Metabolic adaptation of white adipose tissue to nutritional and environmental challenges

<strong>Summary of main findings</strong> When adipose tissue is present in excessive amounts, as in obesity, it predisposes to a number of pathologies. Obesity is a complex, multifactorial condition as it influences many endogenous genetic, endocrine, and inflammatory pathways. Excess dietary intake is one of the important factors which are responsible for the increasing prevalence of obesity. For the understanding of the reciprocity between consumed diet and excessive amounts of adipose tissue, it is essential to investigate underlying functioning. In this thesis, I have addressed three important aspects that play a role in the development of diet induced obesity and its pathologies with a focus on adipose tissue metabolism. Does a body weight set-point exist? How is the diet-induced metabolic response affected by housing at thermoneutrality? Does oxygen restriction induce inflammation in white adipose tissue? <p style="margin-left: 36pt;"> The first aspect investigated was the existence of a body weight set point. A body weight set point is defined as a pre-determined or preferred level of body weight which is preserved by an internal feedback control mechanism. In <strong>chapter 2</strong>, a dietary intervention with none, one, or two diet alterations of purified diets was performed in C57BL/6J mice to investigate if a long lasting effect on body weight persistence was present. Diets contained equal protein content and source of ingredients but differed in the fat-to-sugar ratio. Therefore, energy content and amount of fat was different for either the low fat diet or the high fat. In the intervention the last consumed diet of the mice determined energy intake, energy expenditure, body weight, body fat stores, circulating hormones and metabolites. These data support the settling point theory as body weight and metabolic parameters ‘settle’ based on current energetic input and output and do not support the set point theory. Next to that it underlines the importance of diet choice in intervention studies focusing on aspects on the crossroads of nutrition and physiology. In <strong>chapter 3</strong>adipose tissue physiology and molecular regulation was further investigated by exposure to more metabolic stress in the form of a weight loss challenge with different purified diets. Diet-induced obese C57BL/6J mice were fed a high fat diet restricted to 70% intake of previous <em>ad libitum </em>high fat diet intake or they were changed to <em>ad libitum </em>low fat diet for 5 weeks. Beneficial effects were seen in both interventions regarding physiological parameters. However, molecular parameters in white adipose tissue differed between the two restriction interventions, with increased activation of mitochondrial carbohydrate and fat metabolism in high fat diet restricted mice. When extrapolated to the human situation this may suggest that a reduction of portion size is the best method for weight loss. It is standard practice to house mice at ambient temperature during physiological intervention studies. Unfortunately mice are then exposed to a temperature below their thermal neutral zone. This implies that their metabolism is chronically increased which is known to influence study outcomes. In <strong>chapter 4</strong>the second question; “how is the diet-induced metabolic response affected by housing at thermoneutrality?”was investigated. A 14-week dietary intervention with two semi-purified diets, a low fat diet and a moderately high fat diet, was performed at 28°C in C57BL/6J mice. This resulted in a large diet-induced difference in bodyweight, adipose tissue mass, adipocyte size, and serum leptin level. But no differential effects of the diets were seen on serum glucose, free fatty acids, triacylglycerides, insulin, a panel of cardiovascular markers, and a number of (metabolic) parameters in liver and muscle. Although adipose tissue mass and adipocyte size was increased significantly, there was no sign of inflammation or dysfunction in the adipose tissue. This study suggests that diet-induced obesity of C57BL/6J mice at thermoneutrality results in a suitable model for the metabolically ‘healthy’ obese (people who are significantly overweight but show none of the usual metabolic problems). Next to that, this study emphasizes the importance of consideration and control of housing temperature for mice, as it has profound effects on study outcomes. The third and last question investigated was if oxygen restriction is able to induce inflammation in white adipose tissue. There is substantial evidence that white adipose tissue becomes hypoxic when excessively enlarged. Due to fast expansion of white adipose tissue the vasculature is not able to keep pace with growth. Next to that, adipocytes are able to increase in size beyond the limit of oxygen diffusion. To investigate if hypoxia was able to induce inflammation in white adipose tissue, the model for healthy obese adipocytes (developed in <strong>chapter 4</strong>) was used and exposed to ambient oxygen restriction (13%) to challenge adipose tissue metabolism. This resulted in the presence of systemic oxygen restriction as shown by increased levels of hemoglobin and hematocrit. Furthermore a switch to glycolytic metabolism, which is indicative for tissue hypoxia, was present. No differences in adipose tissue macrophage infiltration (as marker for inflammation) were found. But, serum branched chain amino acids and adipokines were affected. Branched chain amino acids were increased in mice exposed to oxygen restriction which shows resemblance with findings in humans where increased levels were found in lean versus obese people. The peptide hormone adiponectin was increased in serum, without differences in WAT expression. On the other hand, the peptide hormones CCDC3 and CCK showed decreased transcript levels in white adipose tissue without significant change in serum levels, although for CCDC3 a trend was seen. Together these results suggest that oxygen restriction does not induce inflammation in adipose tissue. However, it does affect adipokine regulation. After performing these studies it was clear that composition of the diet has a major influence on outcome parameters of physiological studies as shown in <strong>chapter 2</strong>. To compare functional effects of different nutrients, it is important to use standardized purified diets. Not only the experimental intervention diet is of importance but also the reference control diet can influence outcomes. For example, when an intervention is performed with a high fat purified diet and the reference diet is chow this will lead to a difficult comparison. The content of chow is variable as it is grain or cereal based (ground corn, ground oats alfalfa meal, soybean meal and ground wheat). Nutritional adequacy is ensured by addition of vitamins, minerals, and fat. However, the exact amount of the various ingredients is frequently kept secret by the manufacturer. Next to that, due to the plant based origin of chow it will contain nutritive (protein, carbohydrate, fat) components but also non-nutritive components (phytochemicals). The content of the chow diet will vary from batch to batch as the nutritive and nonnutritive value will change between harvests. When using a chow reference diet in comparison to a purified diet you will never know exactly what you are comparing, i.e. difference in amino acids or effects of phytochemicals etc. Therefore, a reference diet for physiology was designed (<strong>chapter 6</strong>) to improve comparison of study outcomes and to increase efficiency of resources and material. A key feature of the diet is the fixed protein concentration, which allows for an exchange of carbohydrate and fat in a high fat version of the diet. To conclude, the work presented in this thesis provides clear insight in factors that are of importance for improvement of translatability of mouse studies to the human situation. It was shown that when investigating the weight balance many parameters, i.e. genetics, metabolic rate, environmental factors like ambient housing temperature and light and cognitive behavior, besides the diet and its composition are able to influence the outcome parameters. As most mouse experiments are performed in a fixed environment with no choices of food and a standard temperature set to 22°C. This is clearly not reflective of humans under free living conditions. However, these fixed conditions are able to result in experiments that unravel underlying mechanisms of weight balance, which form the basis for discovering a solution to the obesity epidemic.