Insulin X10 revisited: a super-mitogenic insulin analogue.

The molecular safety of insulin analogues has received a great deal of attention over the last year. In particular, attention has been directed to the mitogenic properties of insulin analogues as compared with human insulin. Understanding the mechanisms implicated in mediating mitogenic effects of insulin is therefore of particular interest. In this review we detail the story of the rapid-acting insulin analogue known as X10, which was the first insulin analogue in clinical development, but ended up being discontinued at an early clinical development stage following findings of mammary tumours in female Sprague-Dawley rats. The molecular characteristics of insulin X10, along with its interaction at both the IGF-1 receptor and the insulin receptor, have provided us with important insights into mechanisms implicated in metabolic and mitogenic signalling of insulin analogues.

Differential aetiology and impact of phosphoinositide 3-kinase (PI3K) and Akt signalling in skeletal muscle on in vivo insulin action.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is a key factor in the development of type 2 diabetes and although some studies indicate that this could be partly attributed to reduced content and activity of various proximal and distal insulin signalling molecules, consensus is lacking. We therefore aimed to investigate the regulation of proximal insulin signalling in skeletal muscle and its effect on glucose metabolism in a large non-diabetic population. METHODS: We examined 184 non-diabetic twins with gold-standard techniques including the euglycaemic-hyperinsulinaemic clamp. Insulin signalling was evaluated at three key levels, i.e. the insulin receptor, IRS-1 and V-akt murine thymoma viral oncogene (Akt) levels, employing kinase assays and phospho-specific western blotting. RESULTS: Proximal insulin signalling was not associated with obesity, age or sex. However, birthweight was positively associated with IRS-1-associated phosphoinositide 3-kinase (PI3K; IRS-1-PI3K) activity (p = 0.04); maximal aerobic capacity (VO2(max)), paradoxically, was negatively associated with IRS-1-PI3K (p = 0.02) and Akt2 activity (p = 0.01). Additionally, we found low heritability estimates for most measures of insulin signalling activity. Glucose disposal was positively associated with Akt-308 phosphorylation (p < 0.001) and Akt2 activity (p = 0.05), but not with insulin receptor tyrosine kinase or IRS-1-PI3K activity. CONCLUSIONS/INTERPRETATION: With the exception of birthweight, 'classical' modifiers of insulin action, including genetics, age, sex, obesity and VO2(max) do not seem to mediate their most central effects on whole-body insulin sensitivity through modulation of proximal insulin signalling in skeletal muscle. We also demonstrated an association between Akt activity and in vivo insulin sensitivity, suggesting a role of Akt in control of in vivo insulin resistance and potentially in type 2 diabetes.

Insulin signaling in human skeletal muscle: time course and effect of exercise.

Activation of early steps in the insulin signaling cascade in human skeletal muscle was investigated using a one-step euglycemic-hyperinsulinemic (approximately 100 pU/ml) clamp in seven healthy young men 3 h after one-legged exercise. Concomitant insulin stimulation (three- to six-fold [P < 0.05]) of thigh glucose clearance, muscle insulin receptor tyrosine kinase (IRTK), insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) was observed in the rested leg. Twenty minutes after cessation of insulin infusion, the level of these parameters returned toward basal. A twofold higher insulin-stimulated glucose clearance in the exercised compared with the rested thigh was accompanied by unaltered maximal IRTK activation and IRS-1 tyrosine phosphorylation, and by a decreased (approximately 50%, P < 0.05) maximal IRS-1 associated PI 3-kinase activation. Prior exercise caused significantly faster insulin-stimulated tyrosine phosphorylation of IRS-1, PI 3-kinase activity, and glucose clearance compared with those in the rested thigh. In conclusion, physiological hyperinsulinemia activates IRTK, IRS-1 tyrosine phosphorylation, and PI 3-kinase in human skeletal muscle. However, increased insulin action after exercise is not caused by potentiation of these steps in the insulin signaling cascade. In contrast, at steady state, paradoxically decreased insulin-stimulated IRS-1-associated PI 3-kinase activity was observed in exercised muscle. Thus, the activity of IRS-1-associated PI 3-kinase and glucose uptake may not always be tightly coupled during insulin stimulation in human muscle.

