Quantitative characterization of insulin-glucose response in Watanabe heritable hyperlipidemic and cholesterol-fed rabbits and the effect of cilazapril

Quantitative Characterization of Insulin-Glucose Heritable Hyperlipidemic and Cholesterol-Fed Cilazapril

Response in Watanabe Rabbits and the Effect of

Bo Zhang, Keijiro Saku, Kyoko Hirata, Rui Liu, Kayoko Tateishi, Masashi Shiomi, and Kikuo Arakawa A great deal of evidence suggests that insulin resistance, via hyperinsulinemia, contributes to hyperlipoproteinemia and coronary atherosclerosis. When Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of familial hypercholesterolemia (FH), are compared with normolipidemic Japanese White (JW) rabbits, an elevated fasting plasma insulin level and a heightened plasma insulin response to an intravenous (IV) glucose challenge are found. To elucidate the mechanism behind this phenomenon, a two-compartment model of the glucose/insulin system was fitted to empirical time courses of glucose and insulin concentrations during an IV glucose tolerance test (IVGTT) by nonlinear least-square regression, and the model parameters such as the glucose utilization rate constant, insulin degradation rate constant, and pancreas sensitivity were determined. WHHL rabbits showed decreased values of glucose utilization and insulin degradation rate constants and slightly higher values of pancreas sensitivity. This suggests that insulin resistance occurs in extrapancreatic tissues, and that this may be attributable to insulin receptor and/or post-insulin receptor abnormalities. Cholesterol feeding did not significantly change glucose tolerance or insulin action in JW rabbits. The effects of an angiotensin-converting enzyme (ACE) inhibitor, cilazapril, on insulin resistance were also examined in WHHL and JW rabbits. A decreased insulin response to an IV glucose challenge and increased glucose utilization and insulin degradation rate constants were observed in WHHL rabbits that had been treated with cilazapril, indicating that cilazapril improved insulin resistance in WHHL rabbits, possibly by increasing the number of insulin receptors. No significant differences were found in glucose tolerance and insulin action in JW rabbits before and after cilazapril administration. In summary, a decrease in insulin degradation probably contributes to insulin resistance in WHHL rabbits. Diet-induced hypercholesterolemia in JW rabbits did not affect glucose-insulin metabolism. Cilazapril administration improved insulin resistance in WHHL rabbits, and increased insulin degradation may have played a role in this effect. Copyright 0 1994 by W.B. Saunders Company

I

T IS WELL ESTABLISHED that high levels of serum cholesterol lead to a high risk of coronary heart disease feeding readily produces (CHD),’ and that cholesterol atherosclerosis in rabbits.’ Recent studies have suggested that insulin resistance is also an independent risk factor for the development of CHD.‘,’ In obese insulin-resistant rats that were atherosclerosis-prone. a variety of treatments that decreased very high insulin levels also inhibited cardiovascular diseases.q~5 Atherosclerosis and its sequelae are the principal long-term complications of type II diabetes mellitus and are accompanied by insulin resistance and hyperinsulinemia.’ There is also direct evidence that insulin resistance is associated with asymptomatic atherosclerosis.” Given this strong association between insulin resistance and atherosclerosis. Watanabe heritable hyperlipidemic (WHHL) rabbits, a well-established animal model of familial hypercholesterolemia (FH), were tested regarding their glucose tolerance and insulin sensitivity as assessed by an intravenous glucose tolerance test (IVGTT). To define the underlying mechanism(s) of insulin/glucose metabolism, a two-compartment mathematical model of the glucose/ insulin system’ was used to interpret results from the From the Departments of Internal Medicine and Biochemistv, Fukuoka University School of Medicine. Fukuoka; and the Institute for Experimental Animals, Kobe University School of Medicine, Kobe, Japan. Submitted December 31, 1992: accepted April 8, 1993. Presented in pati at the Eleventh International Symposium on Drugs Affecting Lipid Metabolism (DALM), May 15. 1992, Florence, Ita@. Address reprint requests to Keijiro Saku. MD, PhD, Depatiment of Internal Medicine, Fukuoka Universil?, School of Medicine. 7-45-l Nanakuma Jonanku. Fukuoka 814-01, Japan. Copyright 0 1994 by W.B. Saunders Compai?v 0026-049519414303-0015$03.OOlO 360

