2.2.2. Models of Type2 induced diabetes-associated
atherosclerosis
The prevalence of Type2 diabetes driven by insulin resistance and
obesity is on an exponential rise worldwide with 700 million individuals
predicted to have Type 2 diabetes by 2045[41]. Metabolic features of
Type2 diabetes include hyperglycemia, hyperinsulinemia, obesity and
dyslipidemia encompassing an increase in LDL, vLDL and a decrease in HDL
levels. Thus, it is pertinent to choose an animal model that at best
encompasses all of these features in order to study the mechanisms and
explore therapeutic approaches to limit macrovascular disease induced by
Type2 diabetes. The majority of the animal models available to study
Type2 diabetes-associated atherosclerosis are either genetically
manipulated or diet induced.
Two of the most common genetic models are generated by crossbreeding
genetic models of Type2 diabetes, such as the leptin (ob/ob) and leptin
receptor (db/db) deficient mice, with atherosclerotic mice such as the
ApoE-/- and LDLR-/- mice. Leptin,
the satiety hormone that is produced by adipose cells and enterocytes of
the small intestine, regulates appetite via receptors in the arcuate
nucleus of the hypothalamus. Ob/ob mice and db/db mice carry mutations
in the leptin gene and the leptin receptor-coding gene respectively,
causing the induction of hyperphagia leading to obesity and Type 2
diabetes[42, 43]. The db/db;ApoE-/- mouse is well
established and exhibits typical features of Type2 diabetes by 20 weeks,
such as increased body weight, hyperglycemia, hyperinsulinemia, elevated
cholesterol levels (triglycerides, LDL and vLDL) with a 3-4 fold
increase in atherosclerotic plaque burden[42, 43]. From a more
diabetic perspective, these mice display higher expression of RAGE,
inflammatory adhesion molecules (such as VCAM-1) and matrix
metalloproteinase (MMP-9) activity[43]. Pharmacological
interventions, such as soluble RAGE and PPAR-agonists have proven to be
effective in their pre-clinical evaluation with this model[43].
Similar atherosclerotic observations were noted in the
ob/ob;ApoE-/- and ob/ob;LDLR-/-mice, however, in these models either hyperglycemia was not evident
(ob/ob; ApoE-/- mice) or hyperlipidemia was too
pronounced masking the diabetic effects (ob/ob;LDLR-/-mice). A modification of the ob/ob;ApoE-/- and
ob/ob;LDLR-/- mice to more closely align with the
human lipoprotein profile is to cross these mice to
ApoB100/100 producing mice which do not make ApoB-48
in their liver[44, 45]. Cholesterol in mice is mostly carried in
ApoB-48 containing vLDL particles and chylomicron remnants whereas
humans do not produce ApoB-48 in the liver[44, 45].
Ob/ob;ApoE-/-;ApoB100/100 and
ob/ob;LDLR-/-;ApoB100/100 mice
exhibit a metabolic phenotype which includes obesity, insulin resistance
as well as atherosclerosis and hypertension[44]. Due to their
specificity in lipoprotein profiles, these models are useful for
studying cholesterol-lowering therapies from a Type2 diabetic/metabolic
syndrome perspective[44].
For research that requires less influence of lipid levels and consistent
features of Type2 diabetes, there are other models with a more
clinically relevant lipoprotein profile that can be considered. The
LDLR-/- mouse that is able to synthesise only
ApoB100/100 protein represents a model more closely
aligned to the human lipoprotein profile. This model exhibits
accelerated atherogenesis[45]. Furthermore, Heinonen et al crossbred
the LDLR-/-;ApoB100/100 mouse with
transgenic mice overexpressing insulin-like growth factor-II in
pancreatic β cells which led to the development of Type2 diabetes
without major hyperlipidemia[46]. These mice demonstrated aggravated
atherosclerotic lesions with increased complications such as lesion
calcification which coincided with an increase in the expression of a
variety of genes involved in calcification (OPN, ALP-2 and BMP-2) and
inflammation (MCP-1)[46]. Moreover, the complexity of the lesion and
increased calcification was more prominent in aged mice, which is in
line with clinical studies that demonstrate calcification in peripheral
and coronary arteries, particularly in older patients with a longer
duration of diabetes, and is considered a strong independent risk factor
for cardiovascular events[46-49]. Another useful genetic model is
the ApoE-/- mouse that has a homozygous or
heterozygous deletion of the insulin receptor substrate (IRS1 and IRS2)
gene. ApoE-/- mice that are IRS1+/-,
IRS2+/- and IRS2-/- mice display a
Type2 diabetes/metabolic syndrome phenotype that includes, insulin
resistance, hyperglycemia, hyperinsulinemia, and impaired glucose
tolerance as compared to IRS2+/+ mice, however lipid
levels were only modestly altered[50-52]. This is consistent with
clinical findings in Type2 diabetic patients that have reduced
expression of IRS2 in their pancreatic β cells and a highly polymorphic
IRS1 gene[53]. These mice have augmented atherosclerotic lesions and
vascular inflammation that is attributed to impaired IRS2 signalling of
the AKT and ERK pathways that lead to the upregulation of
pro-atherogenic MCP-1[54]. Lastly, another note-worthy genetic model
of T2D-induced atherosclerosis is the heterozygous glucokinase (GK)
knockout mouse on the ApoE-/- background. GK is the
rate-limiting enzyme of glucose-stimulated insulin secretion, thus is it
not surprising that GK+/-/ApoE-/-mice on a western diet show significant glucose intolerance and impaired
glucose-stimulated insulin secretion, a phenomenon that was consistent
over time[55]. Despite plasma lipid levels being comparable to the
ApoE-/- mouse,
GK+/-/ApoE-/- mice have
significantly accelerated and highly developed atherosclerotic lesions,
strongly suggesting that lesions are driven by hyperglycemia and insulin
resistance[55].
