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Final Report Summary - ADIPONRF2 (Targeting the Keap1/Nrf2 pathway in adipose tissue for obesity prevention and treatment.)

The ADIPONRF2 project aims to study the role of the Keap1/Nrf2 pathway in adipose tissue function and differentiation under basal or stress conditions, such as high-fat diet (HFD)-induced obesity and insulin resistance. The Keap1/Nrf2 pathway lies central to the regulation of a battery of antioxidant and cytoprotective genes. As obesity is characterized by chronic oxidative stress in adipose tissues, it was hypothesized that manipulation of the Keap1/Nrf2 pathway could affect the development of obesity and its detrimental metabolic effects. To this end, genetic mouse models and cell culture systems with gain or loss of Nrf2 function have been used. This project can also reveal novel roles of the Keap1/Nrf2 pathway, beyond the antioxidant and cytoprotective ones as Nrf2, being a transcription factor, can possibly crosstalk directly and indirectly with other pathways as well. The importance of this project lies in the fact that it addresses the obesity/type 2 diabetes epidemic which has become a major health and socioeconomic problem in westernized societies. As small molecule pharmacological activators of the Nrf2 pathway have already been used in clinical trials, such as sulforaphane in the form of broccoli sprout extracts (PMID: 22045030), CDDO-Me (bardoxolone) (PMID: 21699484) and dimethyl-fumarate (PMID: 22992073), the Keap1/Nrf2 pathway can be considered an attractive target for pharmacological intervention/prevention in the setting of obesity and type 2 diabetes.

