Effect of insulin on glycerol production
in obese adolescents
CHILDSY ROBINSON, WILLIAM V. TAMBORLANE, DAVID G. MAGGS,
STAFFAN ENOKSSON, ROBERT S. SHERWIN, DAVID SILVER,
GERALD I. SHULMAN, AND SONIA CAPRIO
Departments of Pediatrics and Internal Medicine, and the Yale Children’s General Clinical
Research Center, Yale University School of Medicine, New Haven, Connecticut 06520;
and the Department of Vascular Surgery, Huddinge University Hospital, Karolinska Institute,
S-105 21 Stockholm, Sweden
glycerol turnover; lipid oxidation; glucose metabolism
is steadily increasing in
adolescents (15) and, according to the National Health
and Nutrition Examination Survey (NHANES) III,
,20% of adolescent males and females are obese (32).
Most obese adolescents will remain obese adults with
an increased risk of metabolic and cardiovascular complications, which may be related to the central deposition of fat that occurs in adolescence (25). In view of
these facts, understanding the early metabolic defects
and the antecedents of obesity-related complications in
obese adolescents is of paramount interest. Most studies on the interactions between glucose and lipid metabolism have been performed in obese adults, most of
THE PREVALENCE OF OBESITY
whom have had long-lasting obesity (8, 16, 19); therefore, any metabolic defect detected in adult obesity may
represent an adaptation to the long-term obesity rather
than being causally related. On the other hand, childhood obesity and adolescent obesity represent ideal
models that offer the unique opportunity to elucidate
early metabolic defects occurring at the time of excessive fat accumulation. Recent studies from our laboratory (11) that have examined in vivo insulin action in
adolescent girls suggest that increased visceral fat,
hyperinsulinemia, and insulin resistance are closely
linked abnormalities that are expressed early in the
natural history of obesity. Similar defects were found
also in preadolescent obese children (9). Such insulin
resistance does not appear to be confined to the glucoregulatory actions of insulin, because elevated fasting
plasma free fatty acid (FFA) levels have also been
reported in obese and nonobese adolescents despite
hyperinsulinemia (9, 11). Indeed, the ability of insulin
to stimulate glucose oxidation is, in part, indirectly
mediated by its capacity to suppress FFA derived from
adipose and possibly intramuscular triglyceride stores
(1, 2, 4, 5). More recently, Boden and colleagues (5, 6)
and Kelley et al. (21) have shown that fat infusion
abolished the ability of insulin to stimulate glycogen
synthase activity in muscle and thus could explain the
impairment in nonoxidative glucose metabolism seen
in obesity. A further study, by Roden et al. (31), suggests
that increased concentrations of plasma FFA induce
insulin resistance through inhibition of glucose transport/phosphorylation activity.
Despite the important interactions between glucose
and FFA metabolism, it is still unclear how lipolysis is
altered in obesity during adolescence, a developmental
stage that appears to be of crucial importance in
adipose tissue cellular proliferation (22). In vivo studies
that have examined the effect of insulin on FFA levels
and turnover in obese adults with long-lasting obesity
concluded that the increased plasma FFA concentration and FFA turnover were the consequence of the
enlarged fat mass rather than impaired insulin sensitivity of the adipocyte (8, 16). FFA turnover, however, may
not necessarily be a reliable indicator of lipolysis,
because FFA can be reesterified within adipose tissue
after triglyceride hydrolysis (27). On the other hand,
glycerol liberated as a result of lipolysis cannot be
reincorporated into triglyceride, because adipose tissue
lacks glycerol kinase (2, 16). Release of glycerol into
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
E737
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Robinson, Childsy, William V. Tamborlane, David G.
Maggs, Staffan Enoksson, Robert S. Sherwin, David
Silver, Gerald I. Shulman, and Sonia Caprio. Effect of
insulin on glycerol production in obese adolescents. Am. J.
Physiol. 274 (Endocrinol. Metab. 37): E737–E743, 1998.—
Impaired stimulation of glucose metabolism and reduced
suppression of lipolytic activity have both been suggested as
important defects related to the insulin resistance of adolescent obesity. To further explore the relationship between
these abnormalities, we studied seven obese [body mass index
(BMI) 35 6 2 kg/m2] and seven lean (BMI 21 6 1 kg/m2 )
adolescents aged 13–15 yr and compared them with nine lean
adults (aged 21–27 yr, BMI 23 6 1 kg/m2 ) during a two-step
euglycemic-hyperinsulinemic clamp in combination with 1) a
constant [2H5]glycerol (1.2 mg · m22 · min21 ) infusion to quantify glycerol turnover and 2) indirect calorimetry to estimate
glucose and net lipid oxidation rates. In absolute terms, basal
glycerol turnover was increased and suppression by insulin
was impaired in obese adolescents compared with both groups
of lean subjects (P , 0.01). However, when the rates of
glycerol turnover were adjusted for differences in body fat
mass, the rates were similar in all three groups. Basal plasma
free fatty acid (FFA) concentrations were significantly elevated, and the suppression by physiological increments in
plasma insulin was impaired in obese adolescents compared
with lean adults (P , 0.05). In parallel with the high
circulating FFA levels, net lipid oxidation in the basal state
and during the clamp was also elevated in the obese group
compared with lean adults. Net lipid oxidation was inversely
correlated with glucose oxidation (r 5 20.50, P , 0.01). In
conclusion, these data suggest that lipolysis is increased in
obese adolescents (vs. lean adolescents and adults) as a
consequence of an enlarged adipose mass rather than altered
sensitivity of adipocytes to the suppressing action of insulin.
