Hypertension, Hypertriglyceridemia, and Impaired Endothelium-dependent
Vascular Relaxation in Mice Lacking Insulin Receptor Substrate-1
Hideki Abe,* Nobuhiro Yamada,‡ Katsuo Kamata,i Tomoyuki Kuwaki,§ Masako Shimada,‡ Junichi Osuga,‡ Futoshi Shionoiri,‡
Naoya Yahagi,‡ Takashi Kadowaki,‡ Hiroyuki Tamemoto,‡ Shun Ishibashi,‡ Yoshio Yazaki,‡ and Masatoshi Makuuchi**Second Department of Surgery, ‡Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan;§
Second Department of Physiology, School of Medicine, Chiba University, Chiba, Japan; and iDepartment of Physiology andMorphology, Institute of Medicinal Chemistry, Hoshi University, Tokyo, Japan

Abstract
Insulin resistance is often associated with atherosclerotic
diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistanceon coronary risk factors in IRS-1 deficient mice, a nonobese
animal model of insulin resistance. Blood pressure andplasma triglyceride levels were significantly higher in IRS-1
deficient mice than in normal mice. Impaired endotheliumdependent vascular relaxation was also observed in IRS-1
deficient mice. Furthermore, lipoprotein lipase activity was
lower than in normal mice, suggesting impaired lipolysis to
be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistanceplays an important role in the clustering of coronary risk
factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance. (J. Clin. Invest.
1998. 101:1784–1788.) Key words: insulin resistance • hypertension • hypertriglyceridemia • insulin receptor substrate-1 •endothelium-dependent vascular relaxation

Introduction
Insulin receptor substrate-1 (IRS-1)1 is the major substrate of
the insulin receptor and insulin-like growth factor (IGF)-1 receptor tyrosine kinase (1, 2). Recent studies have established ahomozygous mouse model for targeted disruption of the IRS-1
gene (3, 4). IRS-1 mutant mice show retarded embryonal andpostnatal growth and develop resistance to glucose-lowering
effects of insulin, IGF-1, and IGF-2. Although blood glucose
levels do not differ significantly between mutant and control
mice either before or after oral glucose loading, serum insulinlevels are significantly higher in homozygous mutant mice than

Address correspondence to Nobuhiro Yamada, M.D., Third Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-3815-5411; FAX: 81-3-56897230.
Received for publication 25 August 1997 and accepted in revised
form 12 February 1998.
1. Abbreviations used in this paper: ACh, acetylcholine; HL, hepatic
triglyceride lipase; IRS-1, insulin receptor substrate-1; LPL, lipoprotein lipase; SNP, sodium nitroprusside.
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
0021-9738/98/04/1784/05 $2.00
Volume 101, Number 8, April 1998, 1784–1788
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Abe et al.

in wild-type or heterozygous mice. Thus, the phenotype of
IRS-1 knockout mice resembles non–insulin-dependent diabetes mellitus at the prediabetes stage with insulin resistance.
Insulin resistance with hyperinsulinemia is often associatedwith clustering of coronary risk factors including hypertension,
glucose intolerance, obesity, hypertriglyceridemia, and low
plasma HDL-cholesterol levels (5–7). This leads to an increased risk of cardiovascular disease, presumably due to promotion of atherosclerosis. However, the mechanisms associated with this clustering of coronary risk factors in subjects
with insulin resistance are not clearly understood. In particular, the specific role of insulin resistance or hyperinsulinemia
needs to be clarified. Recently, we developed an animal modelof hyperinsulinemia by transplanting a second pancreas into
normal rats in order to determine whether increased plasma
insulin levels are related to the clustering of coronary risk factors and accelerated progression of atherosclerosis (8). The
model shows an endogenous insulin level twice that of normal
rats, which influences the process of atherosclerosis by enhancing accumulation of cholesterol esters in the arterial wall.
However, this level of insulin shows no relationship to dyslipidemia or hypertension. This study examines whether this clustering syndrome is affected by insulin resistance in nonobese
IRS-1 deficient mice.

