Write an important dietary factor associated with the obesity epidemic that is a major risk factor of metabolic diseases, such as type 2 diabetes and cardiovascular diseases. Introduction Recently , obesity rates increased worldwide and become a major health burden. This increase in obesity is strongly associated with modern habits notably dietary intake and their interaction with genetic predisposition. Thus the consumption of sugary beverages is attributed to be a major source of added sugar and energy in the population and an important dietary factor associated with the obesity epidemic that is a major risk factor of metabolic diseases, such as type 2 diabetes and cardiovascular diseases. Thereby , a clinical study show that even a short-term high-fructose consumption was associated with increased De Novo Lipogenesis and liver fat in healthy men fed weight-maintaining diets Among these beverages , sucrose is a disaccharide consisting of 50% fructose and 50% glucose and is applied as a sweetener to enhance the taste and palatability of these sugary drinks . it is therefore urgent to understand the mechanisms which link the consumption of sugary drinks, obesity and associated diseases. Research findings suggest that rodents fed sweetened beverages containing sucrose or fructose usually induce body weight gain and glucose intolerance. Given this significant impact of these caloric sweeteners on body weight , another consistent study had proved that high fructose intake improves selectively adipogenesis in mice model , potentially by redirecting substrate utilization toward lipogenesis. Plus, the enzymes involved in lipogenesis are acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS)) and glucose/fructose metabolism .As known , the consumption of fructose containing beverages is shown to be associated with non-alcoholic liver steatosis and a preclinical study demonstrate that the exposure to high fructose diet in mice , exhibit higher levels of hepatic lipogenesis as evidenced by increased FAS . Therefore , in this study we will focus on the caloric sweetener ‘’Sucrose’’ found in humans’ sweetened beverages .In order to determine the effect of high sucrose intake on lipogenesis , we used a suitable type of mice (C3H) exposed to a control diet and to sucrose-sweetened-water and many measurements were conducted , including body weight , body composition , plasma glucose and triglycerides , and the hepatic expression of the enzyme FAS * Q. Qi and L. Qi, “Sugar-sweetened beverages, genetic risk, and obesity,” New England Journal of Medicine, vol. 368, no. 3, pp. 286-287, 2013. * J. Ma, N. M. McKeown, S.-J. Hwang, U. Hoffmann, P. F. Jacques, and C. S. Fox, “Sugar-sweetened beverage consumption is associated with change of visceral adipose tissue over 6 Years of follow-up,” Circulation, vol. 133, no. 4, pp. 370–377, 2016. * V. S. Malik, B. M. Popkin, G. A. Bray, J.-P. Després, and F. B. Hu, “Sugar-sweetened beverages, obesity, type 2 diabetes mellitus, and cardiovascular disease risk,” Circulation, vol. 121, no. 11, pp. 1356–1364, 2010. * R.B. Kanarek, N. Orthen-Gambill, Differential effects of sucrose, fructose and glucose on carbohydrate-induced obesity in rats, J. Nutr. 112 (1982) 1546–1554 *T. Kawasaki, A. Kashiwabara, T. Sakai, K. Igarashi, N. Ogata, H. Watanabe,K. Ichiyanagi, T. Yamanouchi, Long-term sucrose-drinking causes increased body weight and glucose intolerance in normal male rats, Br. J. Nutr. 93 (2005) 613–618. *M.E. Bocarsly, E.S. Powell, N.M. Avena, B.G. Hoebel, High-fructose corn syrup causes characteristics of obesity in rats: increased body weight, body fat and triglyceride levels, Pharmacol. Biochem. Behav. 97 (2010) 101–106. **Consuming fructose-sweetened beverages increases body adiposity in mice * Diet high in fructose promotes liver steatosis and hepatocyte apoptosis in C57BL/6J female mice: Role of disturbed lipid homeostasis and increased oxidative stress *Effect of a High-Fructose Weight-Maintaining Diet on Lipogenesis and Liver Fat *G Baiges-Gaya, S Fernández-Arroyo… – The Journal of …, 2021 – Elsevier. Hepatic metabolic adaptation and adipose tissue expansion are altered in mice with steatohepatitis induced by high-fat high sucrose diet G Lee, JH Han, HJ Maeng, S Lim – … Obesity & Metabolic Syndrome, 2020 – ncbi.nlm.nih.gov. Three-month daily consumption of sugar-sweetened beverages affects the liver, adipose tissue, and glucose metabolism. nih.gov Cited by 22 BT Steffen, DR Jacobs, SY Yi, SJ Lees… – … Journal of Obesity, 2023 – nature.com. Long-term aspartame and saccharin intakes are related to greater volumes of visceral, intermuscular, and subcutaneous adipose tissue: the CARDIA study. nature.com Cited by 1 1.1.Animals and diets The study was approved by the French National Animal Committee on the use of laboratory animals. 21 male C3H mice, aged 5 weeks upon arrival at the laboratory, were housed in standard laboratory rooms, with controlled temperature and lighting (22 ±2 °C and a 12- hour light/dark cycle). In order to adapt to the laboratory conditions ; mice had ad libitum access to standard chow diet and water for one week .After adaptation , in the next 3 weeks , the animals were fed with classical diet that provided 14.6 KJ/g of energy [14% protein , 76% carbohydrate (80% starch , 13% sucrose and 6%cellulose) and 10% fat.(Table S1) and with sucrose-sweetened-water containing 20% of sucrose. In this study , we used the C3H genotype of mice because they are susceptible to diet-induced obesity which make them a suitable model for studying the impact of dietary factors on lipid metabolism. 2.2 Design of the protocol The mice were divided randomly into three weight-matched groups (n = 7/group). At the age of 8 weeks , these three groups were provided with different diets :1) The fasting group (F) was fed with the classical diet and then undergo an overnight fasting before the sacrifice (13% sucrose). 