.1042/CS20070387
Importance of the high-molecular-mass isoform
of adiponectin in improved insulin sensitivity
with rosiglitazone treatment in HIV disease
Saima QURASHI∗ , Dennis C. MYNARCIK∗ , Margaret A. MCNURLAN†,
Hongshik AHN‡, Robert FERRIS∗ and Marie C. GELATO∗
∗
Department of Medicine, Stony Brook University, Stony Brook, NY 11794, U.S.A., †Department of Surgery, Stony Brook
University, Stony Brook, NY 11794, U.S.A., and ‡Department of Applied Mathematics and Statistics, Stony Brook University,
Stony Brook, NY 11794, U.S.A.
A
B
S
T
R
A
C
T
The present study was designed to investigate the relationship of isoforms of adiponectin to insulin
sensitivity in subjects with HIV-associated insulin resistance in response to treatment with the
thiazolidinedione, rosiglitazone. The two isoforms of adiponectin, HMW (high-molecular-mass)
and LMW (low-molecular-mass), were separated by sucrose-gradient-density centrifugation. The
amount of adiponectin in gradient fractions was determined by ELISA. Peripheral insulin sensitivity (Rd) was determined with hyperinsulinaemic–euglycaemic clamp, whereas hepatic sensitivity
[HOMA (Homoeostasis Model Assessment) %S] was based on basal glucose and insulin values.
Treatment with rosiglitazone for 3 months resulted in a significant improvement in the index of
hepatic insulin sensitivity (86.4 +
− 15 % compared with 139 +
− 23; P = 0.007) as well as peripheral
+
insulin sensitivity (4.04 +
0.23
compared
with
6.17
0.66
mg
of glucose/kg of lean body mass per
−
−
min; P < 0.001). Improvement in HOMA was associated with increased levels of HMW adiponectin
(r = 0.541, P = 0.045), but not LMW adiponectin. The present study suggests that the HMW
isoform of adiponectin is important in the regulation of rosiglitazone-mediated improvement in
insulin sensitivity in individuals with HIV-associated insulin resistance, particularly in the liver.
INTRODUCTION
Adipose tissue plays an important role in the regulation
of insulin action. Not only is the obesity-related increase
in adipose tissue associated with insulin resistance [1,2],
but loss of body fat or lipoatrophy is also associated
with insulin resistance [3,4]. The discovery of a number
of physiologically active proteins secreted by adipose
tissue, adipokines, suggests a possible mechanism by
which adipose tissue has an impact on insulin sensitivity.
Since its discovery in 1995 [5], plasma concentrations
of the adipokine, adiponectin (or ACRP 30) have been
associated with insulin sensitivity. Adiponectin levels
are lower in obese individuals [6,7] and also lower in
subjects with diabetes [8]. Not only are adiponectin levels
lower in individuals with insulin resistance, but improvement in insulin sensitivity through weight loss results
in increased circulating concentrations of adiponectin
[8]. A close relationship of the levels of circulating
adiponectin with the degree of insulin resistance has been
demonstrated in populations with diminished insulin
sensitivity, including obese and diabetic subjects [9],
Pima Indians with an increased incidence of diabetes and
obesity [10] and individuals with HIV-associated insulin
resistance [11]. Evidence of a more direct, causal role of
adiponectin in the regulation of insulin sensitivity has
Key words: adiponectin, HIV, insulin resistance, thiazolidinedione.
Abbreviations: BMI, body mass index; HMW, high-molecular-mass; HOMA, Homoeostasis Model Assessment; HOMA-IR,
HOMA-insulin resistance; LMW, low-molecular mass; TZD, thiazolidinedione.
Correspondence: Dr Margaret A. McNurlan (email [email protected]).
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S. Qurashi and others
been provided by animal studies demonstrating a direct
effect of adiponectin on insulin sensitivity in wild-type
mice and in mouse models of insulin resistance [12–14].