Aim1 of this project was to characterize the role of Nrf2 in adipogenesis and in the bioenergetics profile in white and brown adipocytes by employing in vitro cell culture models. To make our models as physiologically significant as possible, primary white and brown mouse preadipocytes cultures were used. It appears that constant activation of the Keap1/Nrf2 pathway either by pharmacologic (sulforaphane, CDDO-Im) or genetic means (use of Keap1 flox/flox hypomorphic mice-model of Nrf2 pathway activation) at least partially inhibits white adipocyte differentiation as assessed by oil-red-O staining and by expression of adipocyte markers (Pparγ, C/ebp etc). As far as the differentiation of brown preadipocytes to mature brown adipocytes is concerned it seems that activation of the Nrf2 pathway did not affect the course of adipogenesis. This discrepancy between white and brown adipocytes is reasonable as the signaling cascades that favor white or brown adipogenesis are different and clearly distinguished from each other. However, there was a trend for mature brown adipocytes to show increased oxygen consumption upon induction of the Keap1/Nrf2 pathway.
Aim2 of this project was to investigate the role of the Keap1/Nrf2 pathway in adipocytes in the setting of high-fat diet-induced obesity using in vivo mouse models. Mice with adipocyte-specific deletion of Nrf2 were generated (Nrf2 flox/flox:AdipoqCre mouse) and the Nrf2 flox/flox mice (lacking AdipoqCre) were used as controls. As a model of Nrf2 pathway activation the Keap1 flox/flox mice were used. These mice were shown to express lower levels of Keap1 compared to the wild-type and thus they consist a model of Nrf2 pathway activation. This is still a useful model as the whole body Keap1 knockout mice die postnatally due to malnutrition because of esophageal hyperkeratosis (PMID: 14517554). The study was performed on male 2-month old albino C57BL6 mice of the Nrf2 flox/flox, Nrf2 flox/flox:AdipoqCre and Keap1 flox/flox genotypes that were fed a standard (10% kcal fat) or a high-fat diet (60% kcal fat) for 6 months. The number of mice used per genotype were n=9-10 and the values that are going to be presented below are means. P<0.05 was considered statistically significant.
Interestingly no difference in body weights and body fat composition was observed between the 2 genotypes (Nrf2 flox/flox, Nrf2 flox/flox:AdipoqCre) in any of the diets during the course of this experiment. However, after this long-term exposure to the high-fat diet the Nrf2 flox/flox:AdipoqCre mice were more glucose intolerant than the Nrf2 flox/flox mice. Moreover, the Nrf2 flox/flox:AdipoqCre mice had higher non-esterified fatty acids levels (1mEq/l) than the control Nrf2 flox/flox (0.75 mEq/L) after the 6-month exposure to the high-fat diet (p=0.03). For the triglycerides levels we have just observed a trend for the Nrf2 flox/flox:AdipoqCre mice to have higher levels. The serum cholesterol levels of the Nrf2 flox/flox:AdipoqCre mice were higher (169.8 mg/dl) than the levels of the control mice on high-fat diet (111.8 mg/dl) (p=0.03). Gene expression analyses in white adipose tissue of mice lacking Nrf2 in adipocytes has showed a trend for increased expression of inflammation markers in these mice compared to the control after the 6-month high-fat diet exposure. Taking these data into account it appears that deletion of Nrf2 in adipocytes eventually led to a worse metabolic phenotype as indicated by the worse glucose tolerance, the higher non-esterified fatty acids and cholesterol levels. However this phenotypic difference was not obvious until after these mice having been for 6 months on the high-fat diet regimen. On the other hand, the genetic model of Nrf2 pathway activation we have used (Keap1 flox/flox mice with hypomorphic Keap1 alleles) exhibited a partial protection against weight gain, a trend towards increased energy consumption and ameliorated glucose tolerance as early as after 3 months on a high-fat diet. However, as this appears to be a model of whole-body Nrf2 pathway activation, the protective effects observed can not be attributed solely to the adipose tissue. In a publication that has emanated from the analysis of these mice (PMID 26701603) we have shown that Nrf2 pathway activation drives a transcriptional program that leads to repressed lipogenesis and gluconeogenesis.
Analysis of the adipose tissue samples on high-fat diet has provided us with a variety of potential Nrf2 target genes or genes indirectly regulated by the Nrf2 pathway. One of these genes that is not directly related to the cytoprotective effects of the Nrf2 pathway is Notch1. The Nrf2-Notch1 axis has been well described in the Kensler lab (outgoing host) in the liver (PMID 24298019, PMID 20628156). To better assess the importance of the Nrf2-Notch pathway in adipocytes, mice that overexpress the Notch intracellular domain in adipocytes (Rosa NICD/NICD:AdipoqCre) were generated. Surprisingly these mice exhibit a lipodystrophic phenotype as they possess minute amounts of white adipose tissue until the age of 1 month and then it is gradually lost. The lipodystrophy is accompanied by a highly insulin resistant diabetic phenotype as the lipids can not be stored in the white adipose tissue and ectopic lipid accumulation in the liver, muscle and other organs is seen with an increase in the circulating triglycerides and non-esterified fatty acids as described in our recent publication (PMID 26137442).
Conclusions and potential impact
The main conclusion of this project is that Nrf2 pathway activation is an effective way to prevent diet-induced obesity. The importance of Nrf2 in adipocytes in this setting was highlighted by the worse metabolic phenotype that was exhibited by the mice that lack Nrf2 in adipocytes after a long-term exposure to high-fat diet. Although these results come from mouse studies, they can be easily translated to clinical trials by using pharmacological Nrf2 pathway activators as the ones described in the “Background-Introduction” paragraph. One of these Nrf2 pathway activators, sulforaphane, can be administered as a broccoli sprout extract (PMID: 22045030) making it a more affordable approach especially in low income countries. Besides their potential practical applications, the results of this project could attract the attention of the research community (basic and clinical) as the use of mouse models and cell lines as well the tissue-specific approach could expand the understanding of the role of the Keap1/Nrf2 pathway in obesity and type 2 diabetes and pave the way for new research projects based on this information.
The project website is . A .eu website was chosen so as to highlight that this research is supported by the European Commission. The facebook page of the project that has also the role of providing information about diet, exercise, obesity and diabetes to the general public is . The researcher Dionysios Chartoumpekis MD, PhD welcomes any inquiries in the following e-mail address: .

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