- Email: [email protected]
N-corpulent
199
Atherosclerosis, 69 (1988) 199-209 Elsevier Scientific Publishers Ireland. Ltd.
ATH 04074
Atherogenesis in two strains of obese rats The fatty Zucker and LA/N-corpulent R.M. Amy ‘, P.J. Dolphin 2, R.A. Pederson 3 and J.C. Russell 4 ’Departments of Pathology and ’ Surgery, University of Alberta, Edmonton, Alberta (Canada), 2 Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia (Canada), and 3 MRC Regulatory Peptide Group, Department Columbia, Vancouver, B. C. (Canada)
of Physiology,
University of British
(Received 6 March, 1987) (Revised, received 25 August, 1987) (Accepted 28 August, 1987)
Two strains of obese rats, the fatty Zucker and the LA/N-corpulent have been compared at 6 months age for the presence of vascular and myocardial disease. Both strains, when obese, exhibit a VLDL hyperlipidemia with elevated triglycerides and moderate elevations of plasma cholesterol concentrations compared to the lean rats of the same strain. The hyperlipidemia is more modest in the fatty Zucker than the corpulent LA/N, and the serum lipid concentrations of the lean Zucker are lower than those of the lean LA/N. Apoliprotein concentrations were similar and elevated in the two obese genotypes compared to the lean genotypes which were also similar to each other. Male and female obese animals of both strains exhibited hyperinsulinemia under fasting conditions and after oral glucose, with obese male LA/N rats exhibiting the most severe hyperinsulinemia. Glucose tolerance was impaired in obese LA/N animals but was normal in lean rats of both strains and fatty Zucker rats of both sexes. The glucose intolerance observed in obese LA/N animals was more severe in the male than in the female rats. Unlike the corpulent rat, which develops atherosclerotic lesions, the fatty Zucker shows no evidence of advanced vascular lesions on scanning electron microscopy. The fatty Zucker also does not develop the myocardial lesions that are frequent in the male corpulent LA/N rat. It is suggested that the initiation of the atherogenic process is dependent upon elevated insulin levels or transient hyperglycemia. Development of the advanced lesions appears to require the presence of hyperlipidemia.
Key words: Atherosclerosis; microscopy
Hyperlipidemia;
Hyperinsulinemia;
This work was supported by the Alberta Heart Foundation and the Medical Research Council of Canada. Correspondence to: Dr. J.C. Russell, Department of Surgery, 12-116 Clinical Sciences Bldg., University of Alberta, Edmonton; Alberta T6G 2G3, Canada, Tel. (403) 432-6359. 0021-9150/88/$03.50
Myocardial
lesions; Scanning electron
Introduction
Occasional spontaneous mutations in laboratory animal colonies have permitted development
0 1988 Elsevier Scientific Publishers Ireland, Ltd
200 of a number of valuable models of human diseases. Mutations in rodents leading to a genetically determined obesity have been prominent in this group. These include the ob/ob mouse, fatty Zucker rat and the corpulent rat originally developed by Koletsky [l-4]. The ob/ob mouse has been regarded as a model for obesity. Similarly, the fatty Zucker rat has been used in studies of obesity, metabolism and insulin resistance [5-81. The corpulent rat gene arose as a mutation in a colony of SHR rats in Koletsky’s laboratory giving, in homozygous rats, an obese hypertensive state. These rats were found to develop a fulminant atherosclerosis with early death [3,4]. Further development work by Hansen resulted in two congenie strains of rats, incorporating the cp gene into standardized parent stocks [9-111. These two strains, the LA/N-cp and SHR/N-cp were repeatedly backcrossed to the parent LA/N and SHR/N strains to remove all extraneous genetic contributions except the corpulent gene. Rats heterozygous for the cp gene (cp/ + ) or homozygous normal ( + / + ), are lean and indistinguishable from the parent strain. Animals homozygous cp (cp/cp) are hyperphagous, hyperlipidemic, insulin-resistant and obese [11,12]. The fatty Zucker rat, in some respects, appears to be very similar to the LA/N-cp rat. If homozygous normal (Fa/Fa) or heterozygous (Fa/fa) it is lean and normal, while if homozygous for the fatty gene (fa/fa) it is obese. The fa/fa rats have been reported to be hyperinsulinemic and exhibit abnormal gut hormone status [7,8,13]. The fa/fa rat has also been reported to be hyperlipidemic [7,14]. There are no reports of cardiovascular disease in this strain. In contrast the LA/N-cp rat, when cp/cp, has been found to develop both atherosclerotic lesions of the major arteries and myocardial lesions [15,16]. The evidence to date would suggest that both the marked VLDL hyperlipidemia [17] and insulin resistance are required for induction of the vascular disease (Russell and Amy, unpublished observations). The two rat strains, the fatty Zucker and LA/N-cp are similar in some respects and complementary, but yet different in the disease that develops. This paper reports a direct comparative study of the two strains, in lipid and apoliprotein status, insulin and glucose metabolism and vascular and myocardial lesion development.
Materials and methods
Animals Fatty Zucker rats were bred at the University of British Columbia in a colony derived from breeding stock supplied by Dr. P. Bechtel, Animal Sciences Laboratory, University of Illinois. The fatty rats will be homozygous fa (fa/fa) while the phenotypically lean will be a 2 : 1 mixture of heterozygotes (fa/Fa) and homozygote normals (Fa/Fa). Thus, they will be represented as Fa/?. The LA/N-cp rats were bred at the University of Alberta in a colony established with stock donated by Dr. C.T. Hansen, Small Animal Section, Division of Research Services, NIH, Bethesda, MD. These rats are derived from the fifth backcross to the LA/N strain. Both cp/cp and + / + rats were bred as previously described [12,15] with the + / + rats bred from demonstrated homozygote normal parents. Matched groups of rats, fatty and corpulent and lean of both strains, were established for the studies of insulin and glucose metabolism. With the exception of the oral glucose tolerance tests, only male rats, 6 months of age, were used in all studies reported here. The rats were fed a standard rat chow (Wayne Lab-Blox, Continental Grain Co., Chicago, IL) ad lib and tap water was available at all times.
Oral glucose tolerance test Rats were fasted overnight and administered 40% glucose by syringe and feeding tube at 1 g/kg. Blood samples were collected at 0, 10, 20, 30 and 60 min following the glucose load. Two samples of 250 ~1 were taken from the tail vein of unrestrained conscious rats into heparinized capillary tubes. The integrated glucose and insulin responses were calculated as the total area under the response curve for insulin or glucose. Glucose was assayed using a glucose oxidase procedure (Beckman Instruments, Fullerton, CA). Insulin was measured using the modification of an RIA previously described [18]. Purified rat insulin was used as standard and for iodination. The antisera were lyophilized anti-human insulin serum (guinea pig).