IVGTT. By model-fitting and parameter identification, values for the system parameters were obtained. ie, the glucose utilization rate constant, pancreas sensitivity constant, and insulin degradation rate constant. Although information can be obtained from the time courses of glucose and insulin concentrations during an IVGTT, parameter identification leads to data reduction that provides a more quantitative approach to determining characteristic system properties.’ Cilazapril is a new angiotensin-converting enzyme (ACE) inhibito?” that has been shown to be more potent as an antihypertensive drug and to have a longer duration of action than captopril,‘? which also has a potent antiatherogenie action in WHHL rabbits,‘” and has also been shown to improve glucose tolerance and insulin sensitivity in hypertensive patientsi Current therapy should aim at overall risk-factor reduction; previous attempts simply to decrease blood pressure with antihypertensive drugs. such as diuretics and certain P-adrenergic-blocking agents, have not been able to prevent CHD because the drugs also cause dyslipidemias and impair tissue insulin sensitivity.‘s.‘h Therefore. the effects of cilazapril on insulin resistance in WHHL rabbits and normal Japanese White (JW) rabbits were examined in the present study. MATERIALS

AND METHODS

Animals

Homozygous WHHL rabbits (n = 6. > 10 months old. weighing between 7.6 and 3.4 kg) were kindly provided by Dr M. Shiomi, Kobe University, Kobe, Japan. Normolipidemic JW rabbits (n = 26, aged-matched to the WHHL rabbits, weighing between 1.5 and 3.4 kg) were purchased from Kyudo. Fukuoka. Japan. WHHL and JW rabbits were maintained individually in a controlled environment with unlimited access to water, and were fed standard chow RC-4 Metabolism, Vol43, No 3 (March), 1994: pp 360-366

INSULIN-GLUCOSE

361

RESPONSE IN WHHL RABBITS

(Oriental Yeast, Tokyo. before the administration

Japan) at a level of 80 g/d per animal of a cholesterol diet or cilazapril.

Experimental Protocols We performed three studies to investigate the interaction between hypercholesterolemia and glucose/insulin metabolism. In study A, we tested glucose tolerance and insulin sensitivity in WHHL rabbits by performing an IVGTI and comparing the results in WHHL rabbits (n = 6) with those in normolipidemic JW rabbits (no = 26. controls), both of which were fed a standard chow. In study B. we investigated the effects of cholesterol feeding, which is known to result in atherosclerosis, on glucose metabolism. A diet of 80 g/d per animal containing 0.5% cholesterol was administered to randornly selected JW rabbits (n = 13) for 3 weeks. Control JW rabbits (n = 11) were fed the same amount of standard chow without added cholesterol. In study C. we investigated the effect of cilazapril on glucose tolerance and insulin sensitivity in WHHL and JW rabbits. A diet of 80 g/d per animal containing 0.0025% cilazapril was administered to WHHL rabbits (n = 3) and JW rabbits (n = 4) for 3 weeks. In studies B and C, the IVGTT was performed before and after administration of cholesterol or cilazapril. Two of the control JW rabbits were used for both studies.