Insulin signaling and insulin sensitivity after exercise in human skeletal muscle.

Muscle glucose uptake, glycogen synthase activity, and insulin signaling were investigated in response to a physiological hyperinsulinemic (600 pmol/l)-euglycemic clamp in young healthy subjects. Four hours before the clamp, the subjects performed one-legged exercise for 1 h. In the exercised leg, insulin more rapidly activated glucose uptake (half activation time [t1/2] = 11 vs. 34 min) and glycogen synthase activity (t1/2 = 8 vs. 17 min), and the magnitude of increase was two- to fourfold higher compared with the rested leg. However, prior exercise did not result in a greater or more rapid increase in insulin-induced receptor tyrosine kinase (IRTK) activity (t1/2 = 50 min), serine phosphorylation of Akt (t1/2 = 1-2 min), or serine phosphorylation of glycogen synthase kinase-3 (GSK-3) (t1/2 = 1-2 min) or in a larger or more rapid decrease in GSK-3 activity (t1/2 = 3-8 min). Thirty minutes after cessation of insulin infusion, glucose uptake, glycogen synthase activity, and signaling events were partially reversed in both the rested and the exercised leg. We conclude the following: 1) physiological hyperinsulinemia induces sustained activation of insulin-signaling molecules in human skeletal muscle; 2) the more distal insulin-signaling components (Akt, GSK-3) are activated much more rapidly than the proximal signaling molecules (IRTK as well as insulin receptor substrate 1 and phosphatidylinositol 3-kinase [Wojtaszewski et al., Diabetes 46:1775-1781, 1997]); and 3) prior exercise increases insulin stimulation of both glucose uptake and glycogen synthase activity in the absence of an upregulation of signaling events in human skeletal muscle.

Cellular mechanism of nutritionally induced insulin resistance in Psammomys obesus: overexpression of protein kinase Cepsilon in skeletal muscle precedes the onset of hyperinsulinemia and hyperglycemia.

The sand rat (Psammomys obesus) is an animal model of nutritionally induced diabetes. We report here that several protein kinase C (PKC) isoforms (alpha, epsilon, and zeta, representing all three subclasses of PKC) are overexpressed in the skeletal muscle of diabetic animals of this species. This is most prominent for the epsilon isotype of PKC. Interestingly, increased expression of PKCepsilon could already be detected in normoinsulinemic, normoglycemic (prediabetic) animals of the diabetes-prone (DP) line when compared with a diabetes-resistant (DR) line. In addition, plasma membrane (PM)-associated fractions of PKCalpha and PKCepsilon were significantly increased in skeletal muscle of diabetic animals, suggesting chronic activation of these PKC isotypes in the diabetic state. The increased PM association of these PKC isotypes revealed a significant correlation with the diacylglycerol content in the muscle samples. Altered expression/activity of PKCepsilon, in particular, may thus contribute to the development of diabetes in these animals; along with other PKC isotypes, it may be involved in the progression of the disease. This may possibly occur through inhibition of insulin receptor (IR) tyrosine kinase activity mediated by serine/threonine phosphorylation of the IR or insulin receptor substrate 1 (IRS-1). However, overexpression of PKCepsilon also mediated down-regulation of IR numbers in a cell culture model (HEK293), resulting in attenuation of insulin downstream signaling (reduced protein kinase B [PKB]/Akt activity). In accordance with this, we detected decreased 125I-labeled insulin binding, probably reflecting a downregulation of IR numbers, in skeletal muscle of Psammomys animals from the DP line. The number of IRs was inversely correlated to both the expression and PM-associated levels of PKCepsilon. These data suggest that overexpression of PKCepsilon may be causally related to the development of insulin resistance in these animals, possibly by increasing the degradation of IRs.

The influence of dietary olive oil and margarine on aortic cholesterol accumulation in cholesterol-fed rabbits maintained at similar plasma cholesterol level.