Lip& and apoliproteins Blood
samples
were
taken
by
open
cardiac
201 puncture of halothane-anesthetized rats following a 12-h fast. The separated sera were preserved with 0.01% (w/v) sodium azide and 0.01% Thimerosal (Sigma Chemical Co., St. Louis, MO) and maintained at 4O C until assayed within 7 days. Lipoproteins were isolated by sequential ultracentrifugation at 10” C in a Beckman L5-50B ultracentrifuge using a 50.3 Ti rotor and Quickseal tubes. Serum density was adjusted by addition of solid sodium bromide and lipoprotein isolated under the following conditions: VLDL at d = 1.006 g/min; IDL at d = g/ml for 1.10 X lo* 1.006-1.030 for 1.19 x lo8 g/mm; LDL at d= 1.030-1.063 g/ml for 1.45 X lo* g/n&; HDL at d = 1.063-1.21 g/ml for 2.7 X 10’ g/mm. Following ultracentrifugation, the lipoproteins were separated from the infranatant by tube slicing. Lipoprotein and serum lipids were analysed by the gas chromatographic total lipid profiling technique of Kuksis et al. [19]. The lipid concentrations are expressed in terms of mass. The data for esterified cholesterol refers to the mass of cholesterol present as cholesteryl ester. Total cholesterol represents the summation of unesterified cholesterol and cholesterol present as cholesteryl esters. Particle diameters were calculated from the lipid composition according to the method of Shen et al. [20]. Electroimmunoassays for Apo B, Apo E and Apo A-I were performed as described previously [21,22]. Agarose gel electrophoresis utilized the method of Maguire et al. [23]. Total protein was measured by the method of Lowry et al. [24].
Histology After blood samples were obtained from the heart, the rats were dissected and the heart removed. The hearts were cut transversely into three pieces, base, midlevel and apex and placed in 10% neutral buffered formalin. The samples were processed by standard histological techniques and embedded in paraffin. Two sections were cut serially from each block transversely across the whole heart. These were stained with hematoxylin and eosin and Masson’s trichrome, respectively. The slides were examined by an experienced anatomic pathologist without knowledge of the identity of the animals. The lesions where characterized as previously described [16]: A: Muscle scar or cell loss with chronic in-
flammatory cell accumulation. B: Necrosis of myocytes with reactive inflammatory cells. C: Nodule of chronic inflammatory cells. D: Muscle scar without chronic inflammatory cells. The number of lesions observed in the three sections from each heart were summed with the two stains used to confirm identification.
Scanning electron microscopy Animals to be sampled for SEM examination of the major vessels are perfusion fixed as previously described [15,16]. The aortic arch and bifurcation, the superior mesenteric artery and celiac plexus were dissected and further fixed in 2.5% glutaraldehyde. The samples were stained with OsO,, dehydrated with a graded ethanol series and triple-point dried. After sputter coating with gold the samples were examined in detail in a Philips SEM505 scanning electron microscope (Philips, Eindhoven, Netherlands).
Statistical analysis Results are expressed as means + standard deviation, except as noted. Statistical analysis was by unpaired t-test, x2-test or Wilcoxon Rank Sum test as appropriate. Results
Lipids and lipoproteins The concentration of lipids in the whole serum of the Zucker and LA/N-cp rats is shown in Table 1. The fa/fa Zucker rats show a significant hyperlipidemia compared to the Fa/? rats. There was an increase in triglycerides and more modest increases in phospholipids and the cholesterol esters and no difference in unesterified cholesterol. The +/+ LA/N rats had higher levels of all lipid species than the lean Zuckers, except for unesterified cholesterol and phospholipids. The differences were all significant (P < 0.01). The cp/cp rats were not only hyperlipidemic with a markedly elevated triglyceride concentration, but showed significantly higher levels of all lipid classes than the fa/fa rats (P < 0.001). The concentrations of lipids in the fa/fa rats were generally intermediate between those of the cp/cp and +/+ rats.
202 TABLE
1
WHOLE
SERUM
Esterified esterified compared
cholesterol refers to the mass of cholesterol present as cholesteryl esters. Total cholesterol is the sum of unesterified and cholesterol. All data are means+ SD. All classes of lipid were present in greater concentration in the obese genotype to the matched lean (P c 0.01) with the exception of unesterified cholesterol in the Zucker rats.
Genotype
LIPID
n
CONCENTRATIONS
IN 6-MONTH-OLD
Unesterified cholesterol
Cholesteryl esters
(mg/lOO
(mg/IOO
ml)
MALE
Esterified cholesterol ml)
(mg/lOO
RATS
Total cholesterol ml)
(mg/lOO
Phospholipids
Triglycerides
(mg/lOO
(mg/loO
ml)
ml)
ml)
Zucker
Pa/? fa/fa
5 5
7.1 + 1.1 8.1 of 1.7
6 6
5.5 f 0.2 14.4 f 1.7
55* 7.5 96+27
33 56
f 4.4 +I16
40 64
f 5.2 *17
37flO 73+17
6.3f 4.4 93 +44
LA/N-cp +/+
cP/cP
TABLE
74+ 137*
3 8.8
43.2* 80.1*
1.9 5.2
48.8f 94.5f
2.0 6.5
33f 109*
1.6 7.8
24.1+ 3.0 181 kl4.2
2
VLDL-LIPID
CONCENTRATIONS
IN 6-MONTH-OLD
All data are means* SD and as described in Table compared to the lean counterpart (P -c 0.01). Genotype
RATS were present
concentrations
Unesterified cholesterol
Cholesteryl esters
(A)
(mg/IOO
(mg/lOO
5 5
144k27 51Ok58
0.54+0.41 2.9 +1.7
1.3 +1.8 6.3 k3.1
0.81 f 1.1 3.8 +1.9
1.4 *1.5 6.7 f3.4
6 6
392 f 57 698+49
0.49 f 0.09 4.6 kO.9
0.71 f 0.32 9.0 f1.2
0.41+ 0.18 5.4 kO.7
0.54 f 0.24 10.0 11.6
ml)
Bsterified cholesterol
at higher
Particle diameter
n
Zucker Pa/? fa/fa
MALE
1. All lipids
ml)
Total cholesterol
(mg/lOO
ml)
(mg/lOO
in the obese
genotypes
Phospholipids
Triglycerides
(mg/lOO
(mg/lOO
ml)
ml)
ml) 2.7 19.4
f 2.6 *11.4
1.3 f 1.3 80.2 *47.1
LA/N-cp +/+ cP/cP
TABLE
CONCENTRATIONS
IN 6-MONTH-OLD
MALE
3.49f 169 f
1.56 9.2
RATS
All data are means f SD and as described in Table 1. With the exception of unesterified were higher in fatty than lean Zucker rats. All lipids in the HDL fraction from cp/cp + / + LA/N rats (P < 0.01).
Zucker Pa/? fa/fa
0.39 2.5
3
HDL-LIPID
Genotype
0.88* 29.1 k
Particle diameter
Unesterified cholesterol
Cholesteryl esters
(A)
(mg/lOO
(mg/lOO
3 3
259 f 27 238*20
2.8k1.5 3.4kO.8
6 6
353 f 26 252+ 6.5
2.9 * 0.2 3.9kO.6
n
ml)
41 78
Esterified cholesterol ml)
f 6 kl7
(mg/lOO
cholesterol and triglycerides all HDL-lipids rats were higher than the concentration in
Total cholesterol ml)
(mg/lOO
Phospholipids
Triglycerides
(mg/lOO
(mg/IOO
ml)
23.9* 3.6 45 *lo
26.7f 4.4 49 *10
20 43
f 4 *11
33.8+ 67.6*
36.8kll.l 76.6* 4.4
19.2&- 1.6 61.7* 4.1
0.92kl.l 0.77 + 0.20
LA/N-cp
+/+ cp/cp
57.8* 115.9*
1.6 7.3
0.05 4.3
ml)
ml)
0.99 kO.52 2.7 kO.8
203 Table 2 shows the VLDL-lipid concentrations for the four rat genotypes. The pattern is similar to that seen for whole serum with the fa/fa rats having lipid concentrations that are roughly halfway between the concentrations of the +/+ and those of the cp/cp rats. Both obese genotypes exhibited VLDL hyperlipidemia, with the VLDL triglyceride constituting the source of the whole serum hypertriglyceridemia. The HDL-lipid concentrations are shown in Table 3. The obese rats, both fa/fa and cp/cp showed consistent small elevations of the HDL lipids and were very similar to each other as were the +/+ and Fa/? rats except that triglycerides and unesterified cholesterol were not elevated in the fa/fa rats. There were no significant differences between the obese and lean rats in the lipid concentrations in the IDL and LDL fractions. As is normal in rats, these fractions contained only minor amounts of all lipid classes. The level and distribution of the apoliproteins is shown in Table 4. There were no significant differences between the Fa/? and + / + rats in apoliprotein concentrations. In both obese genotypes the levels were higher than in the lean genotypes and the fa/fa rats showed significantly greater concentrations of all three apolipoproteins compared to the cp/cp LA/N rats. Insulin resistance Figures 1 and ance curves for following an oral
TABLE
and glucose tolerance 2 show the plasma glucose tolermale rats at 4 months of age glucose load. As is shown in Fig.
