Community Research and Development Information Service - CORDIS

Final Report Summary - METABOLICSYNDROME (Role of the chylomicron and HDL pathways in the development of obesity and insulin resistance)

The main research objectives of the project, as stated in Annex I of the application, is to decipher the cross-talk between the chylomicron and HDL metabolic pathways with adipose and hepatic tissues in the development of diet-induced obesity, type II diabetes and NAFLD. The project is composed of two main objectives:

Objective 1: To determine how expression or lack of expression of apolipoproteins or lipoprotein modifying enzymes in transgenic and knock-out mice affect diet-induced obesity, and type II diabetes.
Objective 2: To identify genes and pathways that are affected by components of the lipoprotein transport system and may promote or prevent adipogenesis and related metabolic perturbations. Below is a brief description of the results gathered during the execution of the study.

Objective 1: 'To determine how expression or lack of expression of apolipoprotein and lipoprotein modifying enzymes in transgenic and knock-out mice affect diet-induced obesity, and type II diabetes'. The outline of the experiments of objective 1 as it is described in the application is described below:

A. Create groups of mice transgenic or deficient for proteins of the chylomicron and HDL pathways with similar body-weights and fasting plasma cholesterol, triglyceride, and glucose levels. Feed mice western-type diet for a period of 24 weeks.
B. Measure fasting plasma cholesterol, triglyceride, and glucose levels every six weeks from the initiation of the experiment. In addition, measure average food consumption and perform glucose tolerance and insulin resistance tests. Determine the ability of these mice to deposit postprandial lipids to the adipose tissue using [14C]-cholesterol ether.
C. At the end of the experiment (week 24) sacrifice mice. Isolate liver samples for fat content determination. Determine body composition of mice by biochemical methods.

Brief description of the work contacted:

During the first reporting period, we focused our studies on the chylomicron metabolic pathways. In particular we focused on apolipoprotein E, and LDLr two key proteins responsible for the clearance of chylomicrons in plasma and the proper metabolism of dietary lipids in the circulation. Our experiments were contacted in experimental mice under the supervision of the University of Patras research animal committee and according to the appropriate European Union (EU) rules and regulations pertaining to the use of experimental mice in research.

Main scientific results:

In this part of our studies, we sought to determine the role of apoE in the development of diet-induced obesity, glucose intolerance and insulin resistance, in vivo. To address this question apoE3knock-in, wild-type C57BL/6, LDLr-deficient (LDLr-/-), and apoE-deficient (apoE-/-) mice were fed western-type diet for a period of fifteen or twenty four weeks during which their plasma lipid and glucose levels, body weight, body composition, glucose tolerance and insulin sensitivity were monitored. We selected to study apoE3 because it is the most common apoE isoform in humans. Our data establish that expression of apoE predisposes mice to diet-induced obesity, hyperglycemia and insulin resistance while deficiency in apoE renders mice resistant to these conditions. Human apoE3 appeared to be more potent than mouse apoE in promoting obesity in response to western-type diet. Furthermore, LDLr-deficient (LDLr-/-) mice were more sensitive to the development of diet-induced obesity and insulin resistance than apoE-/- mice, but still more resistant than wild-type C57BL/6 mice in response to western-type diet. Gavage administration of olive oil containing the non-hydrolyzable [3H]-cholesteryl-hexadecyl-ether to mice raised the possibility that deficiency in the LDLr and apoE reduces the direct delivery of postprandial non-hydrolyzed lipids to the liver one of the major tissues involved in glucose uptake from the circulation. A similar trend was also observed in the delivery of non-hydrolyzed dietary lipids to adipose tissue. Further histological studies in sections from hepatic tissue suggested that mice deficient in apoE are resistant to the deposition of dietary lipids to the liver and the development of nonalcoholic steatohepatitis. Taken together, our data establish that apoE is a key mediator of diet-induced obesity in response to western-type diet. A part of this work is already published in Karagiannides et al., FEBS J 275:4796-809 and summarised in a review article by Kypreos et al., FEBSJ 276:5720-8. Based on this work the principal investigator of the proposal, Dr Kyriakos E. Kypreos, was invited by FEBSJ to organise the mini-review series with the general title 'Mechanisms of obesity and related pathologies', FEBS J 276:5719. In addition, the role of apoE in the development of diet-induced nonalcoholic fatty liver disease (NAFLD) was also investigated. ApoE-deficient, LDLr-deficient and control C57BL/6 mice were fed western-type diet (17.3 % protein, 48.5 % carbohydrate, 21.2 % fat, 0.2 % cholesterol, 4.5 Kcal/g) for 24 weeks and their sensitivity towards NAFLD was assessed by histological and biochemical methods. We report that apoE-/- mice are less sensitive to diet-induced NAFLD compared to control C57BL/6 mice. In an attempt to identify the molecular basis for this phenomenon biochemical and kinetic analyses revealed that apoE-/- mice displayed a significantly delayed post-prandial triglyceride clearance from their plasma. In contrast to apoE-/- mice, LDLr-/- mice fed western-type diet for 24 weeks developed significant accumulation of hepatic triglycerides and NAFLD suggesting that the apoE-mediated hepatic triglyceride accumulation in mice is independent of the LDLr. Our findings suggest a new role of apoE as key peripheral contributor to hepatic lipid homeostasis and the development of diet-induced NAFLD (FEBS J. 2011, 278(17):3119-29).

Objective 2:'To identify genes and pathways that are affected by components of the lipoprotein transport system and may promote or prevent adipogenesis and related metabolic perturbations'.