Methods
Animals and animal treatment. Female mice homozygous for targeted disruption of the IRS-1 gene and female wild-type mice thatwere offspring of heterozygous mice were examined. The genotype ofthe mice was confirmed by PCR. All mice were fed normal laboratorychow ad libitum. Body weight was measured before examination. Homozygous mice weighed significantly less than wild-type mice as described (Tables I and II) (3), and mesenteric fat mass was not apparently different between IRS-1 deficient and wild-type mice.
Measurement of blood pressure. Blood pressure was measured at
the tail artery in 21 homozygous mice and 18 wild-type mice (age, 58 mo)under restraints using an automatic sphygmomanometer machine (9).
Basal blood pressure was measured under restraints in 16 homozygous mice and 18 wild-type mice (age, 58 mo) using a perfluorocarbon cannula (KZ1101; Kent Scientific Co., CT) inserted into the femoral artery under Nembutal-Na anesthesia (10). After 6 h ofcannulation, pulsatile blood pressure was measured under conscious
and unrestrained conditions in a quiet environment after a minimum
30-min acclimatization period between 20:00 and 24:00. Blood pressure was measured every 2.5 s by a peak detector (AP-611G; NihonKohden, Tokyo, Japan). The data were stored on a tape recorder in
addition to heart rate measured using a tachometer (AT-601G; Nihon-Kohden), and then stored in a computer (PC9801RX; NEC, Tokyo, Japan). The mean value of each variable over a 1-h segmentcomposed of 1,440 sample points was calculated.
Vascular reactivity studies. 9 homozygous and 11 wild-type mice(age, 56 mo) were decapitated, and a section of the thoracic aorta between the aortic arch and diaphragm was removed and placed in oxy-

Table I. Restrained and Unrestrained Arterial Blood Pressure
and Pulse Rate of IRS-1 Deficient and Wild-Type Mice
Homozygous

Wild-Type(n 5 21)15.662.5*147611*(n 5 16)18.862.6*
132614‡89616‡
110612*43619
634677(n 5 18)
22.665.6
123624(n 5 18)
26.164.1
119615
79612
99610
40620
652641

Restrained study
Body weight
Systolic BP
Unrestrained study
Body weight
Systolic BP
Diastolic BP
Mean BP
Pulse pressure
Pulse rate

Blood pressure was measured at a tail artery in 5–8-mo-old mice in a restrained condition. Furthermore, pulsatile blood pressure was measured
under conscious and unrestrained conditions in a quiet environment.
Data were expressed as mean6SD. * P , 0.01, ‡ P , 0.05 vs. wild-type.

genated, modified Krebs-Henseleit solution (KHS). KHS consistedof 118.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO3, 1.8 mM CaCl2,
1.2 mM NaH2PO4, 1.2 mM MgSO4, and 11.0 mM dextrose. The aorta
was cleaned of loosely adhering fat and connective tissue and cut intorings (3 mm long). The tissue was placed in a well-oxygenated (95%
O2/5% CO2) bath of 10 ml KHS at 378C with one end connected to atissue holder and the other to a force-displacement transducer (TB612T; Nihon-Kohden). The tissue was equilibrated for 60 min underresting tension of 1.0 g, which is optimal for inducing the maximal
concentration in all vessels used. KHS in the bath was replaced every
20 min (11, 12).
Relaxation response to acetylcholine (ACh; Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan) and sodium nitroprusside (SNP; Sigma
Chemical Co., St. Louis, MO) was expressed as a percentage of decreased tension in contractile force induced by 3 3 1028 to 1 3 1027 M
U46619, a derivative of thromboxane A2. For relaxation studies, theaortic rings were precontracted with 3 3 1028 to 1 3 1027 M U46619.
This concentration produced 85–90% of the maximal response. Aortic rings, which were weighed at the end of each experiment, wereprecontracted with 3 3 1028 to 1 3 1027 M U46619 to ensure thatthey could develop a tension of z 100 mg/mg tissue in both agematched wild-type and homozygous mice. When U46619-induced
contraction reached a plateau, ACh (1029 to 1025 M) and SNP (1029