4
APOLIPROTEIN LEVELS AND MONTH-OLD MALE RATS AI1 data mg/lOO Genotype
n
DISTRIBUTION
IN
ml, means + SD. Whole serum Apo A-l
Apo B
Apo E
5 5
44.2+ 2.8 219 +55
34.2f 5.3 53.8 + 12.1
19.7* 4.2 67.5 + 18.0
6 6
49.2+ 4.3 123.7 + 7.2 *
43.8& 26.4&
11.3* 25.0*
Zucker
Fa/? fa/fa LA/N-q
i/+ cP/cP
* P < 0.005 vs fa/fa.
4.7 2.1 *
1.0 1.4 *
6-
??
4
40
20
60
Minutes Fig. 1. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in obese male LA/N (cp/cp) (n = 5) and obese male Zucker (fa/fa) rats (n = 5). In this and subsequent oral glucose tolerance figures, an asterisk denotes significant differences to at least the 0.05 level.
1 the male cp/cp LA/N rats developed significantly higher glucose levels compared to the + / f males (P < 0.001 at 60 min) or the fa/fa male rats (Fig. 2). Obese female LA/N rats exhibited mild glucose intolerance, but this condition was less severe than that in cp/cp LA/N males (Figs. 1 and 3). Lean female rats of both strains had similar and normal glucose and insulin responses to the glucose load (Fig. 4). Obese rats of both strains exhibited severe fasting and glucose-stimulated hyperinsulinemia as shown in Figs. 1 and 3,
204
0 3
z
100
-
z m a
50
-
+---+ -
I
cplcp
Female
falfa
Female
J
1
20
40
60
Minutes
Fig. 2. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in lean male LA/N ( + / + ) (n = 5) and lean male Zucker (Fa/Fa) rats (n = 5).
II B
I I
I
20
40
60
Minutes
Fig. 3. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in obese female LA/N (cp/cp) (n = 5) and obese female Zucker (fa/fa) rats (n = 5).
compared to the lean rats (Figs. 2 and 4). The integrated glucose responses of all lean groups were similar as were the responses of fatty Zucker rats of either sex and corpulent LA/N female rats (Fig. 5). The insulin responses of the lean groups were approximately 10% of those of fatty Zuckers or female cp/cp animals. The integrated glucose response of the cp/cp male rats was significantly greater ‘than that of the cp/cp female rats (P < 0.005). The integrated insulin response of the cp/cp male rats was 65% greater than that of all other obese groups including cp/cp females (Fig. 5) and this was highly significant (P < 0.001). Scanning electron microscopy Six male fa/fa Zucker rats, 6 months of age were perfusion-fixed and arterial samples examined by SEM. Normal endothelium was observed on most of the endothelial surface as shown
in Fig. 6. This view of a section of aortic arch shows a normal surface of endothelial cells with visible marginal folds. The one significant finding in the arteries from the fa/fa rats was the consistent presence of extensive areas of endothelium with a ‘knobly surface’ as shown in Fig. 7. Some significant area with this abnormal endothelium was found in the samples from each rat. Figure 8 shows one such area at higher magnification, and suggests that the gross appearance is due to swelling of the individual endothelial cells. The appearance is quite different from that of the raised nuclei frequently seen in cp/cp rats and illustrated in Fig. 9. In only one of the fa/fa rats a small area of mild desquamation was found. This is shown in Fig. 10 at moderately high magnification. Individual endothelial cells are missing, but
205 250
2o A
rn
Corrxilent Zucker
-
+I+ Female FalFa Female
1
FMFM
Corpulent Obese 30rB
I
I
40
20
Lean
+t 1 0
25 -
60
Zucker
Minutes
Fig. 4. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in lean female LA/N ( + / + ) (n = 5) and lean female Zucker (Fa/Fa) rats (N = 5).
20 -
15 -
there is no evidence of significant adherence of blood cells or fibrin to the subendothelial layer. In contrast to our previous reports on the cp/cp rat, no advanced lesions of any kind were found in the fa/fa rat arteries. Specifically significant areas of advanced desquamation, raised nuclei, polygonal cells, adherent macrophages or thrombi, were entirely absent in all 6 fa/fa rats. Myocardial lesions The hearts from 6 rats of each genotype at 6 months age were examined for myocardial lesions. Table 5 summarizes the findings. Type A lesions (scar or cell loss with chronic inflammatory cell accumulation) were common in cp/cp rats, absent in +/+ and Fa/? rats and both very small and infrequently present in fa/fa rats. Figure 11A shows a very small type A lesion from an fa/fa rat in comparison to Fig. 11B illustrating a typical
10 -
5-
FMFM
FMFM
Fig. 5. The integrated glucose. (A) and insulin (B) responses to 1 g/kg oral glucose in male and female lean and obese LA/N and Zucker rats using the data shown in Figs. 1-4. Statistical significance is indicated as follows: * P < 0.05, corpulent vs lean; + P < 0.05, male vs female; + P < 0.01, corpulent/fatty vs lean.
and much larger lesion in the heart of a cp/cp rat. The overall incidence in the cp/cp rats was, in addition, significantly greater. Type B lesions were only rarely found in cp/cp rats. Type C lesions
Fig. 6. Scanning electron micrograph of the aortic graph of a 6-month-old male fa/fa rat. The flat smooth individual endothelial cells are delineated by the slightly raised marginal folds. The alternating light and dark bars in the data panel at the bottom are indicated as 10 pm in length. The original magnification was X 1200.
(nodules of chronic inflammatory cells) were found only rarely in fa/fa rats and frequently in cp/cp rats. Type D lesions, that represent old organised scars without chronic inflammatory cells, were found only in cp/cp rats. The incidence was significant compared to the absence of lesions found in the fa/fa rats (P < 0.01). Discussion While the fatty Zucker rats are both phenotypically
Fig. 8. Higher magnification of a portion of the field of view of Fig. 2 showing the altered morphology of individual endothelial cells. The original magnification was X 1200.
significant differences between them both metabolically and in pathogenesis. The obesity appears to be more severe in the cp/cp rat and they can be unequivocably distinguished from the +/+ rats at 21 days while this is only possible with the fa/fa at 35 days [S]. The lipid and apoliprotein results reveal a major difference between the two obese genotypes. Both show a VLDL hyperlipidemia. However, the severity of the hyperlipidemia and the absolute concentrations of the serum lipids is much greater in
and LA/N-corpulent obese rats, there are
Fig. 7. Portion of the aortic arch of a 6-month-old fa/fa male rat showing an area of abnormal swollen or raised endothelial cells. The original magnification was x 300.