E738
LIPOLYSIS IN OBESE ADOLESCENTS
plasma should therefore be a more reliable index of
lipolysis than plasma FFA. In this study, we used
[2H5]glycerol kinetics to determine whether there is
increased lipolysis in obese adolescents compared with
lean adolescents and adults. Differences in rates of
lipolysis were analyzed with and without adjustments
for variations in total body fat mass.
METHODS
Table 1. Clinical characteristics
No. of subjects
Gender, M/F
Age, yr
Ht, cm
Wt, kg
BMI, kg/m2
BSA, m2
Fat mass, kg
Obese
Adolescents
Lean
Adolescents
Lean
Adults
7
4/3
13.6 6 0.6
165 6 3
96 6 8*
35 6 2*
2.02 6 0.1*
29 6 0.7*
7
5/2
13.1 6 0.4
156 6 2
50 6 2
21 6 1
1.47 6 0.04
13 6 3
9
4/5
23.8 6 1.2
167 6 2
62 6 3
23 6 1
1.69 6 0.05
15 6 2
Values are means 6 SE. M, males; F, females; BMI, body mass
index; BSA, body surface area. * P , 0.005 vs. lean adolescents and
adults.
Ra 5 (IEinf / IEpla 2 1) 3 f
where Ra is the rate of appearance of glycerol (µmol/kg), IEinf
is the isotopic enrichment of the infusate [in atoms % excess
(APE)], IEpla is the isotopic enrichment of plasma (APE) at
isotopic equilibrium, and f is the isotope rate of infusion
(µmol · kg21 · min21 ). This calculation was made with the
knowledge that glycerol enrichments reach a steady state in a
relatively short time (34), and we observed stable plasma
glycerol enrichments during the last 30 min of the baseline
period and both insulin clamp steps; therefore, all calculated
rates of glycerol turnover pertain to steady-state conditions.
Whole body glycerol fluxes were adjusted for adipose tissue
mass.
During the insulin clamp study, the amount of glucose
required to maintain euglycemia provides an index of insulinstimulated glucose metabolism. The glucose infusion rate
(mg · m22 · min21 ) was calculated at 20-min intervals and
corrected for deviations from the target plasma glucose level,
as previously described (12). The average rate of glucose
infusion during the last 30 min of each step of the clamp was
used for comparison. Respiratory gas exchange rates were
measured by a computerized open-circuit indirect calorimetry
(Deltatrac; Sensor Medics, Helsinki, Finland) with a ventilated hood system. Oxidation rates for carbohydrate, fat, and
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Subjects. We studied three groups of subjects: seven obese
adolescents [mean age 13.6 6 0.6 yr, mean body mass index
(BMI) 35 6 2 kg/m2], seven lean adolescents (age 13.1 6 0.4
yr, BMI 20.5 6 1.1 kg/m2 ), and nine lean adults (age 23.8 6
1.2 yr, BMI 23 6 1 kg/m2 ) (Table 1). All obese adolescents had
a BMI .95th percentile specific for age and sex (based on
percentile curves for girls and boys computed from the first
NHANES, 1971 to 1974) (17). All seven obese adolescents had
at least one obese parent (BMI . 30), and one subject had a
parent with type II diabetes and morbid obesity. Tanner stage
of pubertal development was assessed by physical exam in
the adolescent subjects; the stages ranged between II and IV
for pubic hair, breast, or genital development. Plasma estradiol, testosterone, and insulin-like growth factor I levels were
obtained and used as additional markers of puberty. All
subjects were healthy and taking no medications. The protocol was approved by the Human Investigation Committee of
the Yale School of Medicine, and informed written consent
was obtained from all subjects and the parents of the adolescent subjects. Subjects were seen on two separate visits for 1)
measurement of fat mass and 2) euglycemic-hyperinsulinemic clamp.
Measurement of fat mass. Bioelectrical impedance analysis
was used to estimate fat-free mass. At the time of measurement, subjects were instructed to fast for 3 h and to abstain
from exercise for 12 h before measurement. The total body
resistance and reactance were measured using a bioelectric
impedance analyzer (RJL 101A, Detroit, MI). Four surface
self-adhesive electrodes were placed according to the procedures recommended by the manufacturer. A standard conduction current of 800 µA and 50 kH was used. The equations
used for calculating the amount of fat-free mass were the
Kushner equation for the adults (23) and the Houtkooper
equation for the adolescents (20).