to 1025 M) were added cumulatively. Each aortic ring was exposed toonly one relaxant agent.
Measurement of plasma lipids and fat loading test. The blood of
2–3, 4–5, and 6–12-mo-old homozygous (n 5 17) and wild-type mice(n 5 21) was collected after a 6-h fast from 9:00 a.m. to 2:00 p.m.
Blood was collected from the retroorbital plexus using heparincoated capillaries and transferred to an ice-cold tube. Plasma concentrations of triglycerides, cholesterol, and FFA were determined
enzymatically. HPLC was applied to show elution patterns with two
distinguished elution peaks of the two major lipoprotein classes, apo B
containing lipoproteins and HDL, which were used to determine
HDL-cholesterol levels (13).
After the 6-h fast, postheparin plasma of 6–12-mo-old homozygous (n 5 12) and wild-type (n 5 16) mice was sampled 3 min after abolus injection of heparin (100 U/kg) into the external jugular vein.
Lipoprotein lipase (LPL) activity was assayed by the method described by Nilsson-Ehle and Schotz (14). Postheparin plasma was assayed in the presence or absence of 1 M NaCl in order to estimate
both LPL and hepatic triglyceride lipase (HL) activity. Lipase activity
in the presence of 1 M NaCl represented HL activity. LPL activity
was calculated by subtracting HL activity from lipase activity in theabsence of 1 M NaCl (15). LPL activity in adipose tissue derived fromthe utero-ovarian region was assayed as described (15).
During the 6-h fast, at which time the mice were given ad libitum
access to water, olive oil (16.7 ml/g body wt) was introduced intragastrically using a cannula to 8–10-mo-old homozygous (n 5 4) and wildtype (n 5 4) mice. After fat loading, z 50 ml of blood was collected
serially at 0, 0.5, 1, 2, 3, and 5 h, and plasma triglyceride levels were
measured enzymatically (15).
Statistical analysis. Data were analyzed by the Mann-Whitney U
test and expressed as mean6SD. P , 0.05 was considered statisticallysignificant.

Results
Blood pressure. Under restrained conditions, systolic arterial
blood pressure was significantly higher in homozygous micethan in wild-type mice (147611 vs. 123624 mmHg, P , 0.01)(Table I), in contrast to unrestrained conditions in which systolic, diastolic, and mean arterial blood pressure were all significantly higher in homozygous mice than in wild-type mice
(Table I). Systolic/diastolic pressure was 132614/89616 mmHg
in homozygous mice and 119615/79612 mmHg in wild-type
mice. Mean blood pressure was 110612 mmHg in homozygous
mice and 99610 mmHg in wild-type mice, P , 0.01. Pulse

Table II. Plasma Lipid Levels in IRS-1 Deficient and Wild-Type Mice

Homozygous mice(months)

Wild-Type mice(months)

2–3
4–5
6–12

2–3
4–5
6–12

Body weight

Triglyceride

Cholesterol

HDL

FFA

g

mg/dl

mg/dl

mg/dl

meq/liter

15.261.6*
15.861.3*
17.862.3*

51.2623.8**
47.9619.3*
53.1622.5*

75.0612.9
76.0611.3
75.466.4——
54.767.6‡——
503.76122.5(n 5 17)(n 5 17)(n 5 17)(n 5 9)(n 5 21)21.062.0
23.861.3
24.561.6(n 5 21)33.1619.8
28.468.0
33.866.7(n 5 21)80.469.0
81.9611.2
79.669.7(n 5 21)——68.968.5(n 5 7)——529.76149.3(n 5 14)

Data were analyzed by the Mann-Whitney U test and expressed as mean6SD. *P , 0.01, ‡ P , 0.02 vs. wild-type.

Metabolic Syndrome in Insulin Resistance1785

Table III. LPL Activities in IRS-1 Deficient andWild-Type Mice

Homozygous mice
Wild-type mice

Figure 1. Vascular reactivity in aortic strip. A section of the thoracic
aorta between the aortic arch and diaphragm was used. The aortic
rings were precontracted with an equieffective concentration of
U46619. When the U46619-induced contraction reached a plateau,
ACh and SNP were added cumulatively. The relaxation response to
ACH (left) and SNP (right) was expressed as a percentage of the decreased tension in contractile force induced by 3 3 1028 to 1 3 1027 M
U46619 (mean6SD).

pressure and pulse rate did not differ significantly between homozygous and wild-type mice.
Vascular reactivity in aortic rings. The mechanisms underlying elevation in arterial blood pressure were examined inorder to elucidate vascular reactivity in aortic rings of IRS-1
deficient mice (Fig. 1). When the U46619 (3 3 1028 to 1 3 1027M)-induced contraction reached a plateau, ACh (1029 to 1025 M)
was added cumulatively. ACh caused concentration-dependent relaxation in aortic strips. This relaxation was significantly reduced in aortic strips from homozygous mice compared with wild-type mice. In contrast, relaxation caused by
SNP (1029 to 1025 M), which is an endothelium-independent
agent and activates soluble guanylate cyclase, did not differ
significantly between homozygous and wild-type mice.