IVGTT After overnight fasting. an IVGTT was performed, starting between 9 and IO AM. Two mL blood was sampled from the marginal auricular vein. 1 mL for the determination of serum total cholesterol (TC) and triglyceride (TG) levels, and 1 mL into a tube containing EDTA for the determination of basal values of plasma glucose and insulin. A solution of 0.6 g glucose/kg body weight was then injected into the marginal vein of the opposite ear. After 5. 10. 15. 20, 30, 45. 60, 75. and 120 minutes. a I-mL blood sample was drawn. Concentrations of glucose and insulin in each sample were determined. Plasma glucose (PG) was assayed by the glucose oxidase procedure (Glucoroder-MK II, Analytical Instruments. Tokyo. Japan) immediately after the IVGTT. and the plasma for insulin measurement was stored at -80°C until assayed. The immunoreactive insulin (IRI) level in plasma was determined by radioimmunoassay (Insulin Kit, Amersham. Tokyo, Japan), as previously reported.” The procedure for using the human Insulin Kit was modifiedlx to make the most precise portion of the standard curve coincide with the range of rabbit insulin levels. Serum TC and TG levels were measured by enzymatic methods. ‘q.2”

Data Analysis and Parameter Calculation Statistical analyses were performed with Student’s I test. Changes in plasma levels of insulin and glucose during the IVGTT within each test group were analyzed with the paired t test. and data between different groups of rabbits were analyzed with the unpaired t test. The association between body weight and basal IRI was measured with the Pearson Product-Moment Correlation in WHHL and JW rabbits. The peak insulin response in JW rabbits was defined as the mean of values obtained at 10, 15, and 20 minutes. The peak insulin response in WHHL rabbits was defined as the mean of values obtained at 30.45, and 60 minutes because of a delayed insulin response in WHHL rabbits. The relative peak insulin response above basal was calculated as the deviation from the basal value. A two-compartment model’ for describing the underlying interaction between glucose and insulin during an IVGTT was applied to evaluate the data. The model assumes that (I) the rate of glucose utilization is proportional to the amount by which glucose

exceeds its stationary

level; (2) the insulin level increases above its stationary value as a result of secretion by p cells that have been stimulated by the increase in glucose above its stationary level; (3) the degradation of insulin begins simultaneously with its secretion, and the rate of degradation is proportional to the amount by which it exceeds its stationary level: and (4) the stationary values of insulin and glucose are reestablished at the end of the IVGTT. Accordingly. the differential equations are as follows: dxidr = kl

dyldt

[x(f) - xs] for 2 < t 5 120 min

= k2.

[x(t) - xs] -

k3

/y(t) - ys].

Eq

1

Eq2

where x(t) is the PG concentration at time t; dxidt is the rate of change of the glucose level; xs is the stationary value of the glucose concentration at the end of the IVGIT; y(t) is the insulin concentration at time t; dy/dt is the rate of change of the insulin level; ys is the stationary value of the insulin concentration at the end of the IVGTT: kl is the relative rate of utilization of glucose: k2 is the pancreas sensitivity constant stimulated by glucose; and k3 is the relative rate of degradation of insulin when the concentration of insulin exceeds its stationary value. The model was fitted to the data of plasma concentrations of glucose and insulin during an IVGTT to determine the parameters of the system. Nonlinear least-squares regression analysis was performed using the NLIN Procedure (Statistical Analysis System, SAS Institute, Cary, NC). The time course of the glucose concentration was expressed as the sum of two exponentials and a constant. The system parameters kl (relative glucose utilization rate) and xs (stationary level of glucose) were determined by fitting equation 1 to the glucose concentrations observed during the IVGTT. Equation 2 was integrated analytically using the Laplace Transform,” and the system parameters k2 (pancreas sensitivity constant), k3 (relative rate of insulin degradation), and ys (stationary value of insulin concentration) were determined by fitting equation 2 to the insulin concentrations observed during the IVGTT, with the predicted glucose value at time t from the fitted equation 1 as the glucose value at time t. The Marquardt iterative method was used during the search for parameters. The parameters are uniquely determined if the initial values are chosen properly. One set of parameters was estimated for each rabbit, and the means of the parameters ? standard errors of each group are presented in the Results. Normality of the distributions of the parameters was verified before the parametric statistical tests. RESULTS