The present study compares the atherogenicity of a standard diet and diets with 10% olive oil or 10% margarine added, in rabbits maintained at a mean plasma cholesterol level of about 20 mM for 13 weeks. Each group consisted of 15 animals. The distribution of cholesterol in plasma between VLDL, IDL, LDL and HDL was similar in the 3 groups. The thoracic aortic cholesterol accumulation was 16.6 +/- 1.6, 11.4 +/- 1.0 (P < 0.05) and 12.6 +/- 1.7 (P > 0.05) nmol/mg wet weight for the group receiving standard diet, diet with 10% olive oil added and diet with 10% margarine added, respectively. There was no significant difference between groups in the occurrence of the atherosclerotic changes in the proximal and distal parts of coronary arteries, abdominal aorta and renal arteries. The occurrence of atherosclerotic changes in the pulmonary arteries was equal in the groups receiving standard diet and diet with 10% margarine added while it was significantly lower (P < 0.05) in the group receiving diet with 10% olive oil added. The atherosclerotic changes at the aortic orifice of coronary arteries were quanticated morphometrically and were most severe in the group on the standard diet. The results indicate a comparable atherogenic effect of 10% olive oil or margarine addition to standard diet on development of atherosclerosis in rabbits maintained at a similar plasma cholesterol level. The study also suggests that supplementation of olive oil or margarine to standard rabbit diet leads to lower cholesterol accumulation in the thoracic aorta compared with standard diet, an effect not modulated by changes in plasma cholesterol concentrations.

Effect of oestrogen replacement therapy on development of experimental arteriosclerosis: a study in transplanted and balloon-injured rabbit aortas.

The mechanism underlying possible protection of oestrogen replacement therapy against cardiovascular disease appears to go beyond beneficial changes in plasma lipoproteins. A direct action of oestrogen on the metabolism of lipoproteins after entering the arterial wall may occur. The present study evaluated whether oestrogen replacement therapy affects the development of experimental arteriosclerosis in immunologically injured (experiment A + B) and balloon-injured (experiment B) aortas in ovariectomized rabbits maintained at a human level of plasma cholesterol; both models involve severe damage to the endothelium with resulting rapid accumulation of lipoproteins in the arterial intima and therefore appear suitable for studying factors directly affecting subendothelial lipoprotein metabolism. In experiment A, dietary cholesterol required to maintain a human level of plasma cholesterol was significantly higher for the oestrogen group than for the placebo group. Similarly, cholesterol accumulation in the aortic grafts was borderline higher for the oestrogen than the placebo group, whilst intimal hyperplasia was without difference between the groups. Due to a modified schedule of cholesterol feeding in experiment B, oestrogen and placebo groups received the same amount of dietary cholesterol, and cholesterol accumulation and intimal hyperplasia were similar in immunologically injured and balloon-injured parts of the aorta in both groups. These results suggest that in the female rabbit maintained at a human level of plasma cholesterol, oestrogen replacement therapy has no direct action on the development of experimental arteriosclerosis when induced by immunological or mechanical injury to the endothelium.

The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions.

A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse antibody Ber-EP4 was used as the primary capture antibody. In order to permit phenotyping of the isolated carcinoma cells the magnetic beads were removed from the carcinoma cells by DN'ase digestion of the DNA linker between the magnetic bead and the secondary antibody. In an ex vivo model system an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found in control experiments with PBMCs without carcinoma cells. Despite a relatively low recovery, the described method will be valuable for the detection of carcinoma cells in cytospin preparations with subsequent phenotyping of the cells for expression of surface antigens. Depending on the chosen antibodies, the method may be useful for the isolation and characterisation of other cell types in various cell suspensions.

Relative importance of ischemic injury and immunological injury on the development of transplant arteriosclerosis in rabbit aortic allografts.