Euglycemic-hyperinsulinemic clamp. Subjects were admitted at 0800 to the Yale General Clinical Research Center for
metabolic study. All studies were performed in a postabsorptive state after an overnight fast of 10–12 h with the subject
lying supine in a quiet room, as described previously (9, 11). A
retrograde cannula was inserted into a vein in the dorsum of
the hand, which was positioned in a heated box for sampling
of arterialized venous blood. A small volume of normal saline
(0.9%) was infused through the intravenous cannula to
maintain patency. A second intravenous catheter was inserted in a contralateral antecubital vein for infusion of
insulin, glucose, and stable isotopes. At the start of a 2-h
baseline period, [2H5]glycerol was infused at a constant rate
of 1.2 mg · m22 · min21; this was continued for the duration of
the study (6 h). After the baseline period, a two-step hyperinsulinemic clamp was initiated. Insulin was administered
intravenously as a primed-continuous infusion at 8 and 40
mU · m22 · min21. Each step lasted for 2 h. Plasma glucose
levels were kept constant (90 mg/dl) using a variable rate
infusion of a 20% dextrose solution. Samples for the measurement of [2H5]glycerol enrichment, plasma glycerol, FFA,
insulin, and C-peptide were collected at 10-min intervals for
the last 30 min of the basal period and each step of the clamp.
Indirect calorimetry using the ventilated hood technique (10)
was also employed during the last 30 min of the basal period
and of each step of the clamp to estimate net rates of
carbohydrate and lipid oxidation.
Determinations. Plasma glucose levels were measured by
the glucose oxidase method with a Beckman glucose analyzer
(Beckman Instruments, Brea, CA). Plasma insulin and Cpeptide were measured by a double-antibody radioimmunoassay. Plasma nonesterified fatty acids (NEFA) were assayed by
a colorimetric method (18) and urinary nitrogen by the
Kjeldahl procedure (26). Plasma glycerol was measured by an
automatic ultrasensitive bioluminescence kinetic assay (19).
Blood for analysis of [2H5]glycerol enrichment was collected
in heparinized tubes and immediately placed in a 4°C ice
bath. The plasma was then separated by centrifugation and
stored at 270°C until time of analysis. Plasma proteins were
precipitated with Ba(OH)2 and ZnSO4, and the resultant
supernatant was passed through anion (Dowex AG1-X8) and
cation (Dowex AG-50-X8) exchange columns. The trimethylsilyl derivative is formed and 2H enrichment determined by a
gas chromatography-mass spectrometry system (HewlettPackard 5985, Palo Alto, CA) described by Wolfe (33).
Calculations: glycerol kinetics. Glycerol rate of appearance
(Ra ) was calculated according to the steady-state equation
(infusion rate divided by enrichment)
E739
LIPOLYSIS IN OBESE ADOLESCENTS
protein before and during the clamp procedure were calculated from the measured O2 consumption, CO2 production,
and urinary nitrogen excretion, as previously described (10).
Statistical analysis. All values are presented as means 6
SE. Repeated-measures analysis of variance (ANOVA) was
performed with a single factor to compare the responses of the
different groups over time (Systat v.5.0; SPSS, Chicago, IL).
One simple ANOVA and two group repeated-measures ANOVA
tests and Tukey’s procedure for multiple comparisons were
used to localize effects found in the initial set of repeatedmeasures analyses.
RESULTS
Table 2. Plasma insulin and C-peptide levels during low- and high-insulin dose clamp studies
Obese Adolescents
Plasma insulin, pmol/l
Basal
8 mU · m22 · min21
40 mU · m22 · min21
Plasma C-peptide, pmol/l
Basal
8 mU · m22 · min21
40 mU · m22 · min21
Plasma glucose, mg/dl
Basal
8 mU · m22 · min21
40 mU · m22 · min21
Lean Adolescents
Lean Adults
117 6 15*
192 6 24 (78 6 18)
552 6 72 (438 6 60)
61 6 8†
108 6 6 (48 6 12)
378 6 18 (318 6 30)
38 6 6
96 6 6 (60 6 12)
372 6 24 (336 6 30)
879 6 111*
836 6 115 (242 6 75†)
691 6 130 (2187 6 115)
466 6 71†
285 6 68 (2180 6 20)
205 6 85 (2261 6 58)
361 6 23
255 6 35 (2106 6 31)
185 6 39 (2175 6 33)
88 6 2
90 6 5
92 6 3
92 6 2
88 6 2
93 6 5
88 6 1
91 6 3
92 6 5
Values are means 6 SE, with changes from baseline in parentheses. * P , 0.01 vs. lean adolescents and adults; † P , 0.05 vs. lean adults.