Study A As shown in Fig 1. the responses of both plasma insulin and PG to an IV glucose load are greater in WHHL rabbits than in JW rabbits. Table 1 presents results from the IVGTT and shows levels of serum lipids. Fasting (basal) levels of plasma IRI (14.6 + 3.5 118.8 f 0.6 FUlmL). ZIRI (410.2 ‘_ 62.9 1’ 171.7 +- 9.0 pU/mL), and the relative peak insulin response above basal (38.2 ? 7.1 v 13.4 ? 6.9 KU/ mL) in WHHL rabbits were significantly (P < .Ol) higher than those in control JW rabbits, whereas fasting PG levels (151 + 8 v 137 r 3 mg/dL) in WHHL rabbits were only

362

ZHANG ET Al

Table 1. Data From the IVGTT and Levels of Serum Lipids in WHHL and Control JW Rabbits (Study A) WHHL (n = 6)

Fasting PG (mg/dL)

Control

151 28

P

Ill = 26)

137 z? 3

NS

Fasting plasma insulin WJlmLl ‘;PG (mg/dL)

8.8 + 0.6

i .Ol

3,003 + 102

2,628 ? 52

< .Ol

ZlRl (&/mL)

410.2 2 62.9

171.7 2 9.0

< ,001

138 ? 22

66 i 4

< .05 i .Ol

ZlRliZPG

14.6 + 3.5

(x103)

Relative peak IRI response 38.2 + 7.1

13.4 ? 6.9

TC (mg/dL)

above basal ($J/mL)*

607.0 ? 64.7

39.2 2 2.7

< ,001

TG (mg/dL)

177.2 & 36.2

45.2 ? 2.6

<.OOl

NOTE. ZPG and ZlRl are the sum of PG and IRI concentrations time point during the IVGTT, respectively. The calculation described

of relative

at each

Data are means + SD.

peak insulin

response

above

basal is

in the Methods.

control rabbits (12.1 -C 1.6 I’ 6.7 t 0.7 kU/mL. P < .Ol). and although the differences were not significant. stationary levels of PG (xs) were slightly higher in WHHL rabbits than in controls (126 t 18 v 98 2 7 mg/dL). These results were in agreement with the changes in fasting plasma concentrations of glucose and insulin (Table 1). The pancreas constant (k2) was higher and the glucose utilization rate (kl) was lower in WHHL rabbits than in control rabbits, but not significantly. The rate of insulin degradation (k3) was significantly (P < .05) lower in WHHL rabbits than in control rabbits (0.28 2 0.16 ~20.91 ? 0.14 per hour, respectively). 0

30

60

90

120

Minutes Fig 1. (A) PG concentrations during the IVGlT in WHHL, cholesterol-fed JW, and standard diet-fed (control) JW rabbits. (B) Plasma IRI concentrations during the IVGTT in WHHL, cholesterol-fed JW, and standard diet-fed (control) JW rabbits. (0) WHHL rabbits (n = 6); (0) control JW rabbits (n = 26); (B) cholesterol-fed JW rabbits (n = 13).

slightly higher than those in JW rabbits, and the differences were not statistically significant. The mean body weight of WHHL rabbits (3.18 2 0.32 kg) is greater than that of JW rabbits (2.71 ? 0.20 kg). No significant correlations, as measured by the Pearson correlation coefficients, between basal IRI levels and body weights were found in WHHL rabbits (r = .2348, P = .65) and JW rabbits (I = .llhO. P = .57), indicating that a higher basal IRI value in WHHL rabbits is not associated with their greater body weight. Significantly higher CIRI/ZPG ((138 -+ 22) x 10” I’ (66 + 4) x lo-‘, P < ,051 in WHHL rabbits reflected tissue resistance to insulin, and the higher CPG (3,003 ? 102 1’ 2,628 + 52 mg/dL, P < .Ol) reflected impaired glucose tolerance in WHHL rabbits. Serum levels of both TC and TG in WHHL rabbits were significantly higher (P < .OOl) than those in control rabbits. System parameters for WHHL and control rabbits are summarized in Table 2. The stationary level of insulin (ys) in WHHL rabbits was significantly higher than that in