Fig. 9. Scanning electron micrograph of aortic arch of a 6-month-old male cp/cp LA/N rat showing area of endothelial cells with raised nuclei. The original magnification was x 1200.
207
Fig. 10. Portion of the aortic arch of a 6-month-old fa/fa male rat showing area of mild desquamation without adherent fibrin or blood cells. The original magnification was x 1620.
the cp/cp rat than in the fa/fa rat. Similarly, the serum lipid concentrations were lower in the Fa/? rat, than in the + /+ LA/N rat. The hyperlipidemia in both these obese rats is largely a hypertriglyceridemia, although the cholesterol concentrations are increased. This pattern is associated, in the cp/cp male rat, with vascular and myocardial lesions [16,17]. However, the female cp/cp, despite an even more severe hypertriglyceridemia (over 1000 mg/lOO ml at 9 months, unpublished observations) does not develop the lesions [17]. Thus, while the VLDL hyperlipidemia appears to be required for cardiovascular lesion induction, it is not in itself sufficient to cause atherosclerotic disease. This finding is apparently not consistent with those of Arbogas et al. [25]. They concluded, on the basis of in vitro studies,
Fig. 11. (A) Light micrograph of small type A lesion ( --t ) in the left ventricle of the heart of an fa/fa male rat 6 months of age. Hematoxylin and eosin, x 125. (B) Type A lesion in the left ventricle of the heart of a 6-month-old male cp/cp LA/N rat. Masson’s trichrome, x 50. The extensive affected areas of this lower magnification field are indicated by arrows.
TABLE
5
MYOCARDIAL LESION OLD MALE RATS
FREQUENCY
IN
6-MONTH-
Data are frequency of occurrence of each lesion type in 3 separated sections from each heart, expressed as means f SD. The lesion types are as defined in the MATERIALS AND METHODS section. Genotype
n
Zucker Fa/? fa/fa
Lesion type A
B
C
D
6 6
0 0.25 + 0.46
0 0
0 0.12 kO.35
0 0
6 6
0 1.83 * 1.60
0 0.16
0 0.67
0 1.33
*0.41
kO.82
+1.03
P < 0.02
N.S.
N.S.
P -= 0.01
LA/N-q +/+
cP/cP
Significance cp/cp
vs fa/fa
that VLDL particles were responsible for endothelial toxicity of serum from diabetic or sucrosefed rats. Thus, it is possible that the damaging effect of the hyperglycemic state is mediated or transmitted through the VLDL. The cholesterol and triglyceride concentrations found in the Zucker rats, fatty and lean, are considerably lower than those reported by Witztum and Schonfeld [26] or Seebach and Kris-Etherton [14]. This difference may be due to differences between colonies of Zucker rats, the age of the animals (and this has not always been specified) or to the very different biochemical techniques used. The lipid concentrations reported by various investigators for Zucker rats are very variable, both within and between studies, suggesting significant variations between animals. It should also be noted that the results reported here were obtained by a gas chromatographic total lipid profile technique whereas previous work was based on chemical or enzymatic assays. The present study has shown that 4-6-monthold Zucker fatty rats of both sexes are normoglycemic but hyperinsulinemic to the same degree (Figs. l-5). This indicates insulin resistance, but not to the extent that this results in glucose in-
tolerance, and is in agreement with earlier studies [8]. Unlike the Zucker strain, corpulent rats exhibit abnormal glucose metabolism as well as hyperinsulinemia. Corpulent female rats exhibited mild glucose intolerance and a similar degree of hyperinsulinemia as the fatty Zucker rat (Fig. 5). In contrast, male corpulent animals demonstrated more severe glucose intolerance and hyperinsulinemia. These results are consistent with a highly insulin-resistant state in the male rats and a more modest impairment in the cp/cp females. This is in contrast to the plasma lipid concentrations were the cp/cp female rats have an extreme VLDL hyperlipidemia and the cp/cp males a more moderate hyperlipidemia. The fatty Zuckers of both sexes have plasma lipid levels resembling those of lean LA/N rats, so that while their lipids are mildly elevated compared to lean Zuckers, this hyperlipidemia is probably not functionally significant. The SEM results from the fa/fa rats are very different from those found previously with the cp/cp rat [16]. The advanced lesions that characterize the cp/cp male rat do not occur in the male fa/fa rat. The areas of abnormal endothelium found in the fa/fa rat are unique to this genotype and may represent an early state of endothelial damage. The absence of more advanced lesions suggests that the processes leading to mature lesions do not occur, perhaps because the plasma lipid levels are not sufficiently high. The abnormal endothelium may be caused by the abnormal insulin metabolism or raised glucose levels. Alternatively, the Zucker rats are known to be chronically infected with mycoplasma, and the vascular abnormality may be an effect of these organisms. Such an origin would explain the absence of similar lesions in the cp/cp rat. The myocardial lesion frequency data show a clear distinction between the obese genotypes. The fa/fa rat, like the Fa/? and + /+ LA/N does not spontaneously develop significant myocardial lesions. The female cp/cp LA/N rat also does not develop myocardial lesions. The females are characterized by a very much less severe insulin resistance compared to comparable cp/cp males. The intravenous glucose tolerance of the cp/cp female is similar to that of the fa/fa Zucker, both resembling the + / + LA/N rat. The pattern sug-
209
gests that the atherogenic process requires an initial event that follows from either the elevated insulin levels or transient hyperglycemia. Such a mechanism is very similar to the response to injury hypothesis suggested by Ross and Glomset [27]. The subsequent response with lipid and apolipoprotein exudation into the vessel wall and cellular responses would then be more or less as proposed by Faggiotto and Ross [28].
13
14
15
Acknowledgements 16
This work was made possible by the excellent technical assistance of Ms. D. Koeslag and Ms. E. Schwaldt.
17
References 18
4
5 6
I
8
9 10
11
12
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22
23
exercise and food restriction, Can. J. Physiol. Pharmacol., 64 (1986) 750. Chan, C.B., Pederson, R.A., Buchan, A.M.J., Tubesing, K.B. and Brown, J.C., Gastric inhibitory polypeptide and hyperinsulinemia in the Zucker (fa/fa) rat: a developmental study, Int. J. Obesity, 9 (1985) 137. Seebach, J.D. and Kris-Etherton, P.M., The effect of vigorous treadmill exercise on plasma lipoproteins and hepatic lipoprotein production in Zucker rats, Atherosclerosis, 57 (1985) 53. Russell, J.C. and Amy, R.M., Early atherosclerotic lesions in a susceptible rat model the LA/N-corpulent rat, Atherosclerosis, 60 (1986) 119. Russell, J.C. and Amy, R.M., Myocardial and vascular lesions in the LA/N-corpulent rat, Can. J. Physiol. Pharmacol., 64 (1986) 1272. Dolphin, P.J., Stewart, B., Amy, R.M. and Russell, J.C., Serum lipids and lipoproteins in the atherosclerosis prone LA/N-corpulent rat, B&him. Biophys. Acta, 919 (1987) 140. Pedersen, R.A. and Brown, J.C., Interaction of GIP, glucose and arginine on insulin and glucagon secretion from the perfused rat pancreas, Endocrinology, 103 (1978) 610. Kuksis, A., Myher, J.J., Geher, K., Hoffman, A.G.D., Breckemidge, W.C., Jones, G.J.L. and Little, J.A., Comparative determination of plasma cholesterol and triacylglycerol levels by automated gas-liquid-chromatographic and autoanalyser methods, J. Chromatogr., 146 (1978) 393. Shen, B.W., Scanu, A.M. and Kezdy, F.J., Structure of human serum lipoproteins inferred from compositional analysis, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 837. Dolphin, P.J., Serum and hepatic nascent lipoproteins in normal and hypercholesterolemic rats, J. Lipid Res., 22 (1981) 971. Krul, E.S. and Dolphin, P.J., Secretion of nascent lipoproteins by isolated hepatocytes from hypothyroid and hypothyroid hypercholesterolemic rats, Biochim. Biophys. Acta, 713 (1982) 609. Maguire, G.F. and Breckemidge, W.C., Agarose gel electrophoresis of plasma lipoproteins using the Durrum cell, Clin. B&hem., 8 (1975) 161.