The development of transplant arteriosclerosis has emerged as a major problem to long-term survival after heart transplantation. The accelerated development of arteriosclerosis in the transplanted arteries, including the aorta, could result either from an ischemic injury in connection with the transplantation, or from an immunological reaction against the transplant, or both. We evaluated histologically and biochemically whether extension of the ischemic period from 1 to 24 hr has any influence on the development of transplant arteriosclerosis, in aorta-allografted rabbits clamped at human levels of plasma cholesterol. One set of rabbits was without immunosuppressive treatment (n = 10 + 9) and another otherwise identical set of rabbits received cyclosporine to achieve blood cyclosporine levels in the human therapeutic range (n = 10 + 12). The number of T lymphocytes in intima suggested that, in the grafts from untreated animals, an immunological injury had arisen, which cyclosporine reduced. A clear trend toward a worsening of the transplant arteriosclerosis was demonstrated as a function of the severity of the ischemic injury, both with and without immunosuppressive treatment. However, the worsening effect of maximal ischemic injury was less than that due to maximal immunological injury. In grafts from cyclosporine-treated animals, the development of transplant arteriosclerosis was significantly less than in grafts from untreated animals exposed to identical periods of ischemia. These results suggest that compared with immunological injury, ischemic injury is of minor importance for the development of experimental transplant arteriosclerosis.

Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears.

The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a variety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefore, phenotypic characterization of disseminated carcinoma cells for expression of protease activators might define the invasive potential of the cells. We present an immunocytochemically enhanced staining method that allows phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytokeratin and of u-PAR intracellularly and on the surface of carcinoma cells. This novel approach can be used for detection and phenotyping of carcinoma cells in blood smears for u-PAR or, presumably, for any other heterogeneously expressed antigen on the surface of the detected cells.

Pattern of malformations in the axial skeleton in human trisomy 21 fetuses.

In the present study, we analyzed the development of the axial skeleton in human trisomy 21 fetuses and defined the fields in the axial skeleton affected in this form of aneuploidy. We investigated 31 human fetuses with trisomy 21, gestational ages 12-24 weeks, on the basis of radiographs of midsagittal tissue blocks of the axial skeleton, comprising the cranial base and the spine. Malformation or agenesis of the nasal bone was present in 19 of 31 fetuses. Nineteen cases had vertebral malformations. Fourteen fetuses had malformations in the cervical region, four in the thoracic and eight in the lumbosacral region. In 1 of 31 fetuses, malformation was seen in the basilar part of the occipital bone. The basisphenoid component appeared scallop-shaped in 30 cases. The pattern of axial skeletal malformations in trisomy 21 fetuses recorded here has not been described previously. Comparison is made with our recent study of trisomy 18, where the pattern of axial skeletal malformations was quite different. It is recommended that axial skeletal radiography should be part of the autopsy of fetuses where chromosome abnormalities are known or suspected.

Pattern of malformations in the axial skeleton in human trisomy 18 fetuses.

We examined and described the development and abnormalities of the axial skeleton in 10 human trisomy 18 fetuses. Whole-body radiographs and radiographs of midsagittal tissue blocks of the cranial base and the spine were studied. In 3 fetuses no spinal radiographs were available. Seven osseous regions or fields along the body axis were analyzed, four in the spine, and three in the cranial base and nasal bones. Malformations occurred in the occipital field in all fetuses. This was a characteristic notching, either unilateral or bilateral, of the basilar part of the occipital bone. Nasal bones were abnormal in 8 cases, either absent or hypoplastic. Malformations were found in the thoracic and/or lumbosacral field in 7 fetuses. A single abnormality was found in the cervical spine in one fetus. The pattern of axial skeletal malformation in trisomy 18 fetuses recorded in the present study has not been described previously. Axial skeletal radiography should be included in autopsies of fetuses when chromosome disorders are present or suspected. The methods applied here are unaffected by autolysis.

Pattern of malformations in the axial skeleton in human triploid fetuses.

