Apolipoprotein E deficiency abrogates insulin resistance in a mouse model of type 2 diabetes mellitus
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Although it is known that lipid metabolism plays a role in insulin resistance in type 2 diabetes and in obesity, the mechanism is still largely unknown. Apolipoprotein E (ApoE) regulates plasma lipid levels and also plays a role in the uptake of lipids into various tissues. To investigate whether the suppression of whole-particle lipoprotein uptake into tissues affects insulin responsiveness and the diabetic condition, we examined the effect of an ApoE (also known as Apoe) gene deletion in MKR mice, a mouse model of type 2 diabetes.
ApoE −/− , MKR, ApoE −/− /MKR and control mice were placed on a high-fat, high-cholesterol diet for 16 weeks. Glucose tolerance, serum insulin, blood glucose, insulin tolerance, tissue triacylglycerol content and atherosclerotic lesions were assessed.
ApoE −/− /MKR and ApoE −/− mice showed significantly improved blood glucose, glucose tolerance and insulin sensitivity. Reduced triacylglycerol content in liver and reduced fat accumulation in liver and adipose tissue were found in ApoE −/− /MKR and ApoE −/− mice compared with control and MKR mice. ApoE −/− and ApoE −/− /MKR mice demonstrated similarly large atherosclerotic lesions, whereas MKR and control mice had small atherosclerotic lesions.
We demonstrated that ApoE deficiency abrogates insulin resistance in a mouse model of type 2 diabetes, suggesting that lipid accumulation in tissue is a major cause of insulin resistance in this mouse model.
KeywordsApoE gene deletion Atherosclerosis MKR mouse model of type 2 diabetes
Peroxisome proliferator-activated receptor
The incidence of type 2 diabetes mellitus is dramatically increasing and represents one of the world’s chief economic and healthcare challenges . Associated with obesity, type 2 diabetes mellitus results from impaired insulin secretion and peripheral insulin responsiveness. Adipose tissue stores and mobilises triacylglycerol, and plays a central role in regulating whole-body metabolism and glucose homeostasis . High circulating NEFA and triacylglycerol, present in obesity and the metabolic syndrome, are associated with impaired insulin sensitivity in skeletal muscle and decreased glucose tolerance [3, 4]. Studies showing an increased triacylglycerol content in liver and muscle have revealed a strong correlation with whole-body insulin resistance [5, 6].
Apolipoprotein E (ApoE) is a 34 kDa glycoprotein that is synthesised by liver, adipocyte, brain and other tissues, and is a ligand for receptors that clear remnants of chylomicrons and VLDL. Lack of ApoE causes accumulation of cholesterol-rich remnants in plasma, leading eventually to atherosclerosis . ApoE-deficient mice (ApoE [also known as Apoe] −/− ) fed a high-cholesterol diet have been used as a mouse model to study atherosclerosis. These mice exhibit reduced lipid accumulation in adipose tissue, improved insulin sensitivity and resistance to the development of obesity [8, 9, 10].
We have previously developed a mouse model of type 2 diabetes by overproducing a dominant negative IGF-1 receptor specifically in skeletal muscle (MKR mice) . Hybrid formation of the mutated IGF-1 receptor with the endogenous IGF-1 and insulin receptors caused impaired insulin and IGF-1 receptor signalling pathways specifically in skeletal muscle. This primary defect in skeletal muscle resulted in hyperinsulinaemia, dyslipidaemia and beta cell dysfunction, leading eventually to hyperglycaemia. Whole-body insulin resistance was associated with impaired insulin sensitivity in skeletal muscle as well as in adipose tissue and liver. Following treatment with WY-14643, a peroxisome proliferator-activated receptor (PPAR)α agonist, or with leptin, MKR mice showed a marked reduction in serum lipids (NEFA and triacylglycerol) and in the lipid content of liver. This was also associated with improved insulin sensitivity and diabetes of the MKR mice [12, 13], suggesting that lipotoxicity plays a major role in the insulin resistance seen in this model.
To investigate whether the suppression of whole-particle lipoprotein uptake affects insulin responsiveness and the diabetic condition, we examined the effect of ApoE gene deletion in MKR mice.
Mouse models and diet
The generation of the MKR mice (FVB/N background) has been described previously . ApoE −/− (FVB/N background) mice were obtained from J. L. Breslow of the Rockefeller Institute (New York, USA)  and were crossed with MKR mice to generate the double gene-disrupted mice (ApoE −/− /MKR). Mice that were heterozygous for any of the alleles ApoE or MKR were not included in the study.