24 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 25 Arbogast, B.W., Berry, D.L. and Newell, C.L., Injury of arterial endothelial cells in diabetic, sucrose-fed and aged rats, Atherosclerosis, 51 (1984) 31. 26 Witztum, J.L. and Schonfeld, G., Lipoproteins in the plasma and hepatic perfusates of the Zucker fatty rat, Diabetes 28 (1979) 509. 27 Ross, R. and Glomset, The patbogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369, 424. 28 Faggiotto, A. and Ross, R., Studies of hypercholesterolemia in the nonhuman primate, Part 11 (Fatty streak conversion to fibrous plaque), Artherosclerosis, 4 (1984) 341.
Atherosclerosis, 69 (1988) 199-209 Elsevier Scientific Publishers Ireland. Ltd.
ATH 04074
Atherogenesis in two strains of obese rats The fatty Zucker and LA/N-corpulent R.M. Amy ‘, P.J. Dolphin 2, R.A. Pederson 3 and J.C. Russell 4 ’Departments of Pathology and ’ Surgery, University of Alberta, Edmonton, Alberta (Canada), 2 Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia (Canada), and 3 MRC Regulatory Peptide Group, Department Columbia, Vancouver, B. C. (Canada)
of Physiology,
University of British
(Received 6 March, 1987) (Revised, received 25 August, 1987) (Accepted 28 August, 1987)
Two strains of obese rats, the fatty Zucker and the LA/N-corpulent have been compared at 6 months age for the presence of vascular and myocardial disease. Both strains, when obese, exhibit a VLDL hyperlipidemia with elevated triglycerides and moderate elevations of plasma cholesterol concentrations compared to the lean rats of the same strain. The hyperlipidemia is more modest in the fatty Zucker than the corpulent LA/N, and the serum lipid concentrations of the lean Zucker are lower than those of the lean LA/N. Apoliprotein concentrations were similar and elevated in the two obese genotypes compared to the lean genotypes which were also similar to each other. Male and female obese animals of both strains exhibited hyperinsulinemia under fasting conditions and after oral glucose, with obese male LA/N rats exhibiting the most severe hyperinsulinemia. Glucose tolerance was impaired in obese LA/N animals but was normal in lean rats of both strains and fatty Zucker rats of both sexes. The glucose intolerance observed in obese LA/N animals was more severe in the male than in the female rats. Unlike the corpulent rat, which develops atherosclerotic lesions, the fatty Zucker shows no evidence of advanced vascular lesions on scanning electron microscopy. The fatty Zucker also does not develop the myocardial lesions that are frequent in the male corpulent LA/N rat. It is suggested that the initiation of the atherogenic process is dependent upon elevated insulin levels or transient hyperglycemia. Development of the advanced lesions appears to require the presence of hyperlipidemia.
Key words: Atherosclerosis; microscopy
Hyperlipidemia;
Hyperinsulinemia;
This work was supported by the Alberta Heart Foundation and the Medical Research Council of Canada. Correspondence to: Dr. J.C. Russell, Department of Surgery, 12-116 Clinical Sciences Bldg., University of Alberta, Edmonton; Alberta T6G 2G3, Canada, Tel. (403) 432-6359. 0021-9150/88/$03.50
Myocardial
lesions; Scanning electron
Introduction
Occasional spontaneous mutations in laboratory animal colonies have permitted development
0 1988 Elsevier Scientific Publishers Ireland, Ltd
200 of a number of valuable models of human diseases. Mutations in rodents leading to a genetically determined obesity have been prominent in this group. These include the ob/ob mouse, fatty Zucker rat and the corpulent rat originally developed by Koletsky [l-4]. The ob/ob mouse has been regarded as a model for obesity. Similarly, the fatty Zucker rat has been used in studies of obesity, metabolism and insulin resistance [5-81. The corpulent rat gene arose as a mutation in a colony of SHR rats in Koletsky’s laboratory giving, in homozygous rats, an obese hypertensive state. These rats were found to develop a fulminant atherosclerosis with early death [3,4]. Further development work by Hansen resulted in two congenie strains of rats, incorporating the cp gene into standardized parent stocks [9-111. These two strains, the LA/N-cp and SHR/N-cp were repeatedly backcrossed to the parent LA/N and SHR/N strains to remove all extraneous genetic contributions except the corpulent gene. Rats heterozygous for the cp gene (cp/ + ) or homozygous normal ( + / + ), are lean and indistinguishable from the parent strain. Animals homozygous cp (cp/cp) are hyperphagous, hyperlipidemic, insulin-resistant and obese [11,12]. The fatty Zucker rat, in some respects, appears to be very similar to the LA/N-cp rat. If homozygous normal (Fa/Fa) or heterozygous (Fa/fa) it is lean and normal, while if homozygous for the fatty gene (fa/fa) it is obese. The fa/fa rats have been reported to be hyperinsulinemic and exhibit abnormal gut hormone status [7,8,13]. The fa/fa rat has also been reported to be hyperlipidemic [7,14]. There are no reports of cardiovascular disease in this strain. In contrast the LA/N-cp rat, when cp/cp, has been found to develop both atherosclerotic lesions of the major arteries and myocardial lesions [15,16]. The evidence to date would suggest that both the marked VLDL hyperlipidemia [17] and insulin resistance are required for induction of the vascular disease (Russell and Amy, unpublished observations). The two rat strains, the fatty Zucker and LA/N-cp are similar in some respects and complementary, but yet different in the disease that develops. This paper reports a direct comparative study of the two strains, in lipid and apoliprotein status, insulin and glucose metabolism and vascular and myocardial lesion development.
Materials and methods
Animals Fatty Zucker rats were bred at the University of British Columbia in a colony derived from breeding stock supplied by Dr. P. Bechtel, Animal Sciences Laboratory, University of Illinois. The fatty rats will be homozygous fa (fa/fa) while the phenotypically lean will be a 2 : 1 mixture of heterozygotes (fa/Fa) and homozygote normals (Fa/Fa). Thus, they will be represented as Fa/?. The LA/N-cp rats were bred at the University of Alberta in a colony established with stock donated by Dr. C.T. Hansen, Small Animal Section, Division of Research Services, NIH, Bethesda, MD. These rats are derived from the fifth backcross to the LA/N strain. Both cp/cp and + / + rats were bred as previously described [12,15] with the + / + rats bred from demonstrated homozygote normal parents. Matched groups of rats, fatty and corpulent and lean of both strains, were established for the studies of insulin and glucose metabolism. With the exception of the oral glucose tolerance tests, only male rats, 6 months of age, were used in all studies reported here. The rats were fed a standard rat chow (Wayne Lab-Blox, Continental Grain Co., Chicago, IL) ad lib and tap water was available at all times.
