Human beings, who have been on earth approximately for 200 thousand years, gave up hunter–gatherer lifestyle and turned to agriculture and animal husbandry 10–12 thousand years ago [1,2]. In the meantime, the integration of modern technology into food, agriculture and livestock industries has facilitated access to foodstuffs and this has caused some changes in human health. Changes in lifestyle (like reduced activity and mobility) and dietary changes have triggered various metabolic diseases [3,4].

Sustainable healthy life is possible with balanced and continuous consumption of essential nutrients, coupled with numerous factors. The dietary components and the proportions of these substances play an important role on the regulation of homeostasis by affecting many tissues in organism [5,6]. Obesity is associated with excessive consumption of food containing high fat, sucrose and/or fructose [7,8]. While dietary high fat intake has long been recognized to trigger obesity-related diseases, studies in recent years have revealed that carbohydrates are responsible for the formation of these diseases [9].

As a result of increasing population, demand for food and hence the demand for sucrose and fructose have gradually increased. Therefore, much more sucrose and fructose are produced day by day [10]. While world sugar production was 250 thousand tons per year in mid-19th century, production quantity of sugar has reached 8 million tons in the 20th century [4]. Today, approximately 170 million tons of sucrose and 15 million tons of HFCS (High Fructose Corn Syrup) are produced annually [11].

High amounts of sucrose and fructose used in diets cause more calorie intake. Higher calorie intake than the organism needs from simple carbohydrates triggers the development of metabolic disorders [12]. Sucrose and fructose have same metabolic energy (3950kcal/kg and 3750kcal/kg, respectively), but fructose has become more preferred than sucrose in production due to being more economical and sweeter (1.73 times sweeter than sucrose). HFCS has been presented as an alternative sweetener to diabetic patients due its low glycemic index. However, subsequent studies have shown that fructose, consumed abundantly via HFCS, is metabolized independently of insulin, triggering triglyceride production and lipogenesis. Chronic activation of lipogenesis pathway induces chronic metabolic disorders [13,14].

Fructose is metabolized independently of insulin in mammals. Furthermore, phosphofructokinase, the rate-limiting enzyme, is not involved in the metabolism of fructose. These factors lead to faster metabolization of fructose in the organism. Recent studies have shown that chronic fructose consumption leads to non-alcoholic fatty liver disease. Some of the researchers claim that in addition to high blood glucose and triglyceride levels, hypertension, abdominal obesity, insulin resistance and glucose intolerance arise with the consumption of fructose [4,12,13].

Scientific debates about the proportion of carbohydrate and fat sources in diet are ongoing. In this study, metabolic changes with consumption of high fat, high sucrose and high fructose diets for 90 days were investigated at molecular, biochemical and histopathological levels in rats. Levels of mRNA and protein of PPARα (Peroxisome Proliferator Activated Receptor alpha), LXRα (Liver X Receptor alpha), ChREBP (Carbohydrate Response Element Binding Protein), and SREBP-1c (Sterol Regulatory Element Binding Protein) were investigated in liver and skeletal muscle. Histology of liver tissue was also assessed.

Obesity and complications related to obesity may be triggered by the consumption of fructose in addition to the consumption of fatty diets with high saturated fatty acid [25–27]. It is general knowledge that high fat in the diet triggers weight gain, but the fatty acid ingredient of the diet is also important [28]. In a study [27], it has been reported that rats consumed chow diet and sucrose solution (35%) for 9 months and their body weights and consumption of chow diet did not change. This finding was believed to be related to the structure of sucrose. Sucrose is metabolized with insulin-dependent pathways due to glucose. Because of its insulin-independent metabolism, fructose does not suppress food intake and creates a hyperlipidemic effect which leads to a significant increase in body weight as noted by this study and also other studies (Table 4) [29].

Fructose is metabolized mainly in the liver since most tissues do not have fructose transport systems. Glucose-6-phosphate formed from glucose cannot be converted to triglyceride as it is regulated by phosphofructokinase, but it is possible for fructose since it becomes ready to convert triglyceride after converting fructose-1-phosphate. This causes lipid production by fructose faster than glucose in the liver. Furthermore, this difference in metabolism results in an increase in circulating triglyceride level due to fructose (Tables 6 and 7) [30,31].

Dietary high calorie intake triggers adipogenesis by suppressing PPARα activity [32]. Studies conducted with different concentrations of fructose solutions have shown that diets with sufficient energy content do not change the activity of PPARα [33–35]. Although studies indicate that the activity of PPARα in muscle tissue is suppressed if the content of sucrose and/or fructose used in the diet is high and feeding time with these carbohydrates is long, in this study, PPARα protein results in both tissues confirmed gene expression results in accordance with literature [33–35].

