(Received for publication, October 2, 1995)
From the
The physiological role of circulating insulin-like growth
factor-II (IGF-II) in adult humans is poorly understood. We recently
generated an IGF-II transgenic murine model of persistent IGF-II
production (plasma IGF-II 30-fold increased above normal) through
overexpression of the transgene driven by the major urinary protein
promoter (Rinderknecht, E., and Humbel, R. E.(1978) J. Biol. Chem. 269, 13779-13784). To determine whether in vivo insulin action is improved in these transgenic mice, we performed
euglycemic insulin (18 milliunits/kg
min) clamp studies in
conscious IGF-II transgenic and in age- and weight-matched control
mice. Plasma glucose and insulin concentrations were significantly
lower in the IGF-II transgenic compared with both control groups.
Despite decreased plasma glucose concentration, basal hepatic glucose
production (HGP) and glucose clearance were increased.
During the
insulin clamp studies in IGF-II transgenic mice compared with control
mice (a) the rates of glucose infusion and glucose uptake were
increased by 65 and
55%, respectively; (b)
glycolysis was increased by
12% while glycogen synthesis was
2-fold higher; (c) while the suppression of plasma free
fatty acid was similar, the increment in plasma lactate concentration
was significantly higher; (d) although HGP was similarly
inhibited by insulin, phosphoenolpyruvate gluconeogenesis was enhanced
and accounted for a larger portion of HGP (64% versus
40%
in control mice).
Our data suggest that the persistence of circulating IGF-II in adult mice to levels commonly observed in adult humans (50-70 nM) causes a marked improvement in peripheral (skeletal muscle) insulin action, which is not due to changes in body composition. These results suggest that circulating IGF-II may exert a regulatory role on insulin sensitivity and body composition in humans.
Insulin-like growth factors I and II are structurally related to
proinsulin (1, 2) and exert growth promoting (3) and metabolic effects (4) . In rodents, IGF-II ()plays an important role during fetal
development(5, 6) , while its gene expression and
circulating concentrations are virtually suppressed
postnatally(7) . Conversely, elevated plasma IGF-II
concentrations have been reported in adult humans (8) with only
a slight decline with aging (9) . IGF-I levels tend to
progressively decrease in middle-aged and old
humans(8, 9) . Recent observations from several
laboratories have spurred interest in the potential physiologic
significance of circulating IGFs in the alterations in body composition
and insulin sensitivity of human aging(10, 11) . These
observations suggest that IGF-II may have a metabolic role in humans.
To examine the potential metabolic function of IGF-II in adulthood, we generated transgenic mice, which overexpress the prepro IGF-II transgene in the liver starting at 3-5 weeks of age(12) . These transgenic mice maintained elevated plasma IGF-II concentrations throughout their lives, in the range commonly observed in young human subjects(8, 12) . Compared with control mice, IGF-II transgenic mice had lower plasma glucose and insulin concentrations and gained less weight between 4 and 18 months of age(12) . The lower body weight was largely due to a marked decrease in fat mass(12) . The metabolic impact of the persistence of high IGF-II levels in adult mice may be interpreted in view of our current understanding of IGFs action in vivo. IGF-II has been shown to stimulate glucose disposal during short term infusions in rodents (13) and humans(14) . Yet, the effect of chronic elevations in the circulating IGF-II concentrations on glucose fluxes have not been examined. Although IGF-II binds with high affinity to the IGF-II/mannose 6-phosphate receptors, there is strong evidence that its growth promoting and metabolic effects are not mediated via this interaction(15) . It is likely that IGF-II exerts its action acting through the IGF-I and/or the insulin receptor depending on the target tissue(15, 16) . In this regard, it is of interest to note that in rodents, IGF-I has differential effects on intermediate metabolism compared with insulin, with more potent effects on protein metabolism and modest effects on lipid metabolism(17) .
Since our previous observation of changes in body composition and plasma glucose concentrations suggested that IGF-II transgenic mice have specific changes in whole body glucose metabolism, we examined tissue sensitivity to insulin and basal glucose kinetics in transgenic and control mice using the insulin clamp technique in combination with tracer infusions. In order to estimate the impact of changes in body composition on insulin sensitivity, we also compared the IGF-II transgenic mice to a group of younger negative control mice matched for body weight. IGF-II transgenic mice had a marked increase in both basal and insulin-stimulated glucose uptake and glycogen synthesis, increased basal HGP, and increased contribution of gluconeogenesis to HGP during the insulin clamp studies. Since the above metabolic effects could not be ascribed to the associated changes in body composition, it is likely that they are the direct consequence of the elevated IGF-II levels.