Oral glucose tolerance test Rats were fasted overnight and administered 40% glucose by syringe and feeding tube at 1 g/kg. Blood samples were collected at 0, 10, 20, 30 and 60 min following the glucose load. Two samples of 250 ~1 were taken from the tail vein of unrestrained conscious rats into heparinized capillary tubes. The integrated glucose and insulin responses were calculated as the total area under the response curve for insulin or glucose. Glucose was assayed using a glucose oxidase procedure (Beckman Instruments, Fullerton, CA). Insulin was measured using the modification of an RIA previously described [18]. Purified rat insulin was used as standard and for iodination. The antisera were lyophilized anti-human insulin serum (guinea pig).
Lip& and apoliproteins Blood
samples
were
taken
by
open
cardiac
201 puncture of halothane-anesthetized rats following a 12-h fast. The separated sera were preserved with 0.01% (w/v) sodium azide and 0.01% Thimerosal (Sigma Chemical Co., St. Louis, MO) and maintained at 4O C until assayed within 7 days. Lipoproteins were isolated by sequential ultracentrifugation at 10” C in a Beckman L5-50B ultracentrifuge using a 50.3 Ti rotor and Quickseal tubes. Serum density was adjusted by addition of solid sodium bromide and lipoprotein isolated under the following conditions: VLDL at d = 1.006 g/min; IDL at d = g/ml for 1.10 X lo* 1.006-1.030 for 1.19 x lo8 g/mm; LDL at d= 1.030-1.063 g/ml for 1.45 X lo* g/n&; HDL at d = 1.063-1.21 g/ml for 2.7 X 10’ g/mm. Following ultracentrifugation, the lipoproteins were separated from the infranatant by tube slicing. Lipoprotein and serum lipids were analysed by the gas chromatographic total lipid profiling technique of Kuksis et al. [19]. The lipid concentrations are expressed in terms of mass. The data for esterified cholesterol refers to the mass of cholesterol present as cholesteryl ester. Total cholesterol represents the summation of unesterified cholesterol and cholesterol present as cholesteryl esters. Particle diameters were calculated from the lipid composition according to the method of Shen et al. [20]. Electroimmunoassays for Apo B, Apo E and Apo A-I were performed as described previously [21,22]. Agarose gel electrophoresis utilized the method of Maguire et al. [23]. Total protein was measured by the method of Lowry et al. [24].
Histology After blood samples were obtained from the heart, the rats were dissected and the heart removed. The hearts were cut transversely into three pieces, base, midlevel and apex and placed in 10% neutral buffered formalin. The samples were processed by standard histological techniques and embedded in paraffin. Two sections were cut serially from each block transversely across the whole heart. These were stained with hematoxylin and eosin and Masson’s trichrome, respectively. The slides were examined by an experienced anatomic pathologist without knowledge of the identity of the animals. The lesions where characterized as previously described [16]: A: Muscle scar or cell loss with chronic in-
flammatory cell accumulation. B: Necrosis of myocytes with reactive inflammatory cells. C: Nodule of chronic inflammatory cells. D: Muscle scar without chronic inflammatory cells. The number of lesions observed in the three sections from each heart were summed with the two stains used to confirm identification.
Scanning electron microscopy Animals to be sampled for SEM examination of the major vessels are perfusion fixed as previously described [15,16]. The aortic arch and bifurcation, the superior mesenteric artery and celiac plexus were dissected and further fixed in 2.5% glutaraldehyde. The samples were stained with OsO,, dehydrated with a graded ethanol series and triple-point dried. After sputter coating with gold the samples were examined in detail in a Philips SEM505 scanning electron microscope (Philips, Eindhoven, Netherlands).
Statistical analysis Results are expressed as means + standard deviation, except as noted. Statistical analysis was by unpaired t-test, x2-test or Wilcoxon Rank Sum test as appropriate. Results
Lipids and lipoproteins The concentration of lipids in the whole serum of the Zucker and LA/N-cp rats is shown in Table 1. The fa/fa Zucker rats show a significant hyperlipidemia compared to the Fa/? rats. There was an increase in triglycerides and more modest increases in phospholipids and the cholesterol esters and no difference in unesterified cholesterol. The +/+ LA/N rats had higher levels of all lipid species than the lean Zuckers, except for unesterified cholesterol and phospholipids. The differences were all significant (P < 0.01). The cp/cp rats were not only hyperlipidemic with a markedly elevated triglyceride concentration, but showed significantly higher levels of all lipid classes than the fa/fa rats (P < 0.001). The concentrations of lipids in the fa/fa rats were generally intermediate between those of the cp/cp and +/+ rats.
202 TABLE
1
WHOLE
SERUM
Esterified esterified compared
cholesterol refers to the mass of cholesterol present as cholesteryl esters. Total cholesterol is the sum of unesterified and cholesterol. All data are means+ SD. All classes of lipid were present in greater concentration in the obese genotype to the matched lean (P c 0.01) with the exception of unesterified cholesterol in the Zucker rats.
Genotype
LIPID
n
CONCENTRATIONS
IN 6-MONTH-OLD
Unesterified cholesterol
Cholesteryl esters
(mg/lOO
(mg/IOO
ml)
MALE
Esterified cholesterol ml)
(mg/lOO
RATS
Total cholesterol ml)
(mg/lOO
Phospholipids
Triglycerides
(mg/lOO
(mg/loO
ml)
ml)
ml)
Zucker
Pa/? fa/fa
5 5
7.1 + 1.1 8.1 of 1.7
6 6
5.5 f 0.2 14.4 f 1.7
55* 7.5 96+27
33 56
f 4.4 +I16
40 64
f 5.2 *17
37flO 73+17
6.3f 4.4 93 +44
LA/N-cp +/+
cP/cP
TABLE
74+ 137*
3 8.8
43.2* 80.1*
1.9 5.2
48.8f 94.5f
2.0 6.5
33f 109*
1.6 7.8
24.1+ 3.0 181 kl4.2
2
VLDL-LIPID
CONCENTRATIONS
IN 6-MONTH-OLD
All data are means* SD and as described in Table compared to the lean counterpart (P -c 0.01). Genotype
RATS were present
concentrations
Unesterified cholesterol
Cholesteryl esters
(A)
(mg/IOO
(mg/lOO
5 5
144k27 51Ok58
0.54+0.41 2.9 +1.7
1.3 +1.8 6.3 k3.1
0.81 f 1.1 3.8 +1.9
1.4 *1.5 6.7 f3.4
6 6
392 f 57 698+49
0.49 f 0.09 4.6 kO.9
0.71 f 0.32 9.0 f1.2
0.41+ 0.18 5.4 kO.7
0.54 f 0.24 10.0 11.6
ml)
Bsterified cholesterol
at higher
Particle diameter
n
Zucker Pa/? fa/fa
MALE
1. All lipids
ml)
Total cholesterol
(mg/lOO
ml)
(mg/lOO
in the obese
genotypes
Phospholipids
Triglycerides
(mg/lOO
(mg/lOO
ml)
ml)
ml) 2.7 19.4
f 2.6 *11.4
1.3 f 1.3 80.2 *47.1
LA/N-cp +/+ cP/cP
TABLE
CONCENTRATIONS
IN 6-MONTH-OLD
MALE
3.49f 169 f
1.56 9.2
RATS
All data are means f SD and as described in Table 1. With the exception of unesterified were higher in fatty than lean Zucker rats. All lipids in the HDL fraction from cp/cp + / + LA/N rats (P < 0.01).