Atherosclerosis was induced by feeding a high-cholesterol diet containing 1.25% cholesterol (wt/wt) and 20% fat (wt/wt) for 16 weeks (D12108; Research Diets, New Brunswick, NJ, USA) starting from 4 weeks of age. Food intake was measured weekly, from 1 to 7 weeks after starting the diet. Food intake was normalised to body weight and expressed as: g food (g body weight)−0.75 day−1. Both male and female mice were followed.
The animal experiments were conducted in accordance with the principles of laboratory care, as designated by the Mount Sinai School of Medicine, Animal Care and Use Committee.
Whole-body fat and lean mass were measured in conscious mice using a quantitative magnetic resonance method (Echo MRI 3-in-1; Echo Medical Systems, Houston, TX, USA).
Serum was obtained from the vein between 10:00 and 12:00 hours in the non-fasting state. Serum levels of NEFA were measured using a fatty acid assay kit (Roche, Indianapolis, IN, USA). Serum insulin levels were determined using a radioimmunoassay kit (Linco Research, St Charles, MO, USA). Serum IL-6 and TNF-α levels were measured by ELISA (R & D Systems, Minneapolis, MN, USA). Blood glucose was measured using a glucose monitoring system (FreeStyle Lite; Abbott, Abbott Park, IL, USA). Serum total cholesterol and triacylglycerol levels were measured by ELISA (BioVision, Mountain View, CA, USA).
Glucose tolerance and insulin tolerance tests
For glucose tolerance tests, mice were fasted for 8 h and blood glucose levels were measured at the indicated time points following an intraperitoneal injection of glucose (2 mg/g body weight). For insulin tolerance tests, mice were injected intraperitoneally with insulin (0.75 U/kg body weight) and blood glucose levels were measured from the tail vein at the indicated time points.
Tissue triacylglycerol assay
Tissue triacylglycerol was extracted with chloroform/methanol as previously described . After hydrolysis with KOH base, triacylglycerol was measured radiometrically using a glycerol kinase assay .
Characterisation of atherosclerosis
Following 16 weeks of a high-cholesterol diet, mice were killed and the aorta removed and perfusion-fixed in phosphate-buffered paraformaldehyde (4% [wt/vol.], pH 7.4). Fat from the isolated aorta was removed under the microscope and the aortas were fixed overnight in 4% paraformaldehyde (pH 7.4). The fixed aortas were cut into four regions: (1) at the region of valve attachment; (2) at the level of the branching of the innominate artery; (3) at the most proximal part of the descending aorta at the level of the subcutaneous artery; and (4) at the descending aorta. Segments of the aorta were paraffin-embedded and stained with haematoxylin and eosin. Four sections per region were used for histomorphometric analysis. The neointimal area, total vessel wall area and medial area in each section were measured using MicroSuite Five (Olympus America, Center valley, PA) and the ratios of neointimal area:medial area and neointimal area:total vessel-wall area were calculated for evaluation of neointimal formation (Fig. 5f). The average of neointimal area:medial area and neointimal area:total vessel-wall area in the above four sections was used to represent the lesion size of each animal.
The left epididymal fat pad and the right lobe of the liver were isolated and fixed overnight in 4% paraformaldehyde in PBS. The tissues were then embedded in paraffin and 5 µm sections were stained with haematoxylin and eosin.
All values are given as mean ± SEM. Statistical differences were determined using one-factor ANOVA followed by a t test. Significance was accepted at p < 0.05.
Cholesterol diet increases body weight of ApoE−/− and MKR mice with no increase in body adiposity
ApoE gene ablation in MKR mice led to reduced liver triacylglycerol content
Effects of ApoE deficiency on serum cholesterol, triacylglycerol, insulin, glucose and insulin sensitivity in MKR mice
Atherosclerotic changes after exposure to high-cholesterol diet
Our study demonstrates that ApoE gene deletion in the MKR mouse model of type 2 diabetes improved glycaemic control, as evidenced by reduced hyperinsulinaemia and hyperglycaemia. ApoE −/− and ApoE −/− /MKR mice showed reduced body adiposity and smaller adipocytes, probably due to reduced triacylglycerol and NEFA content in fat tissue . These reductions were attributed to impaired adipocyte differentiation in ApoE −/− mice, based on in vitro findings showing that levels of adipocyte fatty acid binding protein (AFABP/aP2) and PPAR-γ were markedly reduced in co-cultures of bone marrow stromal cells and 3T3-L1 cells .