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Plasma insulin, C-peptide, FFA, and glycerol concentrations. Fasting plasma insulin concentrations were
higher in the obese adolescents than in either of the
lean groups (P , 0.0001, Table 2). During the clamp,
although absolute insulin levels were higher in the
obese group at each time point, there was no significant
difference in the relative change from baseline between
any of the groups (Table 2). Basal C-peptide concentrations were also significantly higher in the obese adolescents compared with lean groups, and they did not
change significantly during the low-dose insulin infusion in the obese group. In contrast, a substantial
suppression of C-peptide was observed in both lean
groups during the low-dose clamp. During the highdose clamp, the change in C-peptide from baseline was
significant in all three groups. Despite elevated plasma
insulin levels, basal plasma FFA concentrations were
significantly higher in obese adolescents than in lean
adults (P , 0.005; Table 3). As shown in Table 3, during
both the low- and high-dose exogenous insulin infusions, circulating plasma FFA concentrations were
significantly elevated in obese adolescents compared
with lean adults (P , 0.005). Although obese adolescents tended to have higher plasma FFA than lean
adolescents throughout the study, the difference was
not statistically significant (F 5 2.25, P 5 0.159).
However, these seemingly similar levels occurred in the
presence of much higher insulin levels in the obese
group, indicating a shift to the right in the doseresponse curve for insulin’s suppression of lipolysis in
obese adolescents. FFA levels at baseline and during
the clamp studies tended to be higher in lean adolescents than in lean adults; however, the difference was
statistically significant only during the 40 mU insulin
clamp (P , 0.01). Basal and insulin-clamped plasma
glycerol levels were not statistically different among
the three groups. The fall in plasma glycerol concentrations was significant during the low and high insulin
infusions in all three groups (P , 0.02 vs. basal, Table 3).
Glycerol kinetics. The values for plasma [2H5]glycerol
enrichment in each group are reported in Table 3. As
can be seen, a steady state of plasma enrichment was
achieved in all three groups before and during each
step of the insulin clamp, and there was no difference
among the groups at any time during the study (F 5
0.9, P 5 0.424).
Figure 1 illustrates rates of glycerol turnover before
and during the clamp study in relation to plasma
insulin concentrations in the three groups of subjects.
Absolute glycerol turnover rates were higher (P , 0.05)
in the obese adolescents in the basal period (320 6 46
µmol/min) than in either lean adolescents (206 6 10
µmol/min) or lean adults (213 6 25 µmol/min) despite
two- to threefold higher fasting insulin levels. During
both the low- and high-dose insulin clamp steps, glycerol turnover rates remained significantly greater in
the obese compared with both lean groups (Fig. 1A). As
indicated in Table 1, fat mass was twofold greater in
obese adolescents than in either of the lean groups (P ,
0.01); we therefore also analyzed glycerol turnover
after normalizing for differences in fat mass. In contrast to absolute glycerol turnover data, basal glycerol
turnover per kilogram fat mass tended to be lower in
obese adolescents (10 6 1 µmol · kg fat mass21 · min21 )
compared with lean adolescents (17 6 4 µmol · kg fat
mass21 · min21 ) and adults (17 6 3 µmol · kg fat
mass21 · min21 ). However, this difference was not statistically significant and fell along the insulin doseresponse curve in nonobese subjects (P . 0.5; Fig. 1B).
Moreover, during insulin infusion, glycerol turnover
rates expressed per kilogram fat mass were suppressed, with no difference in responses noted among
the three groups. It should also be noted that plasma
glycerol turnover rates among the groups were also not
significantly different when expressed per square meter of body surface area (data not shown), another index
of relative adiposity.
E740
LIPOLYSIS IN OBESE ADOLESCENTS
Table 3. Plasma glycerol enrichment, plasma glycerol and free fatty acid levels during insulin infusions
Insulin Infusion Time, min
8
230
215
90
105
40 mU · m22 · min21
120
210
215
240
8.08 6 0.68 12.07 6 2.1 12.34 6 1.64 11.54 6 1.56 14.93 6 2.15 14.87 6 2.15 15.47 6 2.10
8.89 6 0.58 13.88 6 1.67 13.37 6 0.96 15.28 6 1.85 18.11 6 1.70 17.88 6 1.03 16.47 6 1.99
9.83 6 1.05 11.55 6 2.43 12.07 6 2.63 12.34 6 1.64 21.45 6 1.60 21.74 6 1.03 19.52 6 1.49
533 6 62†
373 6 63
272 6 55
530 6 56*
366 6 57
228 6 49
66 6 11*
72 6 14*
52 6 15*
67 6 14*
74 6 14*
50 6 15*
489 6 63*
345 6 63
223 6 56
313 6 34*
246 6 34‡
129 6 30
49 6 8*
57 6 8*
37 6 8*
244 6 30*
244 6 30‡
133 6 26
49 6 8*
65 6 4*
36 6 7*
342 6 31*
233 6 31‡
133 6 28
Values are means 6 SE. APE, atoms % excess. * P , 0.02 vs. basal values and significantly higher than lean adults; † P , 0.02 vs. lean adults;
‡ P , 0.05 vs. lean adults.