Study B As shown in Fig 1, the responses of PG and plasma insulin to an IV glucose load were similar in both cholestcrolfed and control JW groups during the study period. No significant changes in IIRI, SPG, or C,IRI/ZPG were found in either group of rabbits before and at the end of the study period, although fasting levels of PG (146 t 16 at week 0 1’ 131 t 20 mg/dL at 3 weeks. P < .05) and plasma insulin (9.1 ? 3.6 v 6.2 t 3.1 kU/mL. P < .Ol) were significantly decreased at the end of the study period in cholesterol-fed rabbits (Table 3). Serum levels of TC and TG Table 2. System Parameters for WHHL and Control JW Rabbits (study A) WHHL In = 6)

Control

(n = 26)

xs

126 + 18

98 i 7

kl

0.98 i 0.07

1.25 i 0.08

k2

1.49 % 0.44

1.11 * 0.14

k3

0.28 2 0.16

0.91 2 0.14*

vs

12.1 r 1.6

6.7 f 0.7t

NOTE. Data are means + SE. Abbreviations:

xs,stationary

end of the IVGTT (mg/dL);

value of the glucose concentration

ys, stationary

tion at the end of the IVGTT (&/mL); glucose

(per hour);

glucose ($J/mL,h); stationary

k2, pancreas

*P < .05. tP < .Ol.

kl, relative rate of utilization sensitivity

constant

k3, relative rate of degradation

value (per hour).

at the

value of the insulin concentrastimulated

of by

of insulin above its

NSULIN-GLUCOSE

RESPONSE IN WHHL RABBITS

363

Table 3. Data From the IVGTT and Levels of Lipids in JW Rabbits

Table 5. From the Data of IVGTT and Levels of Serum Lipids in

With and Without Cholesterol Feeding (study B)

WHHL and Control JW Rabbits Before and After Cilazapril

-

Administration

Cholesterol-Fed(n = 13) Control(n = 111

WHHL(n=

Fasting PG tmg/dL) Pre

146 + 16

131 ? 16

Post

131 Ic_20”

123 5 12

Pre

9.1 i 3.6

9.8 + 3.1

Post

6.2 t 3.lt

9.8 -c 1.6

2,704 t 313

2,548 f 216

Post

2,551 t 277

2,404 + 162

Post

176.8 2 52.8

174.4 ? 42.1

148.3 1- 76.2

165.0 -t 37.1

(~10~)

Pre

66 + 22

69+

Post

59 2 33

69 t 14

Pre

151 2 22

137 2 4

Post

141 + 13

132+

16

Pre

12.2 t 4.9

7.2 k 3.6

Post

10.8 ? 4.2

6.7 2 3.0

Pre

2,988 * 383

2,610 k 45

Post

2,965 t- 393

2.885 t 221

Pre

382.6 f 107.9

163.5 k 46.1

Post

231.2 rt 121.1*

126.6 t 34.0

XIRI (r.dJ/mL)

IIRI/‘,PG

(~10~)

Pre

130 + 42 81 -t 45”

Post

TC (mg/dL) Pre

37.4 f 2.5

38.8 + 4.3

Post

893.9 i: 80.6$

34.6 * 4.3

47.7 * 2.4

42.2 -t 3.8

Post

118.3 f 17.4$

44.4 + 5.9

63 i 18 44-t13

TC (mg/dL)

TG (mg/dL) Pre

15

\PG (mg/dL)

\IRI ($J/mL) Pre

Control(n = 4)

Fasting plasma insulin ($/mL)

ZPG (mg!dL) Pre

3)

Fasting PG (mg/dl)

Fasting plasma insulin (@/mLI

llRl/XPG

(study C)

Pre

556.6 ? 10.4

38.3 -t 21.3

Post

644.6 t 26.9

53.6 f 10.4

TG ImgidL) Pre

NOTE. Data are means f SD. \PG and llRl are the sum of PG and IRI concentrations at each time point during the IVGTT, respectively.