We examined the axial skeleton in 15 human triploid fetuses (10 with XXX and 5 with XXY sex chromosomes). All fetuses 14-29 weeks of gestational age (GA), underwent whole-body radiography, permitting analysis of the nasal bone and the spine. From 9 of these, detailed radiographs were taken of midsagittal blocks of the cranial base and the spine, permitting detailed analysis of the cranial base. NASAL BONE: Of 14 fetuses, where the nasal bone was seen on lateral projection, it appeared short in 10 cases. SPINE: The spine was normal in 7 of 15 fetuses; malformations occurred in 8. These were osseous fusions between 2 or more vertebral bodies, most frequently in the cervical and thoracic regions, and disproportions in the sizes of the cervical bodies. Fusions occurred in 5 cases alone, and in one case in combination with disproportions of vertebral size. Disproportions alone occurred in 2 cases. CRANIAL BASE: Malformation of the basilar part of the occipital bone was found in 5 of the 9 fetuses investigated. Of 9 fetuses, bilateral ossification centers of the postsphenoid bone occurred in 7, and shell-like ossification centers in 2. There was no difference in the type of malformations in the different axial fields related to genotype (XXX and XXY). CONCLUSION: The most remarkable findings in the axial skeleton of triploid fetuses are vertebral fusions in 6 of 15 cases; clefts of vertebral bodies, previously reported as common findings in trisomy fetuses, are not demonstrated.

Abnormal timing in the prenatal ossification of vertebral column and hand in Crouzon syndrome.

We report on a radiographically examined fetus (gestational age 13 weeks) with Crouzon syndrome caused by a mutation in the gene encoding the fibroblast growth factor 2 (FGFR2). We found an approximately 2-week delay in vertebral body and hand ossification with normal vertebral arch ossification, suggesting that regionally delayed skeletal maturation might be a manifestation of FGFR2 mutation syndromes. The findings support other studies indicating that different signaling pathways control skeletal maturation in vertebral bodies and vertebral arches.

Pathology of the heart in AIDS. A study of 60 consecutive autopsies.

Cardiac disease and cardiac death in AIDS patients is seldom reported. In recent years minor cardiac abnormalities have been demonstrated, especially by echocardiography. Cardiac pathology in AIDS patients is here reported from 60 consecutive autopsies where the heart was investigated either using single samples of ventricular myocardium (the first 21 cases) or by an examination of the whole heart (the last 39 cases). Myocarditis according to the Dallas criteria was seen in 25 of 60 cases (42%), and in seven of these cases a probable pathogen (Toxoplasma gondii, cytomegalovirus, fungi) was demonstrated. Diffuse myocardial fibrosis was seen in 40 of 60 cases (67%) and is considered to be partly due to repair after myocyte necrosis/myocarditis. A myocardium thus weakened might not be able to meet an increase in functional demand, and in 15 of the 39 cases (38%) where an examination of the whole heart was performed, there was dilation and/or hypertrophy of the right ventricle. This is in agreement with our knowledge that the main diseases and main causes of death in AIDS patients are pulmonary. Survival time in AIDS is increasing due to ever improving symptomatic treatment, and the results of this study indicate that the prevalence of especially right-sided heart failure will increase.

Postmortem axial skeletal radiography can reveal fetal CNS malformations.

The routinely performed autopsy of a macerated fetus will often be of dubious value, particularly as regards the examination of the central nervous system (CNS). Former studies have demonstrated a close relationship between certain CNS malformations and axial skeletal malformations revealed radiographically. In the present report a postmortem examination of a severely macerated fetus demonstrates a transsphenoidal encephalocele. A supplementary histological examination confirmed this condition and furthermore revealed absence of the pituitary gland. The findings were related to a strongly elevated serum alphafetoprotein level in the 18th gestational week. This report emphasizes the value of postmortem axial skeletal radiography of autolyzed fetuses suspected for CNS malformations.

Luteinizing hormone-releasing hormone and innervation pathways in human prenatal nasal submucosa: factors of importance in evaluating Kallmann's syndrome.