Glucose metabolism. Glucose infusion rates changed
minimally in all three groups during the low-dose
infusion. In contrast, glucose infusion rates increased
in all three groups during the high-dose insulin infusion, but to a lesser degree in the obese adolescents
(140 6 10 mg · m22 · min21 ) compared with lean adolescents (220 6 20 mg · m22 · min21 ) and lean adults (305 6
12 mg · m22 · min21 ) (P , 0.01). As shown in Table 4,
glucose oxidation rates at baseline and during the
lower insulin dose infusion were similar in all three
groups. However, during the high-dose insulin infusion, glucose oxidation rates were 25% greater in the
lean adults than in both adolescent groups (P , 0.05)
(Table 4). Of note, as shown in Table 4, basal fat
oxidation rates were greater in both obese and lean
adolescents than in lean adults (P , 0.01). Insulin
infusion suppressed fat oxidation rates by 80 and 40%
in the lean adults and lean adolescents, respectively,
during the higher dose (P , 0.05). In marked contrast,
in the obese adolescents, no change from baseline in fat
oxidation rates occurred at any step of the clamp study.
Fat oxidation correlated inversely with glucose oxidation (r 5 20.50, r2 5 0.25, P 5 ,0.001) in the three
groups.
DISCUSSION
In the present study, we have evaluated insulin
action on lipolysis in obese adolescents and compared
their response with responses observed in groups of
lean adolescents and lean adults. To assess lipolysis we
Table 4. Glucose and fat oxidation rates in basal state
and during 8 and 40 mU · m22 · min21 clamp
Obese
Adolescents
Fig. 1. Absolute glycerol turnover (µmol/min, A) and glycerol turnover per kg fat mass (µmol · kg fat mass21 · min21, B) as a function of
plasma insulin in obese adolescents (s), lean adolescents (r), and
lean adults (k). Absolute glycerol turnover rate in obese adolescents
was significantly greater than rates in both lean groups at baseline
and at the end of each step of insulin clamp, P , 0.05.
Glucose oxidation,
µmol · kg
LBM21 · min21
Basal
8 mU · m22 · min21
40 mU · m22 · min21
Fat oxidation, µmol · kg
LBM21 · min21
Basal
8 mU · m22 · min21
40 mU · m22 · min21
Glucose infusion rates,
mg · m22 · min21
8 mU · m22 · min21
40 mU · m22 · min21
10 6 2
10 6 2
17 6 3
5 6 2†
5 6 1†
4 6 0.8†
50 6 7
140 6 10‡
Lean
Adolescents
Lean
Adults
11 6 2
14 6 3
19 6 4*
13 6 3
12 6 2
24 6 3*
7 6 2†
6 6 2†
4 6 1†
3 6 0.8
2.0 6 0.7
0.7 6 0.2
37 6 12
220 6 20§
56 6 7
305 6 12
Values are means 6 SE. LBM, lean body mass. * P , 0.05 vs.
baseline; † P , 0.01 vs. lean adults; ‡ P , 0.01 vs. lean adolescents
and adults; § P , 0.05 vs. lean adults.
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[2H5 ]glycerol, APE
Obese adolescents
7.73 6 0.81 8.11 6 0.74
Lean adolescents
8.63 6 0.75 8.98 6 1.00
Lean adults
10.59 6 1.45 11.49 6 0.74
Plasma glycerol,
µmol/l
Obese adolescents
107 6 29
Lean adolescents
116 6 19
Lean adults
93 6 23
Plasma free fatty acid,
µM
Obese adolescents
860 6 83†
871 6 91†
Lean adolescents
809 6 83
781 6 92
Lean adults
567 6 73
577 6 80
0
mU · m22 · min21
LIPOLYSIS IN OBESE ADOLESCENTS
adolescents is superimposable on curves of the two lean
groups, indicating a normal rather than an increased
ratio of lipolysis to insulin. Although our study suggests
that the antilipolytic effects of insulin are normal in
juvenile obesity, it is important to acknowledge that
mobilization of lipid stores in response to epinephrine
has recently been found to be reduced during the
dynamic phase of fat accumulation (7). Further studies
are, however, needed to clearly establish whether alterations in the regulation of lipolysis contribute to the
development of obesity.
Even though individual adipocytes may be normally
sensitive to insulin in obese adolescents, the enlarged
fat mass resulted in a net increase in the rates of
lipolysis before and during the insulin clamp procedure. In parallel with the increased circulating plasma
FFA concentrations, we have found increased net lipid
oxidation rates in the basal state and during the clamp
in the obese adolescents compared with lean adults.
The coexistence of increased fat oxidation and decreased glucose metabolism suggests that the defects in
glucose metabolism in obese adolescents at least in part
result from substrate competition between glucose and
FFA, as proposed by the Randle cycle (30).