Diet-induced insulin resistance is extensively studied in models of
Type2 diabetes associated atherosclerosis. In particular, a high-fat
western diet (comprising cholesterol (0.2% total cholesterol) + total
fat (21% by weight; 42% kcal from fat) + high saturated fatty acids
(>60% of total fatty acids) + high sucrose (34% by
weight)) in ApoE-/- or LDLR-/- mice
increases lesions throughout the aortic tree and exhibits hyperglycemia,
hyperinsulinemia and hypertriglyceridemia. Interestingly, in mice fed a
diabetogenic diet where 35% of the energy is sourced from fats,
atherosclerotic lesions were inconsistent with the
LDLR-/- mice being more susceptible to diabetes[56,
57]. LDLR-/- mice on a diabetogenic diet had severe
dyslipidemia and increased atherosclerotic lesions, albeit with modest
increases in glucose and insulin levels. On the contrary, diabetogenic
diet-fed ApoE-/- mice displayed no significant
differences between in lipids, glucose and atherosclerotic lesion sizes
as compared to chow-fed ApoE-/- mice[56, 57]. In
comparing neointimal formation, ApoE-/- mice fed a
western diet exhibited larger lesions, accompanied by higher glucose and
insulin levels, as compared to mice on a diabetogenic diet that
maintained euglycemia but developed insulin resistance[58].
Treatment of these mice with Rosiglitazone, a thiazolidinedione class of
PPAR agonist, led to a marked reduction in macrophage content and
neointimal formation[58].
Due to ongoing discrepancies in lesion size between diet-induced and
genetically manipulated mouse models, research groups have continually
strived to modify diet composition to more closely reflect the human
Type2 diabetic cardio-metabolic risk profile. Indeed, a diabetogenic
diet supplemented with 0.15% cholesterol in the
LDLR-/- mice further enhances obesity, insulin
resistance, systemic inflammation and atherosclerosis with increased
macrophage accumulation[59, 60]. Additionally, Neuhofer et al found
that by altering complex carbohydrates with a high-fat, high-sucrose
diet and the inclusion of 0.15% cholesterol, they were able to further
augment atherosclerotic lesion size and increase adipose tissue
inflammation, hallmarks often seen in human obesity and insulin
resistance, thus creating a more robust model of metabolic syndrome with
diet-induced obesity[61]. Furthermore, supplementing rodent diets
with high fructose to emulate consumption of sugary drinks in humans and
the impact on atherosclerotic lesions has been investigated by several
groups. ApoE-/- mice fed a high fructose diet
exhibited a pro-atherogenic state with insulin resistance, independent
of hypercholesterolemia[62]. This cohort of mice exhibited increased
vascular inflammation (VCAM-1), overproduction of NADPH-oxidase induced
ROS and significant outward vascular remodelling[62].
Supplementation of liquid fructose to western-diet fed
LDLR-/- mice demonstrated similar results with the
mice developing increased atheromatous plaque driven by increased
monocyte/macrophage infiltration and local inflammation. Importantly,
atherosclerotic plaque size strongly correlated with plasma lipid
levels[63]. Another method that has been described to model the
metabolic syndrome associated with diabetes is the incorporation of
low-dose STZ along with high fat feeding. Although this model has not
been validated in terms of atherosclerotic lesions, it has been shown to
develop cardiac inflammation and dysfunction, which combines both early
and late stages of the disease[64, 65].