The studies lasted
170 min and included an 80-min basal period for assessment of basal
turnover rates and a 90-min euglycemic clamp period. 80 min before
starting the insulin infusion, a prime-continuous infusion of high
performance liquid chromatography-purified
[3-H]glucose (DuPont NEN; 10-µCi bolus, 0.1
µCi/min) was initiated and maintained throughout the remainder of
the study. [U-
C]lactate (5-µCi bolus/0.25
µCi/min) was infused during the last 10 min of the study. Plasma
samples for determination of [
H]glucose specific
activity were obtained from the tail vein at 40, 60, 70, and 80 min
during the basal period and at 40, 60, 70, 80, and 90 min during the
clamp period. Steady state conditions for the plasma glucose
concentration and specific activity were achieved within 40 min in both
the basal and clamp periods of the studies. Plasma samples for
determination of plasma insulin concentrations were obtained at time
-30, 0, 40, 60, 90 min during the study. The total volume of
blood withdrawn was
0.9 ml/study; to prevent volume depletion and
anemia, a solution (1:1 (v/v)) of
1.2 ml of fresh blood (obtained
by heart puncture from a littermate of the test animal) and heparinized
saline (10 units/ml) was infused. All determinations were also
performed on portal vein blood obtained at the end of the experiment.
At the end of the insulin infusion, mice were anesthetized
(pentobarbital 60 mg/kg body weight, intravenously), the abdomen was
quickly opened, portal vein blood was obtained, and the liver was
freeze-clamped in situ with aluminum tongs precooled in liquid
nitrogen. The time from the injection of the anesthetic until
freeze-clamping of the liver was less than 45 s. All tissue samples
were stored at -80 °C for subsequent analysis.
The study protocol was reviewed and approved by the Institutional Animal Care and Use Committees of the Albert Einstein College of Medicine.
Glycogenolysis was calculated as the difference between the HGP and
the gluconeogenesis. The percent of the hepatic glucose 6-phosphate
pool directly derived from plasma glucose was calculated as the ratio
of [H]UDPG and plasma
[3-
H]glucose specific activities. Thus, this
ratio also measures the percent contribution of plasma glucose to the
glucose-6-phosphatase flux (i.e. glucose cycling). Since total
glucose output is equal to the sum of the HGP plus glucose cycling (and
Glucose cycling = [
H]UDP-Glc specific
activity/plasma [3-
H]glucose specific activity
total glucose output), the equation can be resolved to
calculate both GC and TGO: TGO = HGP/(1 -
[
H]UDPG specific activity/plasma
[3-
H] glucose specific activity) and GC =
[
H]UDPG specific activity/plasma
[3-
H]glucose SA
TGO(22) . It
should be pointed out that this tracer methodology measures
PEP-gluconeogenesis, which represents the great majority of the
gluconeogenic flux under most experimental conditions. However, it may
underestimate the overall gluconeogenic rate under experimental
conditions in which non-PEP gluconeogenesis is significantly increased.
Figure 1: Rates of HGP during the basal period and during the insulin clamp studies in conscious mice. I, age-matched control (n = 9); II, weight-matched control (n = 5); III, IGF-II transgenic mice (n = 7). *, p < 0.01 versus basal; &, p < 0.01 versus I and II under the same conditions.
Figure 2: Rates of glucose disappearance during the basal period and during the insulin clamp studies in conscious mice. I, age-matched control (n = 9); II, weight-matched control (n = 5); III, IGF-II transgenic mice (n = 7). *, p < 0.01 versus basal; &, p < 0.01 versus I and II under the same conditions.
Figure 3:
Pathways of intracellular glucose disposal
during the insulin clamp studies in conscious mice. Rates of glycolysis
were derived from the rate of conversion of [3-H]
glucose to
H
O(12) . Rates of glycogen
synthesis were estimated by subtracting the rate of glycolysis from the R
. I, age-matched control (n = 9); II, weight-matched control (n = 5); III, IGF-II transgenic mice (n = 7). &, p < 0.01 versus I and II under the same conditions.
The [H]- and
[
C]UDP-Glc specific activities, the
[
C]PEP, and the [
H]glucose
specific activities were used to calculate the contribution of plasma
glucose-derived (direct pathway in Table 3) and PEP-derived
glucose 6-phosphate (indirect pathway in Table 3) to the hepatic
glucose 6-phosphate pool. Table 3displays the hepatic glucose
fluxes during the insulin clamp studies. While the direct pathway was
similar in all groups, the portion of the hepatic glucose 6-phosphate
pool formed via gluconeogenesis was
42% higher in the IGF-II
transgenic mice. Total glucose output and glucose cycling were similar
in all groups (Table 3). However, in transgenic mice,
gluconeogenesis was significantly higher and glycogenolysis was
significantly lower than in negative control mice (Table 3).
