The world-wide epidemic of obesity is due to complex, multifactorial changes in lifestyle and environmental conditions. In addition to an excess of calorie intake and composition of the diet, genetically determined pathways of energy storage and utilisation contribute to the development of obesity. The role of genetic factors in the pathogenesis of obesity is undeniable with familial clusters giving individuals in our modern "obesogenic" environment markedly different risks of becoming obese [21, 22]. The present study investigates the fatty acid composition of tissues and the metabolic responses to high-fat diets rich in either n-3 or n-6 PUFA in mice, selected for high body weight (DU6), leading to rapid obesity development, or selected for high treadmill performance, leading to a lean phenotype. This study also investigates whether different metabolic responses may be linked to changes in insulin signalling pathways in liver, muscle and adipose tissue. The mouse lines used in the present study reflect the range of genetic predisposition to obesity development commonly observed under physiological conditions .
It is most likely that selection of these mouse lines produced metabolic phenotypes by polygenic variants of genes as described in human populations . The mice lines used in this study reveal distinct metabolic phenotypes. The DU6 mice display an "energy storage phenotype" that is characterised by high body weight, increased food intake, larger fat depots, higher liver weight and higher plasma FFA, leptin and insulin levels. In contrast, the DUhTP mice display an "energy usage phenotype" that is characterised by lower body weight, less food intake, smaller fat depots, lower liver weight and lower plasma FFA, leptin and insulin levels. The findings of the present study also suggest that there is a strong genetic contribution to the pattern of fatty acid composition of body tissues. The selection lines showed clear differences in the fatty acid composition of liver and muscle even under standard chow feeding conditions. These data are supported by studies in pigs which showed a significant heritability of fatty acid composition in muscle, especially for the concentrations of n-3 and n-6 PUFA . Interestingly, in liver, the amount of oleic acid was lower in DUhTP mice in comparison with DU6 mice which may be related to the reduced capacity to synthesise oleic acid from stearic acid as indicated by the decrease in delta-9 desaturase index. In muscle, palmitoleic acid content was also lower in DUhTP. This difference in the SCD index between selection lines appears to have a genetic origin. Furthermore the capacity for the de novo synthesis of long-chain n-3 PUFA, DHA and DPA, in muscle was elevated in DUhTP mice fed standard chow compared to DU6 mice. This may lead to increased insulin sensitivity, an association which has been previously observed in human skeletal muscle containing increased proportions of PUFA with twenty and twenty-two carbons . Increased n-3 PUFA content in muscle cells was identified as a key driver of inner mitochondrial membrane properties, thereby affecting membrane-associated proteins . Carnitine palmitoyltransferase activity was increased by dietary n-3 PUFA suggesting an enhanced lipolytic capacity in skeletal muscle and enhanced mitochondrial membrane fluidity . It is therefore tempting to speculate that the increased n-3 PUFA concentrations in muscle may contribute to a metabolic status in DUhTP that leads to the energy usage phenotype and to the ability to adapt more successfully to a HFD. In contrast, DU6 mice have significantly less long-chain n-3 PUFA concentrations in muscle, they have lower insulin signalling capacity (unpublished results) and probably less oxidative capacity to burn fatty acids in skeletal muscle resulting in the energy storage phenotype.
Different responses to n-3 HFD or n-6 HFD
The mechanisms underlying diet-induced obesity are still not fully understood, but are influenced by a range of genetic factors . The present study investigated metabolic responses to isocaloric high-fat diets enriched with either n-3 PUFA or n-6 PUFA in two long-term selected lines of mice. The metabolic responses of the animals were defined by measuring key markers of lipid and glucose metabolism in plasma, liver, muscle and adipose tissue and by determining fatty acid composition of liver and muscle. In agreement with many previous studies, fatty acid composition of body tissues was mainly determined by dietary fat intake in both lines of mice. [39, 40]. However, the specific metabolic responses in body tissues to a HFD enriched with n-3 or n-6 PUFA differed markedly between the two selection lines. Major effects are discussed below, separately, for lipid and glucose metabolism. Different responses to HFD occurred despite similar calorie intake per bwt0.75 between the feeding groups and between the selection lines. Thus, our data suggest that mice with a predisposition for obesity development, the energy storage phenotype, or to leanness, the energy usage phenotype, respond differently to isocaloric HFD rich in n-3 PUFA or n-6 PUFA. However, the formulation of the diets used in the present study had two limitations. Firstly, differences in protein content of the control diet and the high-fat diets could have potentially influenced some metabolic processes. Secondly, the higher content of myristic acid in n-3 HFD may have modified some of the biological effects. Although unlikely, the magnitude of such potential influences could not be addressed in the present study.