Postheparin plasma

Adipose tissue

mmol FFA/h/ml

mmol FFA/h/g

24.265.4*(n 5 16)36.0613.9(n 5 12)3.361.7‡(n 5 7)5.762.3(n 5 7)

LPL activities in postheparin plasma and adipose tissue were measured
in 6–12-mo-old mice. Data were analyzed by the Mann-Whitney U test
and expressed as mean6SD. *P , 0.01, ‡ P , 0.05 vs. wild-type.

Plasma lipid levels. Plasma triglyceride levels after the 6-h
fast in homozygous mice were significantly higher than in wildtype mice at each measurement (Table II). This difference
was 1.6-fold in 2–3-mo-old mice (homozygous/wild-type:51.2623.8/33.1619.8 mg/dl) and 1.7-fold in 4–5-mo-old mice(homozygous/wild-type: 47.9619.3/28.468.0 mg/dl). Plasmacholesterol and FFA levels did not differ significantly between
the two groups. HPLC analysis of the lipoprotein profiles revealed a significant decrease in plasma HDL-cholesterol levels(homozygous/wild-type: 54.767.6/68.968.5 mg/dl, Table II).
Fat loading test. Lipolysis of plasma triglycerides was examined by loading exogenous triglycerides as dietary fat (Fig.
2). The increase in plasma triglyceride level was significantly
greater in homozygous mice than in wild-type mice throughout
the experimental period. Plasma triglyceride peaked 2 h after
loading in both wild-type and homozygous mice. The peakheight in homozygous mice was significantly higher than inwild-type mice (P , 0.02).
LPL activity in postheparin plasma after the 6-h fast wassignificantly lower in homozygous mice than in wild-type mice
(Table III), whereas HL activity did not differ significantly between the two groups (data not shown). LPL activity in adipose tissue was also significantly lower in homozygous micethan in wild-type mice (Table III).

Discussion

Figure 2. Plasma triglyceride levels after fat loading test. After a 6-h
fast, olive oil (16.7 ml/g body wt) was loaded intragastrically to homozygous (n 5 8) and wild-type (n 5 8) mice. Before and after fat
loading, blood was collected serially, and plasma triglyceride levels
were measured. The relative increase of plasma triglyceride levels after fat loading compared with that before fat loading was expressed
as mean6SE.
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Abe et al.

Several researchers have suggested recently that insulin resistance contributes to the pathogenesis of atherosclerosis (5–7,
16). In this study, we observed IRS-1 deficiency–induced insulin resistance to lead to increased blood pressure and plasma
triglyceride levels, implicating involvement of insulin resistance in both of these coronary risk factors. IRS-1 is the major
substrate of the insulin receptor kinase and mediates the signal
pathways of insulin and IGF-1 (1, 2), therefore the phenotypeof IRS-1 deficient mice reflects both defects of insulin and
IGF-1 signaling. IRS-1 deficient mice demonstrate growth retardation and resistance to the glucose-lowering effect of insulin (3, 4). Recent studies have shown insulin resistance in IRS-1
deficient mice to differ among tissues (17). IRS-1 plays a central role in the signaling pathways of insulin in muscle and adipose tissues. Furthermore, IRS-2 has been demonstrated tocompensate for IRS-1 deficiency in the liver of IRS-1 deficientmice. Thus, insulin resistance in IRS-1 deficient mice appears
to be primarily due to resistance in muscle and adipose tissues.