LXRα is reported to be mainly expressed in the liver and it is a reasonably important transcription factor on lipogenesis. LXRα is responsible for converting more carbohydrates to lipid and regulating the expression levels of ChREBP and SREBP-1c [8,36,37]. This transcription factor, determined as a glucose sensor by cell culture studies, is used as a potential therapeutic target in diabetes mellitus, dyslipidemia and cardiovascular diseases [38]. In accordance with the literature, the activity of LXRα depends on the amount of dietary energy and content of sucrose and fructose. In this study, activity of this transcription factor was at similar rates with other studies [8,35]. Both liver and skeletal muscle activity of LXRα gene was found to notably increase, especially in Hfru group. On the other hand, protein amounts of this transcription factor did not significantly change in any of the groups in the study. Although LXRα is an increased transcription factor in liver due to feeding, it is thought that its activity may be restricted by some post translational mechanisms such as miR-613 [39]. It has been reported that miR-613, which has been shown to be active by SREBP-1c promotor control, performs this function by some kind of negative feed-back mechanism [39,40]. In muscle tissue, the amount of LXRα protein in Hfru group confirmed gene expression results. Activation of LXRα in muscle tissue is an important factor for carbohydrate and lipid metabolism [41]. Some researchers claim that activity of this transcription factor is increased in skeletal muscle with lipid and carbohydrate related disorders [42]. This study shows that fructose is an important energy source that triggers lipogenic mechanisms through LXRα in liver as well as in muscle tissue. Skeletal muscle protein results suggest fructose may be more dangerous than sucrose in terms of disorders that may develop due to changes in lipid metabolism.

It is known that most of the genes associated with glucose metabolism in the liver are activated via ChREBP. Lipogenesis is largely mediated through ChREBP and SREBP-1c [43–45]. Experimental animal studies have shown that expression levels of lipogenic genes are reduced in response to glycosylation of ChREBP gene, resulting in decreased lipid production at significant levels [46]. It is conceivable that the amount of ChREBP protein may be low due to miRNA-613 in Hfat and Hsuc groups [40,47]. Significantly increased ChREBP activity in relation to the consumption of fructose in liver has also been shown in many studies [48–50]. In the case of excess production of ChREBP protein, the genes responsible genes expression levels for fatty acid and triglyceride synthesis are increased and hepatic steatosis is triggered [51]. In this study, it was determined that the amount of ChREBP protein in the Hsuc and Hfru groups was significantly higher than the other groups, although there was no difference on gene expression. There is not sufficient information about ChREBP activity in muscle tissue. It is thought that high amount carbohydrates in diets may have regulated the metabolic activity in skeletal muscle through ChREBP protein [52,53].

In the liver, SREBP-1c was upregulated about 4-fold in all experimental groups and it is understood that this upregulation may occur regardless of the energy source, it can increase if the dietary energy is high [44,54]. However, the amount of SREBP-1c protein is the highest in Hfru group and the lowest in Hsuc group (P<0.05) indicating that there was no equal protein translation with gene expression and this may have been controlled by post-translational mechanisms. It is reported that feed-back mechanisms, such as insig-1 and insig-2 which control the production of SREBP-1c proteins, play an important role for the control of lipid synthesis by insulin-mediated pathways [55]. It is also known that SREBP-1c mediated lipogenesis with dietary high fructose. It is triggered with the absence of miRNAs such as miR-145-3p, miR-7-1-5p, miR-33, and miR-613 [56,57]. Excess fat stored in muscle tissue may be associated with SREBP-1c and some post-translational factors activities [58]. It was seen in the current study that high fructose induced lipogenesis by increasing SREBP-1c-stimulatation in muscle tissues. With high fructose diets, SREBP-1c gene and protein may show activity in many tissues without insulin requirements [59,60].

Chronic consumption of fructose can lead to metabolic disorders such as insulin resistance, hypertriglyceridemia, and hypertension [61]. The fact that long-term consumption of high-energy diets deteriorates glucose regulation has been identified in a number of studies [62,63]. Depending on the diet, insulin mechanisms are continuously activated by glucose and trigger lipogenesis. In Hfru group, detected high plasma glucose and triglyceride concentration confirmed that fructose triggered lipogenesis (Tables 6 and 7 and Fig. 4g, h). Hfru group plasma glucose results were found to be the same as many other studies [62–64]. On the other hand, there was no difference in plasma insulin levels among the experimental groups. Although animals were hyperglycemic in Hfru group, it is thought that the reason of similar amount of insulin may have been be related to the percentage of fructose in diet and the feeding period.