Zucker Pa/? fa/fa
0.39 2.5
3
HDL-LIPID
Genotype
0.88* 29.1 k
Particle diameter
Unesterified cholesterol
Cholesteryl esters
(A)
(mg/lOO
(mg/lOO
3 3
259 f 27 238*20
2.8k1.5 3.4kO.8
6 6
353 f 26 252+ 6.5
2.9 * 0.2 3.9kO.6
n
ml)
41 78
Esterified cholesterol ml)
f 6 kl7
(mg/lOO
cholesterol and triglycerides all HDL-lipids rats were higher than the concentration in
Total cholesterol ml)
(mg/lOO
Phospholipids
Triglycerides
(mg/lOO
(mg/IOO
ml)
23.9* 3.6 45 *lo
26.7f 4.4 49 *10
20 43
f 4 *11
33.8+ 67.6*
36.8kll.l 76.6* 4.4
19.2&- 1.6 61.7* 4.1
0.92kl.l 0.77 + 0.20
LA/N-cp
+/+ cp/cp
57.8* 115.9*
1.6 7.3
0.05 4.3
ml)
ml)
0.99 kO.52 2.7 kO.8
203 Table 2 shows the VLDL-lipid concentrations for the four rat genotypes. The pattern is similar to that seen for whole serum with the fa/fa rats having lipid concentrations that are roughly halfway between the concentrations of the +/+ and those of the cp/cp rats. Both obese genotypes exhibited VLDL hyperlipidemia, with the VLDL triglyceride constituting the source of the whole serum hypertriglyceridemia. The HDL-lipid concentrations are shown in Table 3. The obese rats, both fa/fa and cp/cp showed consistent small elevations of the HDL lipids and were very similar to each other as were the +/+ and Fa/? rats except that triglycerides and unesterified cholesterol were not elevated in the fa/fa rats. There were no significant differences between the obese and lean rats in the lipid concentrations in the IDL and LDL fractions. As is normal in rats, these fractions contained only minor amounts of all lipid classes. The level and distribution of the apoliproteins is shown in Table 4. There were no significant differences between the Fa/? and + / + rats in apoliprotein concentrations. In both obese genotypes the levels were higher than in the lean genotypes and the fa/fa rats showed significantly greater concentrations of all three apolipoproteins compared to the cp/cp LA/N rats. Insulin resistance Figures 1 and ance curves for following an oral
TABLE
and glucose tolerance 2 show the plasma glucose tolermale rats at 4 months of age glucose load. As is shown in Fig.
4
APOLIPROTEIN LEVELS AND MONTH-OLD MALE RATS AI1 data mg/lOO Genotype
n
DISTRIBUTION
IN
ml, means + SD. Whole serum Apo A-l
Apo B
Apo E
5 5
44.2+ 2.8 219 +55
34.2f 5.3 53.8 + 12.1
19.7* 4.2 67.5 + 18.0
6 6
49.2+ 4.3 123.7 + 7.2 *
43.8& 26.4&
11.3* 25.0*
Zucker
Fa/? fa/fa LA/N-q
i/+ cP/cP
* P < 0.005 vs fa/fa.
4.7 2.1 *
1.0 1.4 *
6-
??
4
40
20
60
Minutes Fig. 1. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in obese male LA/N (cp/cp) (n = 5) and obese male Zucker (fa/fa) rats (n = 5). In this and subsequent oral glucose tolerance figures, an asterisk denotes significant differences to at least the 0.05 level.
1 the male cp/cp LA/N rats developed significantly higher glucose levels compared to the + / f males (P < 0.001 at 60 min) or the fa/fa male rats (Fig. 2). Obese female LA/N rats exhibited mild glucose intolerance, but this condition was less severe than that in cp/cp LA/N males (Figs. 1 and 3). Lean female rats of both strains had similar and normal glucose and insulin responses to the glucose load (Fig. 4). Obese rats of both strains exhibited severe fasting and glucose-stimulated hyperinsulinemia as shown in Figs. 1 and 3,
204
0 3
z
100
-
z m a
50
-
+---+ -
I
cplcp
Female
falfa
Female
J
1
20
40
60
Minutes
Fig. 2. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in lean male LA/N ( + / + ) (n = 5) and lean male Zucker (Fa/Fa) rats (n = 5).
II B
I I
I
20
40
60
Minutes
Fig. 3. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in obese female LA/N (cp/cp) (n = 5) and obese female Zucker (fa/fa) rats (n = 5).
compared to the lean rats (Figs. 2 and 4). The integrated glucose responses of all lean groups were similar as were the responses of fatty Zucker rats of either sex and corpulent LA/N female rats (Fig. 5). The insulin responses of the lean groups were approximately 10% of those of fatty Zuckers or female cp/cp animals. The integrated glucose response of the cp/cp male rats was significantly greater ‘than that of the cp/cp female rats (P < 0.005). The integrated insulin response of the cp/cp male rats was 65% greater than that of all other obese groups including cp/cp females (Fig. 5) and this was highly significant (P < 0.001). Scanning electron microscopy Six male fa/fa Zucker rats, 6 months of age were perfusion-fixed and arterial samples examined by SEM. Normal endothelium was observed on most of the endothelial surface as shown
in Fig. 6. This view of a section of aortic arch shows a normal surface of endothelial cells with visible marginal folds. The one significant finding in the arteries from the fa/fa rats was the consistent presence of extensive areas of endothelium with a ‘knobly surface’ as shown in Fig. 7. Some significant area with this abnormal endothelium was found in the samples from each rat. Figure 8 shows one such area at higher magnification, and suggests that the gross appearance is due to swelling of the individual endothelial cells. The appearance is quite different from that of the raised nuclei frequently seen in cp/cp rats and illustrated in Fig. 9. In only one of the fa/fa rats a small area of mild desquamation was found. This is shown in Fig. 10 at moderately high magnification. Individual endothelial cells are missing, but
205 250
2o A
rn
Corrxilent Zucker
-
+I+ Female FalFa Female
1
FMFM
Corpulent Obese 30rB
I
I
40
20
Lean
+t 1 0
25 -
60
Zucker
Minutes
Fig. 4. The glucose (A) and insulin (B) responses to 1 g/kg oral glucose in lean female LA/N ( + / + ) (n = 5) and lean female Zucker (Fa/Fa) rats (N = 5).
20 -
15 -
there is no evidence of significant adherence of blood cells or fibrin to the subendothelial layer. In contrast to our previous reports on the cp/cp rat, no advanced lesions of any kind were found in the fa/fa rat arteries. Specifically significant areas of advanced desquamation, raised nuclei, polygonal cells, adherent macrophages or thrombi, were entirely absent in all 6 fa/fa rats. Myocardial lesions The hearts from 6 rats of each genotype at 6 months age were examined for myocardial lesions. Table 5 summarizes the findings. Type A lesions (scar or cell loss with chronic inflammatory cell accumulation) were common in cp/cp rats, absent in +/+ and Fa/? rats and both very small and infrequently present in fa/fa rats. Figure 11A shows a very small type A lesion from an fa/fa rat in comparison to Fig. 11B illustrating a typical
10 -
5-
FMFM
FMFM
Fig. 5. The integrated glucose. (A) and insulin (B) responses to 1 g/kg oral glucose in male and female lean and obese LA/N and Zucker rats using the data shown in Figs. 1-4. Statistical significance is indicated as follows: * P < 0.05, corpulent vs lean; + P < 0.05, male vs female; + P < 0.01, corpulent/fatty vs lean.
and much larger lesion in the heart of a cp/cp rat. The overall incidence in the cp/cp rats was, in addition, significantly greater. Type B lesions were only rarely found in cp/cp rats. Type C lesions
Fig. 6. Scanning electron micrograph of the aortic graph of a 6-month-old male fa/fa rat. The flat smooth individual endothelial cells are delineated by the slightly raised marginal folds. The alternating light and dark bars in the data panel at the bottom are indicated as 10 pm in length. The original magnification was X 1200.