Post

117.3 + 56.7

29.4 k 7.8

226.4 + 39.2

56.4 + 5.7

NOTE. Data are means + SD. ‘CPG and XIRI are the sum of PG and IRI

Abbreviations: Pm, pre-study period; Post, post-study period.

concentrations at each time point during the IVGTT, respectively. Abbreviations: Pre, pre-study period; Post, post-study period. P < .05.

were significantly (P < .OOl) increased in cholesterol-fed rabbits at the end of the study period. System parameters for rabbits in study B are listed in Table 4. Changes in the system parameters were not significant in either group of rabbits during the study period.

Table 4. System Parameters for JW Rabbits With and Without Cholesterol Feeding (study 8) Cholesterol-Fed(n = 131

Control

(n = 11)

XS

Pre

102 + 9

98-t

12

Post

90 * 4

88 i

6

Pre

1.25 + 0.11

1.32 i 0.12

Post

1.20 * 0.14

1.38 2 0.08

Pre

0.97 ? 0.14

1.25 + 0.27

Post

1.46 ? 0.37

0.97 + 0.18

Pre

0.82 + 0.22

0.87 + 0.14

Post

0.62 2 0.09

0.72 + 0.09

Pre

6.1 + 1.1

7.9 t 0.7

Post

4.1 2 1.0

7.7 ? 0.8

kl

k2

k3

vs

NOTE. Data are means -t SE. Abbreviations: xs, stationary value of the glucose concentration at the end of the IVGTT (mg/dL); concentration

vs. stationary value of the insulin

at the end of the IVGTT (@/mL);

kl, relative rate of

utilization of glucose (per hour); k2, pancreas

sensitivity constant

stimulated by glucose ($/mL.h);

k3, relative rate of degradation

of

insulin above its stationary value (per hour); Pre, pre-study period: Post, post-study period.

Stu~v

c

Results of the IVGTT and the serum levels of lipids in WHHL and control JW rabbits before and after cilazapril administration are shown in Table 5. Insulin responses to an IV glucose load in WHHL rabbits after cilazapril treatment were greatly decreased (ZIRI, 382.6 ? 107.9 I’ 231.2 ? 121.1 kU/mL, P < .05), whereas glucose concentrations during the IVGTT in WHHL rabbits were similar before and after administration of cilazapril. In control JW rabbits, administration of cilazapril decreased insulin responses (XIRI, 163.5 ? 46.1 1’ 126.6 * 34.0 kU/mL) and impaired glucose tolerance (XPG, 2,610 + 45 I’ 2,885 + 221 mg/dL), although only slightly. After cilazapril treatment, serum levels of TC and TG in both groups were increased. but not significantly. System parameters for both groups of rabbits are listed in Table 6. In control rabbits, stationary levels of glucose (xs) were increased after cilazapril administration (88 ? 12 1’ 162 2 27 mg/dL), whereas the stationary levels of insulin (ys). utilization rate of glucose (kl), and degradation rate of insulin (k3) did not change greatly. Stationary levels of insulin in WHHL rabbits were decreased after cilazapril administration (12.2 ? 2.8 1’ 8.7 + 1.3 pLJ/mL). The rate constants ofglucose utilization (kl, 1.07 + 0.07~’ 1.60 c 0.28 per hour) and insulin degradation (k3. 0.31 2 0.24 1’ 0.61 + 0.31 per hour) in WHHL rabbits were increased after cilazapril administration. DISCUSSION

In study A, hyperinsulinemia level and a heightened insulin

(an elevated fasting insulin response to an IV glucose

364

ZHANG ET AL

Table 6. System Parameters for WHHL and Control JW Rabbits Before and After Cilazapril Administration WHHL (n = 3)