The outline of the experiments in objective 2 as it is described in the original application is described below:

A. Create groups of mice deficient or transgenic for apoE and other select proteins of the lipoprotein system that will be identified in objective 1 to promote or prevent the development of obesity. Feed mice western-type diet for 24 weeks.
B. Perform gene analyses to identify genes and pathways that are affected by protein components of the chylomicron and HDL pathways.
C. Use adenovirus-mediated gene transfer in order to validate the functional role of select genes in obesity, type II diabetes and hepatic lipid accumulation.

Brief description of the work contacted:

During the second reporting period, we focused our studies on the HDL metabolic pathway. In particular we focused on: apolipoprotein A-I, and LCAT two key proteins responsible for biogenesis of HDL in plasma. ApoA-I interacts with ABCA1 to create HDL, while LCAT is the enzyme that converts the free cholesterol of HDL into esterified thus contributing to the maturation of HDL. Our experiments were contacted in experimental mice under the supervision of the University of Patras animal research committee and the Hellenic department of agriculture, and according to the EU rules and regulations pertaining to the humane use of experimental mice in research.

Brief description of the results:

In this part of our studies, we sought to determine the potential relationship between low HDL and obesity, type II diabetes and NADLD in patients with metabolic syndrome. During the biogenesis of HDL, lipid free or minimally lipidated apoA-I interacts functionally with the lipid transporter ABCA1 to form immature discoidal HDL which are then converted into mature spherical particles by the action of lecithin:cholesterol acyl transferase (LCAT). Based on this, we investigated the mechanistic relationship between low and dysfunctional HDL and diet-induced NAFLD development using mouse models. To address this question we employed male apoA-I-deficient (apoA-I-/-) mice that lack classical apoA-I containing HDL and male deficient (LCAT-/-) mice that have immature discoidal HDL. Mice were fed with western-type diet for 24 weeks, and then biochemical and genetic analyses were performed. Then mice were sacrificed, tissues were isolated and histological analyses were performed. Mice were fed the standard western-type diet for 24 weeks and then histological and biochemical analyses were performed. ApoA-I-/- mice showed increased diet-induced hepatic triglyceride deposition and disturbed hepatic histology while they exhibited reduced glucose tolerance and insulin sensitivity. Quantification of FASN-1, DGAT-1, and PPAR? mRNA expression suggested that the increased hepatic triglyceride content of the apoA-I-/- mice was not due to de novo synthesis of triglycerides. Similarly, metabolic profiling did not reveal differences in the energy expenditure between the two mouse groups. However, apoA-I-/- mice exhibited enhanced intestinal absorption of dietary triglycerides, accelerated clearance of postprandial triglycerides, and a reduced rate of hepatic very low density lipoprotein triglyceride secretion. In agreement with these findings, adenovirus-mediated gene transfer of apoA-IMilano in apoA-I-/- mice fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and an improvement of hepatic histology and architecture. Similar to apoA-I-/- mice, LCAT-/- mice were characterised by increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. Adenovirus-mediated gene transfer of LCAT in LCAT-/- mice that were fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and a great improvement of hepatic histology and architecture.

Since expression of apoA-I is absolutely essential for the formation of HDL while LCAT is essential for the maturation of discoidal HDL into spherical, our data establish that the HDL metabolic pathway is a central contributor to the deposition of dietary triglycerides to the liver and to the development of NAFLD and other related metabolic dysfunctions associated with metabolic syndrome. Our data further support that the coexistence of reduced HDL levels and NAFLD in an individual with metabolic syndrome may not be a mere coincidence, rather it underlays a strong causative relationship between these two conditions. This observation extends the role of HDL beyond atherosclerosis, to the development of NAFLD, a pathological component found in patients with metabolic syndrome. Importantly, our findings raise the interesting possibility that expression of beneficial forms of apoA-I or LCAT by gene therapy approaches may find therapeutic applications for the treatment of NAFLD in the future.

Implications and long-term benefits of the project:

In this research proposal, we determined the metabolic and molecular pathways as well as genes mediating the observed phenotypic differences in our experimental mice. Our data indicate that the lipoprotein transport system is a key modulator of processes associated with obesity, diabetes and NAFLD development in mice. In particular, apoE is a key protein in depositing lipids to the adipose and hepatic tissues. In addition, our studies indicated that the deposition of dietary lipids to the adipose and hepatic tissues were not the result of de novo biogenesis of lipids in these tissues. Rather the lipids were delivered exclusively from the diet via the lipoprotein metabolic system. The use of adenovirus mediated gene transfer of apoA-I and LCAT identified these two important proteins of the HDL metabolic pathway as potential new therapeutics for the treatment of NAFLD. The novelty of our research proposal lays on the hypothesis supported by our data that 'peripheral stimuli' initiating from the lipid-charged lipoprotein metabolic system in plasma and/or the brain may act as positive or negative extracellular signals associated with obesity and related metabolic perturbations such as diabetes and NAFLD. Overall, the reults of these proposal provided novel mechanistic insights and 'peripheral stimuli' (such as HDL protein components for example) that modulate extracellular processes associated with the deposition of dietary fat to peripheral tissues, leading to the development of obesity, and its related metabolic dysfunctions. In the future we will further work on the translation of these novel pharmacological targets that we identified in this proposal into new pharmaceuticals for the treatment of these conditions.

Related information

Reported by

UNIVERSITY OF PATRAS
1, University of Patras Medical School
TK. 26500 RIO, PATRAS
Greece
See on map
Follow us on: RSS Facebook Twitter YouTube Managed by the EU Publications Office Top