Plasma cholesterol levels can vary depending on diet ingredients. Nevertheless, the results have been found to be compatible with similar studies in which plasma cholesterol levels of rats exposed to high-energy diets for 90 days did not significantly change during the study [56,65]. Plasma HDL and LDL results were similar to those found in the relevant literature [66–68]. It was observed in the study that carbohydrate content of diet was not as important as fat content for HDL and LDL regulation. But the amount of energy sources could affect these parameters and insulin resistance, diabetes and other metabolic disorders begin with hyperlipidemia [69,70]. Plasma triglycerides were significantly higher in Hfru group and it confirmed that fructose was hyperlipidemic.

AST and ALT may be within limits, even if pathology develops in liver. It was reported in a study that fructose-fed rats had lower AST and ALT values than control animals [71]. But, the ratio of AST and ALT levels may be a more decisive criterion to have an idea of liver damage while investigating the effects of diets on the liver. It is reported that in humans, this ratio is greater than 2 with alcohol-induced liver damage [72]. Also, some studies show that consumption of high fructose led to an increase in AST/ALT ratio [73,74]. In this study, AST/ALT ratio was the highest in Hsuc and Hfru groups. However, there was no significant difference between the Hsuc, Con and Hfat groups (Table 6). Hfru group's AST/ALT ratio had the highest value. Therefore, it was observed that AST/ALT ratio may be a determining criterion for detecting damage to the liver due to high energy intake. This was also confirmed by the histopathological findings of the study. There was no significant change in plasma GGT levels due to high energy intake for 90 days.

In the present study, correlations between protein and biochemical parameters showed that dietary carbohydrate levels significantly effect the metabolic activity in liver and also skeletal muscle at molecular levels. Biochemical markers in plasma may give important information about liver physiology via ChREBP and LXRα activities. LXRα, ChREBP and SREBP-1c proteins activities in skeletal muscle were found significantly correlated with AST/ALT ratio. The correlation between these proteins and biochemical markers suggests an idea of metabolic activity and physiology of muscle tissue. This means liver health and skeletal muscle metabolism closely related. Some of the biochemical parameters (glucose, LDL, triglyseride) in plasma give information about protein levels of SREBP-1c in skeletal muscle. Thus, it is possible to have an idea about skeletal muscle metabolic activity.

Liver weights in Hfru group were significantly higher than the liver weights in the other experimental groups, possibly due to fat deposition of liver. Fatty liver and degeneration of hepatocytes were detected in Hsuc group, but it was better than Hfru. The reason for this was the rate of sucrose used in diet and the feeding period. On the other hand, Haubert et al. detected microscopically severe degeneration with fatness in liver in rats fed with a diet containing 70% sucrose [75]. These results show that the rate of sucrose in diets is also important for metabolism related diseases. Ackerman et al. claimed that compared to sucrose, fructose has negative effects on fatty liver that are stronger and speedier and causes degeneration of hepatocytes [76]. Also, Vilà et al. found that consumption of 10% fructose solution in addition to normal diet for just 2 weeks caused necrotic areas in the liver in Sprague-Dawley rats [50]. It was microscopically determined that the rats in Hfru group had the most damaged livers. The most severe form of degeneration, balloon degeneration, was clearly identified in the Hfru group and fattening of liver in this group was more severe than others. Sucrose and fructose have been found to cause significant inflammation as well as destructive effects in hepatocytes. This may make liver tissue more susceptible to secondary bacterial infections and increase oxidative damage. A common finding in the experimental groups (Hfat, Hsuc and Hfru) was the stagnation of erythrocytes in the central veins and sinusoidal regions. It is thought that degenerative and necrotic changes in hepatocytes may cause the foreground by forming an anoxic and hypoxic environment in liver tissue.

This study found that fructose triggers lipogenesis more than sucrose and sunflower oil. This is thought to be due to the fact that fructose consumption does not particularly affect insulin secretion. Results have showed that fatty liver disease and related complications can be caused by using 27% fructose solution beside normal diets in rats in 90 days. Furthermore, the feeding period can be shortened by using high concentration fructose solution for metabolic disorder model studies.

LXRα and SREBP-1c are important molecular markers for lipogenesis in both liver and muscle tissue. The activities of SREBP-1c in these tissues are closely related to biochemical parameters as triglyceride production and body weight changes with lipogenesis. It has been understood that the relationship between ChREBP and lipogenesis needs to be further investigated depending on the feeding with fructose. Further studies are needed to understand the relationship between ChREBP and PPARα in muscle tissue. SREBP-1c is a transcription factor on lipogenesis pathway and can be activated by many energy sources, but checking protein levels of this transcription factor is more important than gene expression levels. On the other hand, AST/ALT can be used as a more specific parameter for diagnosis about liver pathology and therefore this ratio may be evaluated together with other parameters.

As a result, it can be said that fructose triggers lipogenesis, weight gain and metabolic disorders more and therefore it is metabolically more dangerous than sucrose and sunflower oil as energy resourcesAbbreviationsHFCS

High Fructose Corn Syrup