Although adiponectin levels have been associated with
insulin sensitivity, this ability has been linked to distinct
macromolecular forms. Adiponectin has a monomeric
structure that exists in a LMW (low-molecular-mass)
form consisting of six monomers which readily trimerizes
to a HMW (high-molecular-mass) form consisting of
eighteen monomers [5]. The majority of adiponectin in
the serum has been shown to be the HMW form [15],
with a sexual dimorphism in distribution with greater
circulating levels of the HMW form in females [16]. When
insulin sensitivity is improved by treatment with TZDs
(thiazolidinediones), circulating levels of adiponectin are
increased [17,18]. Scherer and co-workers have demonstrated that improved insulin sensitivity is associated
specifically with increased blood levels of the HMW form
of adiponectin [19,20]. Moreover, direct application of
the TZD pioglitazone to human adipose tissue in vitro
results in increased levels of HMW adiponectin specifically [21]. Further confirmation of the physiological
importance of the HMW form of adiponectin in the
regulation of insulin action is provided by the demonstration that genetic mutations preventing HMW formation are associated with an increased risk of diabetes [22].
In light of the importance of HMW adiponectin in
improving insulin sensitivity [19–21], the present study
was undertaken to examine the relationship of the HMW
form of adiponectin to insulin sensitivity in subjects with
HIV-associated insulin resistance and further to examine
the changes in adiponectin associated with improvement
in insulin sensitivity following treatment with the TZD
rosiglitazone.
MATERIALS AND METHODS
sensitivity in a subgroup of these subjects has been
described previously [23].
Insulin sensitivity
Peripheral insulin sensitivity (glucose disposal or Rd)
was determined as the rate of glucose infused to maintain
euglycaemia during an insulin infusion (hyperinsulinaemic–euglycaemic clamp, [24]). Patients were admitted
to the General Clinical Research Center the night before
the study. Beginning at 07:00 hours, study subjects
received an intravenous infusion of 1.2 m-units of
insulin (Humulin; Eli Lilly)/kg of body weight per min.
Dextrose (10 %) was administered intravenously to
maintain plasma glucose at 90 mg/dl.
Insulin sensitivity was also assessed with HOMA
(Homoeostatis Model Assessment), which is based on
fasting insulin and glucose [25,26]. HOMA is more
a measure of hepatic sensitivity to insulin rather than
insulin sensitivity of peripheral tissues. HOMA%S
(HOMA insulin sensitivity) was calculated from
fasting blood glucose and insulin with a computer
model (http://www.dtu.ox.ac.uk/) and expressed as a
percentage [27]. HOMA-IR (HOMA-insulin resistance)
is the reciprocal of %S, and therefore similar conclusions
are reached if the results are expressed as HOMA-IR.
The plasma glucose levels were analysed by the glucose oxidase method, assessed with a Beckman Glucose
Analyzer II. Insulin levels were analysed by radioimmunoassay (Diagnostic Products).
Viral load
Plasma samples of HIV RNA were frozen and analysed
by the New York state-approved Department of Pathology at Stony Brook. The licensed assay has a lower limit
of detection of 400 copies of RNA/ml of plasma. Samples with less than 400 copies of RNA/ml of plasma were
assayed with an ultra-sensitive assay with a lower limit
of less than 40 copies of RNA/ml of plasma.
Subject characteristics
Subjects with HIV-associated insulin resistance were
enrolled into the study if they had demonstrable insulin
resistance but not overt diabetes. Subjects were not
enrolled if they had had any acute illness in the 3 months
preceding the study, a random (non-fasting) glucose
level of greater than 200 mg/dl, liver function assessment
three times normal or a change in HIV medication in the
3 months prior to the study.