Another interesting finding of the current study is
the lack of feedback inhibition of endogenous insulin
secretion in the obese group. This is indicated by the
significantly greater changes in and percent suppression of plasma C-peptide levels in both lean groups
compared with the obese group. Thus, in contrast to
nonobese individuals, at a very early stage of obesity
the b-cell appears to be blind to the circulating levels of
insulin and does not promptly decrease its secretion as
occurs in lean individuals, even during a very low
insulin infusion. Insulin and C-peptide are secreted in
an equimolar fashion and, unlike insulin, C-peptide is
not extracted by the liver and therefore is a better
reflection of ‘‘true’’ insulin secretion (28, 29). It is
conceivable that these higher plasma C-peptide levels
are due to an altered metabolic clearance rate in obese
compared with lean children. However, studies in obese
adults have indicated that the metabolic clearance of
C-peptide is unaltered by the induction of hyperinsulinemia. Thus inadequate feedback suppression may account for the prevailing hyperinsulinemia of juvenile
obesity, as also described in studies by Elahi et al. (13)
in obese Pima Indians. This chronic elevation in insulin
levels may favor accumulation of triglycerides in muscle,
which in turn will interfere with glucose storage and
oxidation, leading to insulin resistance. It is conceivable that alterations in skeletal muscle metabolism
might play a role in the etiology of obesity. This is,
however, an area that clearly needs further study.
Our present study also provides information about
insulin’s effects on lipolysis and fat oxidation rates in
the insulin resistance of puberty. We found no differences between the lean adolescents and adults with
regard to the ability of insulin to suppress glycerol
turnover, an index of lipolysis. In contrast, suppression
of lipid oxidation was impaired in lean pubertal subjects vs. lean adults despite equivalent insulin concen-
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used the appearance rate of glycerol in plasma, because
it is thought to be a more reliable index of lipolysis than
that of FFA turnover (5, 27). In the postabsorptive
state, glycerol turnover in absolute terms was enhanced and less suppressed by insulin in obese adolescents compared with either lean group. To correct for
differences in body composition, we also analyzed glycerol flux per kilogram of adipose tissue. With this
analysis, basal glycerol flux adjusted per fat mass
tended to be lower in obese adolescents than in either
lean adolescents or lean adults, but the difference was
not significant and was appropriate for the level of
insulin present. Thus, when the enlarged fat mass and
increased insulin levels of the obese adolescents are
taken into account, the elevated fasting glycerol turnover rates are normalized.
Although absolute basal glycerol turnover rates were
elevated in obese adolescents, their basal plasma glycerol levels were not significantly different from those of
lean adolescents. The reason for this is not clear, but it
may be due to a poor relationship between plasma
levels and turnover rates or to a different glycerol
clearance. Previous studies in obese adults found increased basal lipolytic rate expressed per lean body
mass, whereas these rates appeared to be decreased
when adjusted for differences in fat mass. Campbell et
al. (8) showed that, despite impaired insulin sensitivity,
the release of FFA from adipose tissues, expressed per
kilogram fat mass, was less in obese vs. lean subjects
after an overnight fast, as the result of a reduction of
lipolysis and an increase in FFA reesterification. Using
a stepwise euglycemic insulin clamp technique in combination with an infusion of [14C]palmitate and indirect
calorimetry, Groop et al. (16) reported that the absolute
rate of basal FFA turnover was greater and less suppressed by insulin in obese compared with lean subjects. To our knowledge none of these studies was
performed in subjects still accumulating fat. Our study,
on the other hand, provides a unique documentation of
fatty acid metabolism and lipolysis during the active
phase of excessive fat accretion.
Our results are also somewhat similar to those of
LeStunff and Bougnères (24), who reported that obese
prepubertal children have greater absolute rates of
glycerol turnover than lean prepubertal children. However, even in these younger subjects, the rates of
lipolysis were significantly lower than those of controls
when corrected for adipose tissue mass. They concluded
that, during the initial dynamic phase of human obesity, the sensitivity of lipolysis to insulin may actually
be increased. However, the greater suppression in
glycerol turnover rates per kilogram of fat mass reported by those investigators is likely to be due to the
higher ambient basal plasma insulin levels compared
with levels seen in the nonobese children. As shown in
Fig. 1B, the lower basal glycerol turnover rates per
kilogram of adipose tissue in obese subjects can be
accounted for by basal hyperinsulinemia. When data
relating glycerol turnover to plasma insulin under
basal and insulin-stimulated conditions are interpolated (Fig. 1B), it is notable that the curve for obese
E741
E742
LIPOLYSIS IN OBESE ADOLESCENTS
We are grateful to all the children for participating in the study.
We thank the nursing staff of the Pediatric General Clinical Research
Center for the excellent care given to our subjects during these
studies. We are indebted to the staff of the Core Laboratory of the
Clinical Research Center for their technical assistance, and we
particularly thank Dr. G. Cline for measurements of [2H5]glycerol. We
are grateful to Nancy Canetti for superb preparation of the manuscript.