2) The control group (C) was provided the classical diet only (13%sucrose). 3) The sucrose-sweetened water group (SSW) had access to the classical diet in addition to the sucrose-sweetened-water (33% sucrose in total ).All diets were available to the animals without restriction. Prior to euthanization, mice were anesthetized by intraperitoneal injection of 50 mg of pentobarbital sodium per kilogram of body weight. 2.3. Plasma/tissue collection and analysis After anesthetization (phenobarbital 50 mg/kg, ), body weight of different groups was measured. The liver and epididymal fat pads were dissected out and weighed to the nearest 0.01g .Thus , the liver was cut into 50-100 mg , then snap-frozen in Trizol® reagent and stored -20°C . Blood was collected in Microvette® tubes , then centrifuged (4000rpm, 4 °C, 10min) and the plasma samples are stored at -20°C . Plasma levels of triglycerides and glucose were measured with Infinite M200 Pro device (wavelength:500 nm) and by using a commercial kit (Randox Glucose PAP and Kit TRIGS Randox) 2.4.Extraction of RNA and real time q-PCR In order to measure the hepatic expression of FAS, liver tissues were homogenized in 1ml Trizol® reagent. After the extraction and the purification of the RNA , a NanoDrop spectrometer is used to identify RNA concentration and to measure RNA purity by measuring absorbance at 260 nm for RNA, 280 nm for proteins and 230 nm for carbohydrate .The Agarose Gel Electrophoresis is then used to assess the quality of RNA through the UV visualization of 18S and 28S ribosomal RNA bands. However ,1µl of total RNA was reverse-transcribed using a high-capacity cDNA archive kit. Real-time PCR was performed using an ABI Real-time PCT System and the SYBR Green PCR Master. The primer sequences of the target genes are listed in Table S2 where 18s is the housekeeping gene and FAS is the gene of interest . Gene expression was calculated as 2−ΔCt and the expression values are expressed relatively to 18S of the liver. 2.5. Statistical analyses Data are expressed as means ± SEMs. One-way analysis of variance (ANOVA) was performed to determine the effects of the different type of diets , and then followed by Tukey’s Post Hoc Test to identify the significant difference among two pairs of group of diets . A P-value <0.001 was considered to be strongly significant ,P-value <0.05 was considered to be statistically significant and a P-value between 0.05 and 0.1 was considered as a tendency. All analyses were performed using GraphPad Prism 9 . Results Body weight , body composition and organ mass : Over 3 weeks of experiment , data revealed insights into the physiological effects of different dietary conditions (standard diet vs sucrose-sweetened-water vs fasting)- on body mass, liver mass, and epididymal fat mass. The ANOVA global test unveils that , there was a trend to have difference in body mass among the groups (p = 0.0667), while both liver mass (p < 0.001) and epididymal fat mass (p = 0.02) exhibit statistical significant differences within the groups . However , post hoc Tukey’s test show that mice fed with SSW showed a trend to have a higher body mass than the fasting group (p=0.07) , and showed to be slightly higher than the control group but statistically not significant (p=0.9) Thus the liver mass was found to significantly differ across all pairwise comparisons C vs. F and SSW vs. F with p-values < 0.001, suggesting notable variations influenced by dietary conditions. . While no significant difference was observed between control and Fasting conditions (p = 0.708), both SSW vs. F (p = 0.003) and SSW vs. C (p = 0.014) revealed statistically significant disparities. Plasma glucose and triglycerides Due to a technical issue, data for one mouse of the fasting group were unavailable in the analysis of blood parameters. However these missing data will not affect the overall interpretation of the trends and significant differences observed in the study. The analysis of blood glucose levels revealed a p-value =0.10, indicating a trend toward a difference among the dietary conditions (Fasting, Control, and Sucrose). The mean differences between Fasting vs. Control (p = 0.9), and Control vs. SSW (p = 0.2) were all non-significant , while the mean differences of blood glucose show a trend to be higher in SSW than the fasting group (p = 0.1) Therefore ,these results suggest that, while there is a subtle trend in blood glucose levels, the specific dietary conditions did not yield statistically significant differences in pairwise comparisons. On the other hand, the analysis of triglyceride levels yielded a p-value of 0.04, indicating a statistically significant difference among the three groups . Tukey’s multiple comparisons test demonstrated a significant mean difference of 1.2 between Control and Fasting group (p = 0.04), signifying higher triglyceride levels in the Control group compared to the Fasting group. However, no significant differences were observed between SSW vs. Fasting (p = 0.8037) and the SSW blood triglyceride had a trend to be higher than the Control group (p = 0.1225) In conclusion , the blood glucose and triglyceride levels have a trend to increase with the consumption of sucrose sweetened beverage . Hepatic expression of the enzyme FAS In the control group , one aberrant result was excluded from the analysis of FAS expression. Among the dietary groups , FAS expression has a tendency to be different (p=0.06). The comparison of the Control group with Fasting group ( p-value= 0.95), indicates no significant difference in FAS expression between these two groups . Meanwhile , when comparing Fasting group against SSW group ( p= 0.07) and Control vs SSW (p=0.142) ,show a tendency of FAS expression to be higher in SSW and to be towards significance but not reaching a strong threshold.
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