(study C) Control

(n = 41

xs Pre

140 k 36

Post

180 k 26

162 5 27

Pre

1.07 t- 0.07

1.11 k 0.11

Post

1.60

k 0.28

1.38 z! 0.30

88-t

12

kl

k2 Pra

1.24 k 0.27

1.05 ? 0.16

Post

1.81 + 1.12

0.76 t 0.23

Pre

0.31 k 0.24

1.09 + 0.47

Post

0.61 t 0.31

0.97 5 0.30

k3

YS Pre Post

12.2 2 2.8

5.3 * 1.0

8.7 + 1.3

6.3 !z 1.4

NOTE. Data are means + SE. Abbreviations:

xs, stationary

value of the glucose

the end of the IVGTT (mg/dL); concentration

ys, stationary

at the end of the IVGTf

(pU/mL);

utilization

of glucose

(per hour):

k2, pancreas

stimulated

by glucose

(pU/mL,h);

k3, relative

insulin

above

Post, post-study

its stationary

value (per hour);

concentration

value

at

of the insulin

kl, relative sensitivity

rate of constant

rate of degradation Pre, pre-study

of

period;

period

challenge) and impaired glucose tolerance (XPG, Table 1 and Fig 1) were observed in WHHL rabbits, an animal model of FH, thus confirming our preliminary investigation.” A cellular resistance to insulin may underlie this abnormality,22-2d and hyperinsulinemia occurs as a response to insulin resistance.th In the present study, the possible location and cellular mechanism of insulin resistance in WHHL rabbits were analyzed using a two-compartment model of the glucose/insulin system.‘The higher pancreatic insulin response to glucose in WHHL rabbits, as expressed by the higher relative peak insulin values above basal in response to a glucose challenge (Fig 1) and by the pancreas sensitivity constant (kZ), which was higher in WHHL rabbits than in control JW rabbits (although the difference was not significant), reflected p-cell compensation for the impaired glucose tolerance. Although the feedback relationship between the p cells and the extrapancreatic glucoseutilizing tissues during the IVGTT makes it difficult to draw absolute conclusions regarding p-cell function, concomitant glucose intolerance and hyperinsulinemia during the IVGTT is indicative of insulin resistance and normal pancreatic function.?’ The decreased glucose utilization rate constant (kl) in WHHL rabbits suggests an impaired ability of insulin to stimulate glucose utilization (insulin resistance). A reduced efficiency of insulin degradation was indicated by the significantly decreased rate constant of insulin degradation (k3). The decreased insulin degradation in WHHL rabbits may play a role in insulin resistance, since a decrease in insulin internalization and degradation has been reported in monocyte@ and adipocytesz7 from patients with noninsulin-dependent diabetes mellitus, and fewer hepatic

insulin receptors and decreased receptor-mediated insulin degradation have also been demonstrated in genetically obese rats,2x both of which arc known to show insulin resistance. Although the cellular mechanisms of insulin resistance in WHHL rabbits are not known, our finding suggests that these may be attributable to insulin receptor and/or postinsulin receptor abnormalities since insulin degradation, which is receptor-mediated,‘” is decreased, and that insulinbinding defects could be the predominant abnormality since insulin regulates its own receptor and changes in the number of insulin receptors (downregulation) due to hyperinsulinemia in WHHL rabbits may influence the amount bound and therefore the amount susceptible to degradation. It is also possible that a membrane defect that affects the ability of the insulin receptor to bind insulin is present in WHHL rabbits. Since the liver is the major target organ responsible for insulin degradationly and since insulin degradation in WHHL rabbits was slower. as suggested by the twocompartment model analysis, the liver might play a significant role in the pathogenesis of insulin resistance in WHHL rabbits. There is a strong possibility that renal function may be impaired in WHHL rabbits, since the kidney is second only to the liver with regard to responsibility for insulin metabolism and degradation3” Therefore, impaired renal function could decrease insulin degradation. However, recent studies have shown a lack of pathologic lesions” in the kidney and a normal renal function, as expressed by normal creatinine levels”? (1.0 2 0.4 mg/dL). in adult WHHL rabbits compared with New Zealand White rabbits (1.2 * 0.9 mg/dL). Further studies are needed to clarify the pathogenesis and cellular mechanism of insulin resistance in WHHL rabbits. Study A showed that serum levels of both TC and TG were significantly higher (P < ,001) in WHHL rabbits. However, hyperinsuhnemia and hypertriglyceridemia do not appear in FH patientssj as they do in WHHL rabbits, which suggests that a strong association exists between hyperinsulinemia and hypertriglyceridemia in WHHL rabbits. In fact, insulin directly stimulates TG synthesis in the liver; increased TG production was observed in patients with non-insulin-dependent diabetes mellitus with moderate hypertriglyceridemia,?J and an increased rate of verylow-density lipoprotein (VLDL) TG production by the liver was found in hyperinsulinemic rats.“5 In addition, when TG concentrations in hypertriglyceridemic patients were decreased with gemfibrozil, insulin concentrations during an oral glucose tolerance test were decreased in proportion to reductions in TG levels, while glucose concentrations were unaffected.‘” A causal relationship may also exist between hyperinsulinemia and hypertriglyceridemia in WHHL rabbits. In study B, cholesterol feeding, which greatly increased levels of serum TC (893.9 ? 80.8 L’ 37.4 + 2.5 mg/dL, P < .OOl) and TG (118.3 2 17.5 15 47.7 * 2.4 mg/dL. P < ,001) was shown to have no marked effect on glucose tolerance and insulin sensitivity. However, cholesterol feed-