Epidemiological studies have demonstrated an association
between obesity, insulin resistance, and hypertension (18, 19).
Subsequent studies have suggested that essential hypertension
even in lean individuals is related to insulin resistance and hyperinsulinemia (20, 21). The mechanism associated with hypertension in insulin resistance with hyperinsulinemia may berelated to skeletal muscle blood flow and relative skeletal muscle fiber type (22), vascular endothelial function (23), increased renal sodium reabsorption secondary to hyperinsulinemia (24), increased activity of the sympathetic nervous
system (25), or increased cellular cation transport (26). We estimated blood pressure in a nonobese animal model of insulin
resistance under restrained and unrestrained conditions. Bloodpressure under both conditions was significantly higher in IRS-1
deficient mice than in control mice. Thus, hypertension appears to occur as a result of pathophysiological abnormalities
caused by IRS-1 deficiency rather than increased sympathetic
activity during the experiment. It is possible that relatively low
body weight in IRS-1 deficient mice may influence blood pressure. However, we found high blood pressure in IRS-1 deficient mice as compared with body weight–matched control
mice. Furthermore, secondary hyperinsulinemia may havebeen responsible for hypertension in the present animalmodel. However, in our previous study, hyperinsulinemia did
not affect blood pressure in either normal Wistar or SHR rats
with two pancreas, despite a twofold increase in plasma insulin
after pancreas transplantation (8). Similar plasma insulin levels were observed in the IRS-1 deficient mice (3) and therefore
are not likely to influence blood pressure. Thus, insulin resistance appears to be a cause of hypertension in IRS-1 deficient
mice, although high insulin levels caused by insulin resistance
may play a role in the elevation of blood pressure.
Endothelium-dependent relaxation of the aorta was reduced in IRS-1 deficient mice, whereas endothelium-independent relaxation did not differ between IRS-1 deficient and control mice. Nitric oxide, a potent vasodilator produced byendothelial cells, has been proposed as the major form of endothelium-dependent relaxing factor which mediates vascularrelaxation in response to ACh (27, 28). A recent study reported that mice lacking the gene for endothelial nitric oxide
synthase show hypertension (29), suggesting that a resulting
impairment in endothelium-dependent vascular relaxation is
involved in the pathophysiology of hypertension. Impaired endothelium-dependent vascular relaxation has been observed in
genetically diabetic rats (30), streptozotocin-induced diabetic
rats (31), and streptozotocin-induced diabetic mice (11). However, the role of insulin resistance in endothelium-dependent
vascular relaxation is not yet known. Since glucose tolerance isnormal in IRS-1 deficient mice (3), insulin resistance may be
responsible for impaired endothelium-dependent relaxation of
the aorta.
High plasma triglyceride and low plasma HDL-cholesterollevels, which are inversely correlated, are common clinicalfindings in the insulin-resistant states which include diabetes
mellitus, obesity, and syndrome X (32). Plasma triglyceride
levels increased 1.6-fold and plasma HDL-cholesterol levels
decreased 0.79-fold in IRS-1 deficient mice. Two underlying
mechanisms may explain the increase in plasma triglycerides
associated with insulin resistance in mutant mice: overproduction of VLDL in the liver and decreased clearance of VLDL
and chylomicrons (33, 34). Impaired insulin action not only
stimulates lipolysis, increasing delivery of FFA to the liver andconsequently increasing production of triglycerides, but also
reduces LPL activity. LPL is an enzyme located on the endothelial surface of adipose tissue and skeletal and heart muscle
which hydrolyzes triglycerides in chylomicron and VLDL (35).
LPL activity of postheparin plasma and adipose tissue of IRS-1
deficient mice showed a significant reduction in this study,
leading to decreased hydrolysis of lipoprotein triglycerides. In
accordance with low LPL activity, a significantly greater increment in plasma triglyceride after administration of fat loading
was observed in IRS-1 deficient mice as compared with normal
mice. Plasma FFA levels are regulated by the transfer of FFA
between plasma and adipose tissue (36, 37), which involves
LPL and hormone-sensitive lipase (38). Lipolysis by hormonesensitive lipase in adipose tissue may be enhanced in the insulin-resistant state of IRS-1 deficient mice (39). However,plasma FFA levels did not increase in IRS-1 deficient mice.
Thus, hypertriglyceridemia in IRS-1 deficient mice may becaused by defective hydrolysis of plasma triglycerides rather
than an increased delivery of FFA to the liver.
The present results suggest that insulin resistance plays anessential role both in the clustering of coronary risk factors
such as hypertension and hypertriglyceridemia and in reduced
endothelium-dependent vascular relaxation. Endothelial dysfunction such as impaired endothelium-dependent vascular relaxation are considered to be closely related to the pathogenesis of atherosclerosis (28). Therefore, not only clustering ofcoronary risk factors but also impaired endothelium-dependent vascular relaxation may be involved in the progression ofatherosclerosis observed in insulin resistance.

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