The purpose of the present study was to delineate the metabolic consequence of the overexpression of the human IGF-II gene in adult mice. This transgenic mouse model was developed through germline insertion of a minigene containing the human prepro-IGF-II cDNA placed under the control of the MUP promoter(12) . Use of this promoter allow one to discern the effects of IGF-II overproduction in adulthood from the noteworthy growth promoting effects during fetal development (5) . In fact, consistent with the natural expression pattern of the MUP promoter, transgene expression was demonstrated in the livers of IGF-II homozygous mice starting at 3-4 weeks of age(12) . While IGF-II levels were almost undetectable in the plasma of negative control mice, they were increased to 35-65 nM in IGF-II transgenic mice(12) . Initial analyses of this model revealed the presence of hypoglycemia and lower body weight in adult homozygous mice(12) . To investigate the mechanisms responsible for the alterations in glucose homeostasis in IGF-II transgenic mice, we compared conscious transgenic mice to age- and weight-matched negative control mice. The use of two control groups was necessary to account for the potential effects of altered body composition per se on the metabolic parameters. Mice were examined under basal (6 h fast) conditions and during insulin clamp studies to assess insulin sensitivity.
In the postabsorptive state, IGF-II transgenic mice were characterized by a moderate decrease in both plasma glucose and insulin concentrations, in agreement with our previous observation in 18 h fasted mice(12) . Whole body glucose homeostasis is maintained by the balance between HGP and peripheral glucose utilization. Thus, postabsorptive hypoglycemia could be the consequence of either decreased HGP or increased glucose disposal. Systemic glucose clearance was indeed markedly increased in IGF-II transgenic mice compared with both negative control groups, but HGP was also increased by 20-34%. This pattern differs from that observed with insulin-induced hypoglycemia in which HGP is decreased and glucose clearance is increased (26) and suggests the presence of a factor stimulating glucose disposal in the absence of a significant inhibitory effect on HGP. In fact, while the increased HGP is consistent with the decrease in circulating insulin and glucose concentrations, the marked stimulation of basal glucose clearance is paradoxical. In view of the known metabolic effects of IGFs on glucose metabolism in rodents(13, 17) , we offer the following interpretation of the results. Circulating IGF-II levels in our homozygous transgenic mice achieved levels sufficient to promote glucose disposal in peripheral tissues (mostly skeletal muscle). The increased disposal of glucose in muscle causes a decline in the plasma glucose concentrations and an appropriate decrease in insulin secretion. The lower plasma insulin concentrations directly or indirectly bring about the increased HGP. In this regard it is likely that in the presence of basal plasma insulin concentrations skeletal muscle glycogen synthase and pyruvate dehydrogenase were partially inactive and that a sizable portion of the incoming glucose was metabolized to lactate through anaerobic glycolysis. The absence of a significant increase in the circulating lactate concentrations may be explained by concomitant increases in both peripheral lactate release and lactate utilization via gluconeogenesis. Finally, although hypoglycemia may also trigger hormonal counter-regulation with increases in plasma glucagon and free fatty acid concentrations, we were unable to demonstrate such an effect in IGF-II transgenic mice. Thus, the postabsorptive hypoglycemia was due to increased systemic clearance of glucose. Overall, this observation in IGF-II transgenic mice closely resembles the physiologic profile of transgenic mice overexpressing the human GLUT4 or GLUT1 protein in insulin target tissues(27, 28, 29, 30) .