Liver triacylglycerol content, which was increased in MKR and control mice fed a high-cholesterol diet, decreased in ApoE −/− and ApoE −/− /MKR mice, a finding also evident in histological examination. Previous studies with MKR mice have shown that leptin , PPARα agonist  and the β3 adrenergic agonists  treatment reduced triacylglycerol content in liver and muscle, and improved insulin sensitivity of MKR mice. However, treatment with thiazolidinediones , which improved insulin sensitivity solely in adipose tissue of MKR mice, was insufficient to improve whole-body insulin sensitivity. In these previous studies, we used the euglycaemic–hyperinsulinaemic clamp technique and demonstrated that when insulin sensitivity improved, the liver was the major organ to show improved insulin responsiveness. Skeletal muscle is very unlikely to play a major role, since the function of the insulin and IGF-1 receptors was interfered with (genetically affected). In the current study, we show that ApoE gene deletion in MKR mice led to reduced lipid accumulation not only in fat but also in liver, resulting in improved insulin sensitivity. Together, these studies suggest that the liver plays a major role in improvement of whole-body insulin sensitivity in MKR mice.
Our studies are in line with studies published by Hofmann et al., who found that ApoE −/− mice have impaired lipid uptake in adipose tissue and improved glucose tolerance . Gao et al. found that ApoE −/− mice prevented the development of genetically induced obesity . These ApoE −/− /KKAy mice had decreased fat accumulation in liver and adipose tissue, and improved insulin sensitivity despite increased plasma NEFA levels . Adipocytes of ApoE −/− /KKAy mice were smaller and the serum cytokine profile (leptin, TNF-α, adiponectin) was improved.
Several molecular mechanisms have been presented to explain the relationship between insulin resistance and increased cellular accumulation of triacylglycerol. In muscle, increased triacylglycerol content leads to increases in diacylglycerol and ceramide that activate a serine kinase cascade, decreasing insulin-stimulated activity of IRS-1-associated phosphatidylinositol-3-kinase [23, 24], leading eventually to reduced glucose transport  and glycogen synthesis . Moreover, increased intramyocellar triacylglycerol, thought to be more prone to oxidative stress, generates lipid peroxidation products that potentially inhibit signalling . Studies have shown that cellular triacylglycerol accumulation in liver as such is not initially toxic [28, 29]; however, intermediate metabolites such as acyl-CoAs and diacylglycerol are determinants of the development of insulin resistance [30, 31]. Studies in mouse models deficient in the enzymes acetyl coenzyme A carboxylase, stearoyl-CoA desaturate, or the elongation of long-chain fatty acids family member 6, which is related to fatty acid and triacylglycerol synthesis in liver, showed decreased lipogenesis in liver and improved insulin resistance and resistance to diet-induced obesity [31, 32, 33, 34].
Several studies have shown that increased secretion of proinflammatory cytokines, decreased secretion of adiponectin, increased circulating levels of NEFA and postprandial hyperglycaemia can all affect endothelial function [35, 36]. ApoE −/− mice with deletion of the gene encoding endothelial nitric oxide synthase showed accelerated atherosclerosis  and ApoE −/− mice with deletion of the receptor for advanced glycation end-products or with the deletion of TNF-α showed reduced atherosclerotic lesions . In our study, ApoE −/− and ApoE −/− /MKR male and female mice had markedly increased atherosclerotic lesions compared with MKR and control mice, despite improvement in insulin sensitivity. These findings strongly suggest that lipid metabolism and increase in inflammatory cytokines in serum play a major role in causing atherosclerosis and that improvement of hyperglycaemia or insulin resistance in liver is not necessarily a prerequisite for the treatment of atherosclerosis. Interestingly, recent outcomes in studies of type 2 diabetes have found that improved blood glucose control was associated with reduced microvascular complications, but was not associated with clear-cut improvement in macrovascular complications [39, 40, 41]. Our study is in line with these findings, showing that improved insulin sensitivity and blood glucose, in the face of an atherogenic phenotype, are unable to reverse macrovascular disease.
We thank N. Mall (Mt Sinai School of Medicine, New York) for help with histological experiments, and W. Jou and O. Gavrilova (NIDDK, Bethesda, MD, USA) for assistance with liver triacylglycerol assays. This study was supported by an American Diabetes Association Mentorship Grant, awarded to Y. Kawashima and D. LeRoith.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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