One of the most important contributing factors to obesity development is the increased consumption of energy-dense foods resulting in higher calorie intake and higher intake of dietary fat . Interestingly, only the DU6 mice showed a significant increase in adipose tissue weight in response to both the n-3 HFD and the n-6 HFD. Body weight increased significantly in DU6 mice fed either n-3 or n-6 HFD due to the high calorie density of these diets, although food intake measured in grams was reduced. The higher body weight of DU6 mice was mainly due to increased fat deposition, emphasising the energy storage phenotype of these animals. The effects of the HFD on parameters of adiposity were almost missing in DUhTP mice. Interestingly, the increase in adiposity in DU6 mice was not caused by changes in food intake; the calorie intake per bwt0.75 was very similar between the different selection lines. These data clearly underscore the biological importance of genetically determined pathways of energy utilisation that contribute to the development of obesity.
Many dietary components and metabolic conditions are known to be involved in nutrient partitioning that shift energy from storage in adipose tissues to energy utilisation in muscle and to muscle protein synthesis [42–45]. A lower capacity to oxidise fat in skeletal muscle and liver in high fat feeding conditions was stipulated as the main driver of shifting energy into storage as fat in obesity-prone rats . Data from the present study suggest that dietary n-3 and n-6 PUFA may play a role in nutrient partitioning. The DUhTP mice fed a HFD showed a remarkable adaptive response. Although body weight was increased in the n-3 HFD group, total cholesterol levels were decreased when DUhTP mice were fed n-3 HFD, indicating a health benefit on lipid metabolism of the n-3 PUFA diet by inhibiting cholesterol synthesis . In addition, in DUhTP there was only a trend of an increase in adipose tissue weights and a slight, but significant increase in plasma leptin concentrations when fed n-3 HFD, conveying further health benefits on lipid metabolism. These intriguing findings warrant further study and may involve n-3 PUFA-mediated changes in peripheral oxidation of fatty acids, especially in individuals with an energy usage phenotype[37, 47, 48].
The main differences in glucose metabolism responses to n-3 HFD and n-6 HFD for the two selection lines are described by the following main findings. Plasma glucose concentrations were increased by both HFD in DU6 mice. In addition, plasma insulin concentrations in DU6 fed n-6 HFD were increased suggesting development of insulin resistance . In contrast, HFD-induced hyperglycaemia and hyperinsulinaemia was absent in DUhTP mice. Interestingly, in both selection lines n-3 HFD reduced hepatic protein expression of a key component of the insulin signalling pathway, the β-subunit of the IR β, while the PKC ζ signalling protein concentration was reduced in liver tissue of DU6 mice only. In contrast, in abdominal adipose tissue, the PKC ζ protein expression was strongly increased by n-3 HFD indicating a higher capacity for glucose uptake. Importantly, this effect was only observed in DUhTP mice. In previous studies it was suggested that the adipose tissue is a key player in modulating insulin-sensitising effects of dietary n-3 PUFA [50, 51]. Although the underlying molecular pathways are still uncertain, our data suggest that up-regulation of PKC ζ within the insulin signalling cascade by dietary n-3 PUFA may explain the insulin-sensitising effects. The present study showed for the first time that PKC ζ may be a target of n-3 PUFA action in a tissue-specific manner. It is increasingly appreciated that the insulin-sensitising effects in adipocytes may be crucial for the health benefits of dietary n-3 PUFA .