A previous study has demonstrated that luteinizing hormone-releasing hormone (LHRH) is localized in the human bilateral vomeronasal organs in the nasal septum during a 4-week period of intrauterine life (22). The purpose of the present study was to elucidate the location of LHRH-expressing cells outside the vomeronasal organs, with special emphasis on the submucosa of the medial wall and roof of the nasal cavity. An additional aim was to study the innervation pathways in the same regions. Both regions can be affected in Kallmann's syndrome, which is characterized by hypogonadotropic hypogonadism (lack of LHRH) and often associated with anosmia. Histological sections of craniofacial regions (49 normal human fetuses, 6-19 weeks) were examined by immunohistochemical techniques for LHRH and for neuronal tissue (protein gene product 9.5, PGP 9.5). LHRH reactions were only seen in the septal submucosa extending from the vomeronasal organs to the olfactory bulb. There was a close spatiotemporal association between the occurrence of LHRH and neuronal tissue. From the rhino-olfactory epithelium separate nerve tissue extended to the olfactory bulb. It is suggested that the medial region of the nasal placode giving rise to the septal wall is always affected in Kallmann's syndrome, and in cases in which the phenotypic features are associated with anosmia, also the more lateral part of the nasal placode, from which the rhino-olfactory region originates, is affected.

Hormone replacement therapy prevents coronary artery disease in ovariectomized cholesterol-fed rabbits.

The prevention of coronary artery disease in women is of considerable importance. We have therefore investigated the influence of oestrogen monotherapy and oestrogen-progestogen replacement therapy on coronary artery disease using a simple morphometric method. We studied sixty-three cholesterol-fed rabbits for nineteen weeks. They were randomized to either ovariectomy (51 rabbits) or a sham operation (12 rabbits). The ovariectomized rabbits were randomized to receive either 17 beta-estradiol, 17 beta-estradiol plus norethisterone acetate, 17 beta-estradiol plus levonorgestrel, or placebo. The rabbits with the sham operation received placebo. The hormone therapies reduced the development of coronary artery disease compared to placebo (p less than 0.0001). Furthermore, the coronary artery disease was attended by atherosclerosis in the more distal parts of the coronary arteries (p less than 0.0001), the thoracic aorta (p less than 0.0001) and the abdominal aorta (p less than 0.0001), and by a reduced relative heart weight (p less than 0.05). We conclude that coronary atherosclerosis can be determined quantitatively by morphometry in rabbit arteries. Estradiol monotherapy reduces coronary atherosclerosis in cholesterol-fed rabbits and the addition of norethisterone acetate or levonorgestrel does not attenuate this beneficial effect.

Aspergillus fumigatus fungaemia and myocarditis in a patient with acquired immunodeficiency syndrome.

Necrotizing myocarditis due to Aspergillus fumigatus was a contributory cause of death in a patient with acquired immunodeficiency syndrome and non-Hodgkin lymphoblastic malignant lymphoma of the Burkitt type. A transient remission of the lymphoma had been obtained by cytostatic treatment. A. fumigatus was isolated from blood two weeks before death, but myocarditis was not diagnosed until autopsy.

Atherosclerosis in Watanabe heritable hyperlipidaemic rabbits. Evaluation by macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables.

The spontaneous development of atherosclerotic disease in 38 homozygous and 34 heterozygous Watanabe heritable hyperlipidaemic rabbits was evaluated by qualitative and quantitative light microscopy in aorta, coronary, pulmonary and renal arteries, by naked eye and macroscopic morphometric estimation of aortic atherosclerosis extent and by biochemical analysis of aortic cholesterol content. No noteworthy atherosclerosis was demonstrated within 19 months in heterozygous rabbits. In homozygous rabbits, atherosclerotic lesions were seen from the age of 4 months and progressed with age. All 19-month-old rabbits had severe atherosclerotic disease. As much as 64% of the variation in atherosclerosis extent/severity could be explained by serum cholesterol and age. A highly significant correlation between the various methods for quantitation of atherosclerosis extent and/or severity was demonstrated, suggesting that quantitative microscopy, macroscopic morphometry and determination of aortic cholesterol content may be equally valid as a measure of atherosclerosis in WHHL rabbits and are therefore interchangeable.