Following assessment of baseline insulin sensitivity,
subjects received 4 mg of rosiglitazone (Avandia;
GlaxoSmithKline) twice daily for 12 weeks. Safety evaluations included assessment of liver function, complete
blood counts, CD4 counts and viral load during the treatment period. The study was approved by the Committee
on Research Involving Human Subjects at Stony Brook
University Medical Center and all subjects gave their
written, informed consent. The improvement in insulin
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2008 Biochemical Society
Adiponectin isolation and analysis
The HMW and LMW forms of adiponectin from 20 μl of
plasma were determined by centrifugation (55 000 rev./
min for 8 h at 4 ◦ C in a Beckman SWT155 rotor and
L8-60M ultracentrifuge) on a step-wise sucrose density
gradient (5–20 %). Twelve gradient fractions of 400 μl
each were harvested from the bottom of the gradient.
The amount of adiponectin in gradient fractions was
determined by ELISA (R&D Systems). The concentration of HMW adiponectin in plasma was determined
by combining the amount (concentration × volume) in
fractions 2–5. LMW adiponectin was determined in a
similar manner from fractions 6–12 (Figure 1).
Statistical analysis
Shapiro–Wilks test was used to test the normality of
the data and the data were log-transformed as needed
HMW adiponectin with rosiglitazone treatment in HIV
litazone treatment minus the log of the value before
rosiglitazone treatment. Statistical analysis was carried
out with SAS statistical software, version 9.1.
RESULTS
Figure 1 Adiponectin concentration in sucrose density
fractions
Adiponectin concentration (μg/ml) in fractions from 0.02 ml of plasma separated
by sucrose-density-gradient ultracentrifugation and assayed by ELISA, as described
in the Materials and methods section. The profiles are from one subject before and
after treatment with rosiglitazone. The adiponectin content of fractions 2–5 was
taken to be the HMW isoform and the adiponectin content of fractions 6–12
was taken to be the LMW isoform.
to ensure normality. Comparisons between pre- and
post-values were assessed with a paired t test. All data are
reported as means +
− S.E.M., except as noted, and P < 0.05
from a two-sided test was considered significant. Pearson
correlation coefficients were calculated to determine
the relationship of changes in variables such as insulin
sensitivity with changes in adiponectin with rosiglitazone
treatment. The changes in each variable with treatment
were assessed as the log of the value following rosig-
Seventeen subjects with HIV-associated insulin resistance
completed the study (Table 1). The study cohort of seven
males and ten females had a mean age of 44 +
− 2 years and
2
a BMI (body mass index) of 27 +
− 1 kg/m . The number
of CD4 cells in these subjects ranged from 47–1330 cells/
mm3 and the viral burden ranged from less than 40 to
42 000 copies of RNA/ml of plasma. Fifteen subjects
were on highly active antiretroviral treatment, whereas
two of the subjects were not on any antiretroviral
medications. Because the study was designed to test the
ability of rosiglitazone to improve insulin resistance,
all of the subjects enrolled in the study were insulin
resistant, i.e. had glucose disposal rates (Rd) of equal to or
less than 6 mg of glucose/kg of lean body mass per min as
assessed with the hyperinsulinaemic–euglycaemic clamp
[24].
Prior to treatment with rosiglitazone, the mean fasting glucose in these subjects was 104 +
− 3 mg/dl and
fasting insulin was 13.5 +
2.9
(Table
2). Peripheral
−
insulin sensitivity (Rd) was 4.04 +
0.23
mg
of glucose/kg
−
of lean body mass per min and insulin sensitivity
from the HOMA%S was 86.4 +
− 15 %. In the insulinresistant-state (pre-rosgliatzone), there was a gender
difference in the concentrations of both isoforms: HMW
adiponectin (males, 0.50 +
− 0.22 μg/ml and females, 1.56 +
−
3.5 μg/ml; P = 0.037) and LMW adiponectin (males,
Table 1 Subject characteristics
M, male; F, female; PI, protease inhibitor; NRTI nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor.