To
directly examine the sensitivity of tissue glucose uptake and HGP to
insulin in IGF-II transgenic mice, we performed euglycemic
hyperinsulinemic (850 microunits/ml) clamp studies in conscious
mice. Whole body glucose utilization (R
) and the
rate of glucose infusion required to maintain euglycemia were
55-65% higher in IGF-II transgenic mice compared with both
negative control groups in the presence of equal plasma glucose and
insulin concentrations. This marked increase in insulin-stimulated
glucose uptake (+180-194 µmol/kg
min) cannot be
solely explained by the increased rate of basal turnover
(+38-59 µmol/kg
min). We also examined the two
major pathways of intracellular glucose disposal. While both glycolysis
and glycogen synthesis were significantly increased in IGF-II
transgenic mice, the increased glycogen deposition accounted for
>70% of the increased R
. This improvement in
the efficiency of glycogen synthesis during hyperinsulinemia was
confirmed at the tissue level. In fact, the rate of
[3-
H]glucose incorporation in skeletal muscle
glycogen was
2-fold increased in the IGF-II transgenic compared
with both negative control groups. Thus, in the presence of
hyperinsulinemia, the excess of glucose taken up in skeletal muscle is
efficiently stored in muscle glycogen. The increased rates of glycogen
synthesis during periods of relative hyperinsulinemia (postprandially)
in IGF-II transgenic mice may provide substrates for the enhanced
gluconeogenesis and HGP during the following postabsorptive/fasting
periods. While basal HGP was significantly increased in the transgenic
mice, its inhibition during the hyperinsulinemic clamp studies was
similar to control. The paucity of type I IGF receptors in the adult
liver (31) may account for the inability of the elevated IGF-II
concentrations to restrain the hepatic production of glucose. The
similar HGP in the presence of equal plasma insulin and glucose
concentrations provide further support for the notion that the
increased HGP under basal conditions is secondary to the lower plasma
glucose and insulin concentrations. However, it is of interest that
while the negative control mice derived 60% of the HGP from glycogen
breakdown, the IGF-II transgenic mice were characterized by a marked
increase in gluconeogenesis, which comprised >60% of HGP. While we
failed to demonstrate significant alterations in basal plasma glucagon
concentrations or in the activity of key hepatic enzymes, such as
glucokinase, glucose-6-phosphatase, and phosphoenolpyruvate
carboxykinase, there was a marked increase in the plasma lactate
concentrations during the insulin clamp studies in the IGF-II
transgenic mice but not in age-matched control mice. Importantly, this
occurred despite a likely increase in hepatic lactate utilization for
gluconeogenesis. Indeed, an increase in the availability of
gluconeogenic precursors has been shown to result in enhanced
gluconeogenesis in the absence of changes in HGP(32) . Thus,
the hepatic overexpression of IGF-II in postpuberal mice leading to a
20-30-fold increase in circulating IGF-II concentrations
increased basal and insulin-stimulated glucose disposal, muscle
glycogen synthesis, and hepatic gluconeogenesis.
Although it is likely that these metabolic changes are due to high circulating IGF-II levels per se, some alternative hypothesis should also be considered. The sustained increase in plasma IGF-II concentrations is expected to lower growth hormone (GH) secretion. A decrease in GH may contribute to some of the observed changes in body composition and insulin sensitivity. Although we did not directly measure GH levels in these mice, the modest and inconsistent alterations in IGF-I and IGF-binding proteins concentrations previously reported in these mice (12) appear to indicate that the GH levels were maintained to near-normal levels. Furthermore, the administration of GH to aging humans has been associated with increased lean body mass and decreased fat mass, suggesting a role of GH deficiency for the relative increase in fat mass in human aging(10) . These effects of the hormone are opposite to those observed in this transgenic model. It is also possible that the marked metabolic impact of the postpuberal increase in IGF-II gene expression is mediated by its effects on body composition. In fact, the body weight and composition of 8-10-month-old IGF-II transgenic mice (12) closely resembles those of younger (4-6-month-old) negative controls. In fact, lean body mass comprises a larger portion (91-97% versus 84%) of body weight in IGF-II transgenic mice compared with age-matched negative control mice. However, the observation that the rates of basal and insulin-stimulated glucose disposal were markedly increased in IGF-II transgenic mice compared with young weight-matched negative controls suggest that other factors contribute to the metabolic changes in this transgenic model. Conversely, it may be speculated that the changes in body composition are the consequence of the sustained metabolic actions of IGF-II in this transgenic model. In fact, since IGF-II displays higher affinity for the IGF-I than for the insulin receptors(33) , it may exert more potent effects on protein anabolism than on lipogenesis compared with insulin(17) . The concomitant decrease in circulating insulin levels is likely to contribute to decreased net lipogenesis. Recent data from transgenic mice with overexpression of the glucose transporter subtype, GLUT 4, in adipose cells suggests that in vivo glucose flux may regulate fat accretion in mice(30) . Thus, a relative increase in the disposal of glucose in skeletal muscle versus adipose tissue may also contribute to decreased fat mass and preserved lean body mass in IGF-II transgenic mice. Finally, the independent hypoglycemic effects of IGF-II are supported by the observation of tumor-associated hypoglycemia in patients with increased IGF-II gene expression in tumor cells(34) . Although there is no conclusive evidence for an age-related decrease in plasma IGF-II concentrations in humans, the reported decline in IGF-I levels (10, 11) is likely to determine an overall decrease in IGF action in human aging(8, 9, 10, 11) .
In conclusion, the postpuberal increase in IGF-II gene expression in mouse liver generating plasma IGF-II levels comparable with those of adult humans causes a marked improvement in glucose homeostasis with increased basal and insulin-stimulated rates of glucose disposal in adult transgenic mice. It is suggested that IGF-II has a physiologic metabolic function in adult humans.