This study was supported by National Institutes of Health Grants
RO1-HD-28016, MO1-RR-00125, MO1–06022, RO1-DK-20495, RO1HD-30671, RO1–49230, and P30-DK-45735.
Address for reprint requests: S. Caprio, Dept. of Pediatrics, 333
Cedar St., Yale Univ. School of Medicine, New Haven, CT 06520.
Received 28 July 1997; accepted in final form 9 January 1998.
REFERENCES
1. Arner, P. Control of lipolysis and its relevance to development of
obesity in man. Diabetes Metab. Rev. 5: 507–551, 1988.
2. Arner, P., J. Bolinder, P. Engfeldt, and J. Östman. The
antilipolytic effect of insulin in human adipose tissue in obesity,
diabetes mellitus, hyperinsulinemia, and starvation. Metab.
Clin. Exp. 30: 753–760, 1981.
3. Arslanian, S., and S. K. Kalhan. Correlations between fatty
acid and glucose metabolism. Potential explanation of insulin
resistance of puberty. Diabetes 43: 908–914, 1994.
4. Bevilacqua, S., K. Bonadonna, G. Buzziqoli, C. Boni, F.
Bioliar, M. Maccori, and E. Ferrannini. Acute elevation of
fatty acid levels leads to hepatic insulin resistance in obese
subjects. Metabolism 36: 502–506, 1987.
5. Boden, G., X. Chen, R. A. DeSantis, and Z. Kendrick. Effects
of insulin on fatty acid reesterification in healthy subjects.
Diabetes 42: 1588–1593, 1993.
6. Boden, G., X. Chen, J. Ruiz, J. V. White, and L. Rossetti.
Mechanisms of fatty acid-induced inhibition of glucose uptake. J.
Clin. Invest. 93: 2438–2446, 1994.
7. Bougnères, P., C. LeStunff, C. Pecquer, E. Pinglier, P.
Adnot, and D. Ricquier. In vivo resistance of lipolysis to
epinephrine. A new feature of childhood obesity. J. Clin. Invest.
99: 2568–2573, 1997.
8. Campbell, P. J., M. Carlson, and N. Nurjhan. Fat metabolism
in human obesity. Am. J. Physiol. 266 (Endocrinol. Metab. 29):
E600–E605, 1994.
9. Caprio, S., M. Bronson, R. S. Sherwin, F. Rife, and W. V.
Tamborlane. Co-existence of severe insulin resistance and
hyperinsulinemia in pre-adolescent obese children. Diabetologia
39: 1489–1497, 1996.
10. Caprio, S., R. A. Gelfand, W. V. Tamborlane, and R. S.
Sherwin. Oxidative fuel metabolism during mild hypoglycemia:
critical role of free fatty acids. Am. J. Physiol. 256 (Endocrinol.
Metab. 19): E413–E419, 1989.
11. Caprio, S., L. D. Hyman, C. Limb, S. McCarthy, R. Lange, R.
Sherwin, J. Shulman, and W. V. Tamborlane. Central adiposity and its metabolic correlates in obese adolescent girls. Am. J.
Physiol. 269 (Endocrinol. Metab. 32): E118–E126, 1995.
12. DeFronzo, R. A., J. D. Tobin, and R. Andres. Glucose clamp
technique: a method for quantifying insulin secretion and resistance. Am. J. Physiol. 237 (Endocrinol. Metab. Gastrointest.
Physiol. 6): E214–E223, 1979.
13. Elahy, D., M. Nagulasperan, J. Hershcopf, D. Muller, J.
Tobin, P. Blix, A. Rubenstein, R. Unger, and R. Andres.
Feedback inhibition of insulin secretion by insulin: relation to
the hyperinsulinemia of obesity. N. Engl. J. Med. 306: 1196–
1202, 1982.
14. Ferrannini, E., E. J. Barrett, S. Bevilacqua, and R. A.
DeFronzo. Effect of fatty acids on glucose production and
utilization in man. J. Clin. Invest. 72: 1737–1747, 1983.
15. Gortmaker, S. L., W. H. Dietz, A. M. Sobol, and C. A. Wehler.
Increasing pediatric obesity in the United States. Am. J. Dis.
Child. 141: 535–540, 1987.
16. Groop, L. C., R. C. Bonadonna, D. C. Simonson, A. S.
Petrides, M. Shank, and R. A. DeFronzo. Effect of insulin on
oxidative and nonoxidative pathways of free fatty acid metabolism in human obesity. Am. J. Physiol. 263 (Endocrinol. Metab.
26): E79–E84, 1992.
17. Hammer, L. D., H. C. Kraemer, D. M. Wilson, R. L. Ritter,
and S. M. Dornbusch. Standardized percentile curves of body
mass index for children and adolescents. Am. J. Dis. Child. 145:
259–263, 1991.