Interestingly, PKC ζ expression was unchanged in adipose tissue when DU6 mice were fed n-3 HFD, but hepatic insulin signalling parameters decreased. Consequently, plasma glucose concentrations increased but without any changes in insulin concentration. It is intriguing that muscle tissue did not respond to the HFD in either of the selection lines suggesting that skeletal muscle may be less responsive to dietary regulating of insulin sensitivity in mice fed HFD. Comparable results were obtained in a study using C57BL/6J mice after an eight-week HFD showing only minor changes in muscular transcriptome . The increase in PKC ζ expression in adipose tissue and the decrease in liver insulin signalling in DUhTP mice offers novel pathways for cellular mechanism of glucose partitioning in response to increased dietary n-3 PUFA. This shift in glucose re-partitioning may also be induced in DU6 mice fed n-3 HFD; however, any effect will be less efficient due to the lack of increased PKC ζ expression in adipose tissue. This notion is supported by our observation of increased plasma glucose concentrations with n-3 HFD in DU6 mice. HFD enriched with n-6 PUFA led to hyperglycaemia and hyperinsulinaemia in DU6 mice indicating insulin resistance which is commonly induced by HFD feeding . The observed decrease in PKC ζ expression in adipose tissue may contribute to development of insulin resistance in the energy storage phenotype, again, demonstrating the important metabolic role of adipose tissue in the regulation of insulin sensitivity. In contrast, DUhTP mice showed only small shifts in hepatic insulin signalling when fed with n-6 HFD which was paralleled by plasma glucose and insulin concentrations within the normal physiological range. The data suggest that the capacity to deposit glucose in insulin-sensitive adipose tissue and liver was maintained in the energy usage phenotype even in the face of a n-6 HFD.
Fatty acid composition
The n-3 and n-6 HFD resulted in different fatty acid composition of liver and muscle in DU6 and DUhTP mice. These changes in fatty acid composition reflect the fatty acid composition of the diet which has been demonstrated previously in studies by Valencak and Ruf  and Poureslami et al. . Diet-induced changes were observed in the content of myristic acid which is increased in both, muscle and liver of each line fed n-3 HFD reflecting the high myristic acid content of this diet. Similarly, the increase in n-3 PUFA concentrations with a simultaneous decrease of n-6 PUFA concentrations in muscle and liver with n-3 HFD feeding was observed in both lines of mice indicating a dominant influence of the dietary fat content. However, some differences seemed to be set by long-term selection of the mice lines indicating a high heritability of fatty acid composition in some tissues. The heritability of body tissues fatty acid composition, especially the deposition of n-3 PUFA, has been widely studied in many species, including pigs, salmon and humans [34, 54, 55]. In DUhTP mice deposition of ALA and EPA was lower in muscle resulting in a smaller sum of n-3 PUFA when the n-3 HFD was fed. The lower content of n-3 PUFA in muscle, most likely located in cell and mitochondrial membranes, suggests a lower susceptibility to peroxidation compared to DU6 , thereby contributing to muscle cell viability and performance in DUhTP. It is tempting to speculate that an increase in the antioxidant capacity in DUhTP mice may also lower susceptibility to peroxidation, thus providing further health benefits of dietary n-3 PUFA . This may contribute to the increased running performance in DUhTP mice and support the energy usage phenotype. An increase in the n-6 PUFA content of body tissues was expected with n-6 HFD feeding, however, this was only observed in DU6 liver and muscle and not in the DUhTP animals indicating again a genetic component of fatty acid deposition. Another remarkable difference in specific fatty acid deposition in muscle was observed regarding the SFA content, especially palmitic and stearic acid. Feeding n-3 HFD increased the content of these SFA in DU6 but not in DUhTP mice. These results may suggest that lipogenic pathways are amplified in the DU6 line because palmitic acid is the end product of de novo fatty acid synthesis. Enhanced capacity for lipogenesis, even when HFD are fed, may contribute to the development of the energy storage phenotype of the DU6 mice.