(nodules of chronic inflammatory cells) were found only rarely in fa/fa rats and frequently in cp/cp rats. Type D lesions, that represent old organised scars without chronic inflammatory cells, were found only in cp/cp rats. The incidence was significant compared to the absence of lesions found in the fa/fa rats (P < 0.01). Discussion While the fatty Zucker rats are both phenotypically
Fig. 8. Higher magnification of a portion of the field of view of Fig. 2 showing the altered morphology of individual endothelial cells. The original magnification was X 1200.
significant differences between them both metabolically and in pathogenesis. The obesity appears to be more severe in the cp/cp rat and they can be unequivocably distinguished from the +/+ rats at 21 days while this is only possible with the fa/fa at 35 days [S]. The lipid and apoliprotein results reveal a major difference between the two obese genotypes. Both show a VLDL hyperlipidemia. However, the severity of the hyperlipidemia and the absolute concentrations of the serum lipids is much greater in
and LA/N-corpulent obese rats, there are
Fig. 7. Portion of the aortic arch of a 6-month-old fa/fa male rat showing an area of abnormal swollen or raised endothelial cells. The original magnification was x 300.
Fig. 9. Scanning electron micrograph of aortic arch of a 6-month-old male cp/cp LA/N rat showing area of endothelial cells with raised nuclei. The original magnification was x 1200.
207
Fig. 10. Portion of the aortic arch of a 6-month-old fa/fa male rat showing area of mild desquamation without adherent fibrin or blood cells. The original magnification was x 1620.
the cp/cp rat than in the fa/fa rat. Similarly, the serum lipid concentrations were lower in the Fa/? rat, than in the + /+ LA/N rat. The hyperlipidemia in both these obese rats is largely a hypertriglyceridemia, although the cholesterol concentrations are increased. This pattern is associated, in the cp/cp male rat, with vascular and myocardial lesions [16,17]. However, the female cp/cp, despite an even more severe hypertriglyceridemia (over 1000 mg/lOO ml at 9 months, unpublished observations) does not develop the lesions [17]. Thus, while the VLDL hyperlipidemia appears to be required for cardiovascular lesion induction, it is not in itself sufficient to cause atherosclerotic disease. This finding is apparently not consistent with those of Arbogas et al. [25]. They concluded, on the basis of in vitro studies,
Fig. 11. (A) Light micrograph of small type A lesion ( --t ) in the left ventricle of the heart of an fa/fa male rat 6 months of age. Hematoxylin and eosin, x 125. (B) Type A lesion in the left ventricle of the heart of a 6-month-old male cp/cp LA/N rat. Masson’s trichrome, x 50. The extensive affected areas of this lower magnification field are indicated by arrows.
TABLE
5
MYOCARDIAL LESION OLD MALE RATS
FREQUENCY
IN
6-MONTH-
Data are frequency of occurrence of each lesion type in 3 separated sections from each heart, expressed as means f SD. The lesion types are as defined in the MATERIALS AND METHODS section. Genotype
n
Zucker Fa/? fa/fa
Lesion type A
B
C
D
6 6
0 0.25 + 0.46
0 0
0 0.12 kO.35
0 0
6 6
0 1.83 * 1.60
0 0.16
0 0.67
0 1.33
*0.41
kO.82
+1.03
P < 0.02
N.S.
N.S.
P -= 0.01
LA/N-q +/+
cP/cP
Significance cp/cp
vs fa/fa
that VLDL particles were responsible for endothelial toxicity of serum from diabetic or sucrosefed rats. Thus, it is possible that the damaging effect of the hyperglycemic state is mediated or transmitted through the VLDL. The cholesterol and triglyceride concentrations found in the Zucker rats, fatty and lean, are considerably lower than those reported by Witztum and Schonfeld [26] or Seebach and Kris-Etherton [14]. This difference may be due to differences between colonies of Zucker rats, the age of the animals (and this has not always been specified) or to the very different biochemical techniques used. The lipid concentrations reported by various investigators for Zucker rats are very variable, both within and between studies, suggesting significant variations between animals. It should also be noted that the results reported here were obtained by a gas chromatographic total lipid profile technique whereas previous work was based on chemical or enzymatic assays. The present study has shown that 4-6-monthold Zucker fatty rats of both sexes are normoglycemic but hyperinsulinemic to the same degree (Figs. l-5). This indicates insulin resistance, but not to the extent that this results in glucose in-
tolerance, and is in agreement with earlier studies [8]. Unlike the Zucker strain, corpulent rats exhibit abnormal glucose metabolism as well as hyperinsulinemia. Corpulent female rats exhibited mild glucose intolerance and a similar degree of hyperinsulinemia as the fatty Zucker rat (Fig. 5). In contrast, male corpulent animals demonstrated more severe glucose intolerance and hyperinsulinemia. These results are consistent with a highly insulin-resistant state in the male rats and a more modest impairment in the cp/cp females. This is in contrast to the plasma lipid concentrations were the cp/cp female rats have an extreme VLDL hyperlipidemia and the cp/cp males a more moderate hyperlipidemia. The fatty Zuckers of both sexes have plasma lipid levels resembling those of lean LA/N rats, so that while their lipids are mildly elevated compared to lean Zuckers, this hyperlipidemia is probably not functionally significant. The SEM results from the fa/fa rats are very different from those found previously with the cp/cp rat [16]. The advanced lesions that characterize the cp/cp male rat do not occur in the male fa/fa rat. The areas of abnormal endothelium found in the fa/fa rat are unique to this genotype and may represent an early state of endothelial damage. The absence of more advanced lesions suggests that the processes leading to mature lesions do not occur, perhaps because the plasma lipid levels are not sufficiently high. The abnormal endothelium may be caused by the abnormal insulin metabolism or raised glucose levels. Alternatively, the Zucker rats are known to be chronically infected with mycoplasma, and the vascular abnormality may be an effect of these organisms. Such an origin would explain the absence of similar lesions in the cp/cp rat. The myocardial lesion frequency data show a clear distinction between the obese genotypes. The fa/fa rat, like the Fa/? and + /+ LA/N does not spontaneously develop significant myocardial lesions. The female cp/cp LA/N rat also does not develop myocardial lesions. The females are characterized by a very much less severe insulin resistance compared to comparable cp/cp males. The intravenous glucose tolerance of the cp/cp female is similar to that of the fa/fa Zucker, both resembling the + / + LA/N rat. The pattern sug-
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gests that the atherogenic process requires an initial event that follows from either the elevated insulin levels or transient hyperglycemia. Such a mechanism is very similar to the response to injury hypothesis suggested by Ross and Glomset [27]. The subsequent response with lipid and apolipoprotein exudation into the vessel wall and cellular responses would then be more or less as proposed by Faggiotto and Ross [28].
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Acknowledgements 16
This work was made possible by the excellent technical assistance of Ms. D. Koeslag and Ms. E. Schwaldt.
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References 18
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