INSULIN-GLUCOSE

365

RESPONSE IN WHHL RABBITS

ing did decrease fasting levels of plasma insulin (P < .Ol). When insulin levels are low, such as during fasting, lipolysis increases and nonesterified fatty acid concentrations, which may play an important role in the control of hepatic VLDL-TG production, increase.35 The association between hypercholesterolemia and insulin metabolism seems quite complex. since FH patients have normal insulin metabolism.“” WHHL rabbits have hyperinsulinemia, and cholesterol-fed rabbits have lower fasting insulin levels. Studies A and B show that inherited hypercholesterolemia in WHHL rabbits and diet-induced hypercholesterolemia have different pattlerns of insulin metabolism. It is interesting that both hyperinsulinemia and low levels of fasting plasma insulin arc associated with high levels of plasma TG. Since high levels of plasma TG due to cholesterol feeding did not produce hyperinsulinemia (study B) [which is a secondary phenomenon to insulin resistanceZ4], insulin resistance in WHHL rabbits appears to be independent of diet. In study C. our data showed that hyperinsulinemia and insulin sensitivity (ZIRI/ZPG) in WHHL rabbits were improved by cilazapril treatment, whereas glucose tolerance was not affected, indicating an improved insulin resistance in WHHL rabbits treated with cilazapril. The beneficial effects of cilazapril on insulin resistance in WHHL rabbits may help explain a previous report that the ACE inhibitor captopril decreased atheromatous plaqueI

in WHHL rabbits. Hyperinsulinemia and insulin resistance both play a role in the expression of elevated VLDL and low-density lipoprotein levels, as well as in the depression of high-density lipoprotein levels.” Hypertension markedly stimulates atherosclerosis in WHHL rabbits,37 which may in part explain why antihypertensive drugs also have antiatherogenic effects. Therefore, the antihypertensive effects of cilazapril, in addition to its effects on insulin resistance, should be emphasized in therapeutic studies aimed at reducing the risks of CHD. Due to limited experimental conditions, blood pressures of WHHL and JW rabbits were not measured in this study. Model parameters in study C showed that insulin degradation in WHHL rabbits was improved by administration of cilazapril (Table 6). Therefore, one possible mechanism by which cilazapril exerts its effect on insulin resistance is that it increases the affinity or number of insulin receptors. Cilazapril treatment produced a tendency for lower levels of insulin and higher levels of glucose in JW rabbits (Table 5), but the mechanism behind this observation is unknown. Our present data showed that decreased insulin degradation probably contributes to insulin resistance in WHHL rabbits. WHHL and cholesterol-fed rabbits have different patterns of glucose-insulin metabolism. Cilazapril administration improved insulin resistance in WHHL rabbits, and increased insulin degradation may play a role in this effect.

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