18. Hawk, P. B. Kjeldahl method. In: Practical Physiological Chemistry. Toronto: Blakison, 1947, p. 814–822.
19. Hellner, J., P. Arner, and A. Lundin. Automatic luminometric
kinetic assay for glycerol for lipolysis studies. Anal. Biochem.
177: 132–137, 1989.
20. Houtkooper, L. B., T. G. Lohman, S. B. Going, and M. C.
Hall. Validity of bioelectrical impedance for body composition
assessment in children. J. Appl. Physiol. 66: 814–821, 1989.
21. Kelley, D. E., M. Mokan, J. A. Simonson, and L. J. Mandarino. Interaction between glucose and fatty acid metabolism in
human skeletal muscle. J. Clin. Invest. 92: 91–98, 1993.
22. Knittle, J. L., K. Timmes, F. Ginsberg-Fellner, A. E. Brown,
and D. P. Katz. The growth of adipose tissue in children and
adolescents. J. Clin. Invest. 68: 239–246, 1979.
23. Kushner, R. F., D. A. Schoeller, C. R. Feld, and L. Danford.
Is the impedance index (ht2/R) significant in predicting body
water? Am. J. Clin. Nutr. 56: 836–839, 1992.
24. LeStunff, S., and P. F. Bougnères. Glycerol production and
utilization during the early phase of human obesity. Diabetes 41:
444–450, 1992.
25. Must, A., P. F. Jacques, G. E. Dallal, C. J. Bajema, and W. H.
Dietz. Long-term morbidity and mortality of overweight adolescents. A follow-up of the Harvard growth study of 1922 to 1935.
N. Engl. J. Med. 327: 1350–1355, 1992.
26. Novak, M. Colorimetric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6: 431–433, 1965.
27. Nurjhan, N., P. Campbell, F. Kennedy, J. Miles, and J.
Gerich. Insulin-dose response characteristics for suppression of
glycerol release and conversion to glucose in humans. Diabetes
35: 1326–1331, 1986.
28. Polonsky, K., B. D. Given, L. Hirsh, E. T. Shapiro, H. Tillil,
C. Beebe, J. A. Galloway, B. H. Frank, T. Karrison, and E.
Van Cauter. Quantitative study of insulin secretion and clearance in normal and obese subjects. J. Clin. Invest. 81: 435–441,
1988.
29. Polonsky, K., and A. H. Rubenstein. C-peptide as a measure
of the secretion and hepatic extraction of insulin. Pitfalls and
limitations. Diabetes 33: 486–496, 1984.
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trations during both steps of the clamp (Tables 2 and 4).
These findings are in agreement with those of Arslanian and Kalhan (3), who reported that increased lipid
oxidation may contribute to the insulin resistance
normally observed during puberty. Of particular interest, in their study Arslanian and Kalhan found a
relationship between plasma insulin-like growth factor
I levels and total body lipid oxidation rates, suggesting
that increased growth hormone secretion during puberty might be responsible for the augmented rates of
lipid oxidation. Attractive as it is, however, this hypothesis needs further evaluation.
In summary, in contrast to the marked resistance to
insulin’s stimulatory effect on glucose metabolism, the
lipolytic rate in adipose tissue of obese adolescents is
normally sensitive to the insulin’s suppressive effects.
Lipolysis, as assessed by the glycerol appearance rate
in plasma in obese adolescents, is increased as a
consequence of the expanded adipose mass, which in
turn results in increased plasma FFA. Inhibition of net
lipid oxidation rates is markedly impaired in early
onset obesity. The augmented supply of fatty acid
substrates and their ability to interfere with glucose
oxidation and storage may be an important component
of insulin resistance in adolescent obesity.
LIPOLYSIS IN OBESE ADOLESCENTS
30. Randle, P. J., P. B. Garland, E. A. Newsholme, and C. M.
Hales. The glucose fatty acid cycle in obesity and maturity onset
diabetes mellitus. Ann. NY Acad. Sci. 131: 324–333, 1965.
31. Roden, M., T. B. Price, G. Perseghin, K. Falk Petersen, D.
Rothman, G. W. Cline, and G. I. Shulman. Mechanism of free
fatty acid-induced insulin resistance in humans. J. Clin. Invest.
97: 2859–2865, 1996.
32. Troiano, R. P., K. M. Flegal, R. J. Kucemarski, S. M.
Campbell, and C. L. Johnson. Overweight prevalence and
E743
trends for children and adolescents. The National Health and
Nutrition Examination Surveys, 1963 to 1991. Arch. Pediatr.
Adol. Med. 149: 1085–1091, 1995.
33. Wolfe, R. R. Radioactive and Stable Isotope Tracers in Biomedicine. New York: Liss, 1983, p. 146–147.
34. Wolfe, R. R., S. Klein, F. Carraro, and J.-M. Weber. Role of
triglyceride-fatty acid cycle in controlling fat metabolism in
humans during and after exercise. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E382–E389, 1990.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on June 17, 2017