Subject
Age (years)
Gender
BMI (kg/m2 )
Viral burden (copies/ml)
CD4 (cells/ml)
HIV medication
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
33
44
52
47
39
39
61
48
36
38
45
30
49
39
51
45
45
M
M
F
M
M
F
F
F
F
M
M
F
F
M
F
F
M
21.2
31.6
27.8
25.8
29.4
20.3
23.0
33.1
27.8
29.3
25.9
23.8
34.9
25.0
22.9
25.9
27.9
< 50
1157
400
1094
< 40
370
4000
< 40
< 40
42 000
1663
2257
< 40
< 40
< 40
16 291
6622
700
303
760
48
650
1330
250
762
1016
400
808
952
341
47
778
367
354
NNRTI, NRTI
PI, NRTI
PI, NNRTI, NRTI
PI, NRTI
PI, NRTI
PI, NNRTI, NRTI
NNRTI, NRTI
PI, NRTI
PI, NNRTI, NRTI
None
NRTI
PI, NRTI
PI, NRTI
NNRTI
PI, NRTI
NNRTI
None
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S. Qurashi and others
Table 2 Changes in insulin, glucose, insulin sensitivity and
adiponectin following treatment with rosiglitazone
Values are expressed as means +
− S.E.M. The percentage of HMW is the proportion
of HMW adiponectin in the plasma, i.e. HMW adiponectin divided by the total
(HMW + LMW). P values are from the paired t test of log-transformed data and
are for the comparison of pre- and post-rosigilitazone.
Prerosiglitazone
Variable
n
Insulin (μ-units/ml)
Glucose (mg/dl)
Rd (mg of glucose/kg of
lean body mass per min)
HOMA (%)
HMW adiponectin (μg/ml)
LMW adiponectin (μg/ml)
Total adiponectin (μg/ml)
Percentage of HMW adiponectin
14
17
16
13.5 +
− 2.9
104 +
−3
4.04 +
− 0.23
Postrosiglitazone
P value
8.75 +
0.016
− 2.0
99 +
2.3
0.025
−
6.17 +
− 0.66 < 0.001
14 86.4 +
139 +
0.007
− 15
− 21
+
17 1.12 +
0.26
3.90
0.79
<
0.001
−
−
+
17 2.34 +
0.36
4.71
0.64
<
0.001 Figure 2 Relationship of HMW adiponectin and hepatic
−
−
+
17 3.46 +
0.57
8.60
1.27
<
0.001
−
−
insulin sensitivity (HOMA) in response to rosiglitazone
17 0.278 +
0.002 HOMA is an index of hepatic insulin sensitivity based on basal glucose and insulin
− 0.03 0.395 +
− 0.04
values [25,26]. HMW adiponectin was determined from an ELISA assay following
fractionation of plasma on a sucrose density gradient. The fold-change in HMW
adiponectin was determined as log (post HMW) − log (pre HMW). The fold-change
1.54 +
− 0.29 μg/ml and females, 2.89 +
− 0.49 μg/ml; in HOMA was determined as log (post HOMA) − log (pre HOMA). The relationship
P = 0.053).
displayed is the Pearson correlation.
Treatment with rosiglitazone resulted in a significant
decrease in both mean fasting glucose (99 +
− 2 mg/dl;
P = 0.025) and insulin (8.75 +
2.0
μ-units/ml;
P = 0.016, levels following treatment with rosiglitazone (r = 0.39,
−
Table 2). Peripheral insulin sensitivity (Rd) improved by P > 0.05), but the improvement in hepatic insulin
51 % with rosiglitazone treatment (from 4.04 +
− 0.26 mg sensitivity, HOMA%S, was significantly associated
of glucose/kg of lean body mass per min to 6.17 +
− 0.66; with increased levels of HMW adiponectin (r = 0.54,
P < 0.001, Table 2) and HOMA%S, an index of hepatic P = 0.045; Figure 2). In contrast, increased levels of
insulin sensitivity, was also improved by a similar amount LMW adiponectin were not significantly associated with
(by 76 %; from 86.4 +
improvement in either peripheral insulin sensitivity,
− 15 % to 139 +
− 23 %; P = 0.007).
The improvement in insulin sensitivity was accom- Rd (r = 0.41, P = 0.11) or hepatic insulin sensitivity,
panied by increases in both isoforms of adiponectin; HOMA%S (r = 0.34, P = 0.24). The data were also examthe HMW isoform increased from 1.12 +
− 0.26 μg/ml ined for the relationship between the increase in insulin
to 3.90 +
0.79
μg/ml
(P
<
0.001)
and
the
LMW
isoform sensitivity and the proportion of the HMW isoform of
−
+
increased from 2.34 +
0.36
μg/ml
to
4.71
0.64
μg/ml adiponectin in plasma, i.e. HMW divided by HMW plus
−
−
(P < 0.001). ANOVA modelling indicated both gender LMW adiponectin following rosiglitazone treatment
(HMW, P = 0.0008; LMW, P = 0.0023) and drug (HMW, (Table 2). The correlations of the change in HMW
P < 0.0001; LMW, P < 0.0001) effects, but no gender/drug with the improvement in peripheral insulin sensitivity
interaction (HMW, P = 0.5377; LMW, P = 0.35) of log- (r = 0.243, P = 0.37) and HOMA (r = 0.44, P = 0.11)
transformed variables, suggesting that although the were not as strong as the correlations with the changes
isoforms differed by gender in the pre-rosiglitazone in plasma levels of the HMW isoform. Interestingly
condition, there was no significant difference in the res- the improvement in peripheral insulin sensitivity (Rd)
ponse to rosiglitazone between the genders. Therefore following treatment was highly correlated with changes
the values on the response to treatment for males and in liver sensitivity (HOMA, r = 0.66, P = 0.01; Figure 3).
females has been combined in subsequent analyses.
Although the blood levels of HMW adiponectin
increased following rosiglitazone treatment for the group DISCUSSION
as a whole, there was individual variation in the response
of HMW adiponectin to rosiglitazone. The variation Subjects recruited into the present study all had HIVin response of adiponectin and a variable response in associated peripheral insulin resistance with a mean
insulin sensitivity allowed an assessment of whether or glucose disposal rate (Rd) of 4.04 +
− 0.26 mg of glucose/kg
not these variables were related. There was no significant of lean body mass per min. For comparison, the Rd
relationship between the improvement in peripheral for a group of subjects of similar age and BMI without
insulin sensitivity (Rd) and the increase in adiponectin HIV infection was 11.1 +
− 1.1 [23]. Insulin resistance is
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HMW adiponectin with rosiglitazone treatment in HIV
Figure 3 Relationship of peripheral insulin sensitivity
to hepatic insulin sensitivity (HOMA) in response to
rosiglitazone
Peripheral insulin sensitivity from the hyperinsulinaemic–euglycaemic clamp (Rd)
was expressed in mg of glucose/kg of lean body mass per min. The fold-changes
in peripheral insulin sensitivity (Rd) were determined as log (post Rd) − log
(pre Rd). Hepatic insulin sensitivity from HOMA was expressed as a percentage.
The fold-changes in hepatic insulin sensitivity were determined as log (post
HOMA) − log (pre HOMA). The relationship displayed is the Pearson correlation.
accompanied by lower plasma concentrations of adiponectin in HIV disease [11] and other insulin resistant states
[9,10]. Although the adiponectin levels in blood are reduced in subjects with HIV-associated insulin resistance,
in the present study there was still evidence of significant
gender dimorphism in the isoforms of adiponectin, with
significantly higher levels of the HMW form of adiponectin in females compared with males. This is consistent
with the gender differences in HMW and LMW isoforms
reported in mice [16], children [28] and older adults [29].
In these subjects with HIV-associated insulin resistance, treatment with rosiglitazone, a TZD, resulted in
a significant improvement in insulin sensitivity assessed
either with a hyperinsulinaemic–euglycaemic clamp or
with HOMA. During the clamp procedure, exogenous
insulin is provided and the response of peripheral tissues
is assessed. The rosiglitazone-mediated improvement in
peripheral insulin sensitivity of approx. 50 % has been
previously reported in a subgroup of these subjects
[23]. The HOMA model, based on fasting insulin and
glucose levels, reflects hepatic insulin sensitivity and was
increased by a somewhat larger amount (76 %).
The improvement in insulin sensitivity following
rosiglitazone treatment was accompanied by significantly
increased blood levels of HMW and LMW isoforms. This
is in agreement with increased levels of total adiponectin
reported in other insulin resistant states [17,18]. However,
since the magnitude of the changes in insulin sensitivity in
the present study (results not shown) and that of Haider
et al. [30] did not correlate with the changes in the levels of
total adiponectin, it seems unlikely that total adiponectin
is important in the regulation of insulin sensitivity.
In contrast with the changes in total adiponectin,
the increase in the amount of the HMW isoform of
adiponectin was significantly related to changes in hepatic
insulin sensitivity as assessed by HOMA (Figure 2). This
is in keeping with reports from others that it is the HMW
isoform of adiponectin that is important in the regulation
of insulin action [19–21], although in the present study
the strongest association was with the plasma concentration of the HMW isoform of adiponectin rather than
the proportion of adiponectin present as the HMW
isoform reported in other studies [19,20]. In the present
study there was an observable gender difference in the
concentration of the HMW isoform of adiponectin in
subjects prior to rosiglitazone treatment, the changes
in levels of HMW in response to rosiglitazone treatment
were similar in both males and females. Although there
are limitations to the present study, including the lack of
a placebo group and the relatively small number of subjects, the present study confirms the importance of
the HMW isoform of adiponectin in hepatic insulin
sensitivity as reported for humans in other insulinresistant conditions [19,20] and animals [12,31].
Given the importance of the HMW isoform of adiponectin in assessing insulin sensitivity, different methodologies have been developed to simplify the determination
of this isoform. Initially, the different isoforms of
adiponectin were separated by sucrose-density-gradient
ultracentrifugation [19,20] or gel filtration [15,32] followed by quantitative Western blot analysis. The method
employed in the present study separated the adiponectin
isoforms by density-gradient centrifugation, but
employed ELISA for quantification of the isoforms since
ELISA correlates well with quantitative Western blot
analysis [33] and provides enhanced sensitivity. There
are a variety of other methods for the measurement of
HMW adiponectin, including non-reducing SDS/PAGE,
which provides both the separation and quantification of
the isoforms of adiponectin [22] and ELISA analysis for
HMW adiponectin after selective removal of the LMW
isoform [34]. However, the most promising technique for
the assessment of HMW adiponectin is the commercially
available ELISA based on a monoclonal antibody to
HMW adiponectin (Fujirebio; [35]).
In conclusion, the present study suggests that the
HMW isoform of adiponectin is important in the regulation of the rosiglitazone-mediated improvement
in insulin sensitivity in individuals with HIV-associated insulin resistance, particularly in the liver.
ACKNOWLEDGMENTS
This work was supported by U.S. National Institutes of
Health grant DK049316 to M. C. G. and #MO1RR10710,
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which supports the General Clinical Research Center at
Stony Brook University Medical Center and the Empire
Clinical Research Investigators Program Award (to
S. Q. and R. F.). We gratefully acknowledge the General
Clinical Research Center nursing, Core Lab staff and the
volunteer subjects who assisted in these studies.
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Received 31 October 2007/22 January 2008; accepted 5 February 2008
Published as Immediate Publication 5 February 2008, doi:10.1042/CS20070387
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The Authors Journal compilation
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2008 Biochemical Society