Following the habituation period, animals were divided into two groups: a control group (n = 23) exposed to a standard chow diet (4RF21, 3.952 kcal/g containing 19.42% proteins, 2.58% lipids, 5.54% fibers, 6.76% ash, 54.61% nitrogen-free extract, Mucedola, Italy) and a free-choice high-fat high-sugar group (fcHFHS, n = 25) exposed to standard chow with normal tap water, combined with availability of saturated fat (“blanc de boeuf”, Vandermoortele, France) and a bottle of 10% sugar dissolved in water (Powder Sugar, Erstein, France), as previously described [28]. The regimen lasted 6 weeks (Fig. 1a). The sucrose concentration was chosen to be comparable to most sweetened beverages consumed by humans, and as a preferred sugar concentration in rats [31]. Animals were weighed once per week and daily food and liquid intake were recorded every 24 h. Group randomization was established based on the initial animal weight. Total calorie intake was then calculated per day for each rat.

Effects of a 6-week free choice high fat high sugar (fcHFHS) diet on hyperphagia and obesity. a Experimental design. b Mean daily caloric intake in fcHFHS versus control (chow) animals. Caloric intake was significantly higher in the fcHFHS group compared to the chow group over the 6 weeks of the diet. c left panel. Linear regression between mean daily sucrose consumption and time (n = 25). Dashed lines represent the 95% confidence bands of the best-fit line. Sucrose consumption progressively increased over weeks in both groups. Right panel. Mean weekly weight gain during the diet (% of the body weight at the start of the diet). The fcHFHS diet led to a significant, rapid, and higher increase in weight gain in rats compared to the chow-control animals. d Mean fat pads weight, BMI, and weight gain at the end of the 6-week regimen. These three obesity hallmarks were increased following the diet. e Graphical representation of OP and OR clusters resulting from Km Clustering based on the 3 obesity criteria mean values measured at the end of the experiment (Fat pad weight, BMI and weight gain). X and y axes represent the first two components of the principal component analysis after dimensional reduction (3 to 2 variables). % = percentage of variance. f Comparison of the 3 obesity criteria between groups. BMI and weight gain were significantly higher in OP than OR rats. The 3 obesity criteria were increased in the 2 clusters when compared with the chow group. g Graphical representation of HSC, HFC, and LPC clusters resulting from Km Clustering based on the 3 palatable food intake-based criteria mean values measured at the end of the experiment (total sucrose intake, total fat intake, and total palatable food intake). Minor geometric forms represent individual values, major geometric forms represent cluster centroids. h Comparison of palatable food intake between clusters. HSC consumed more sucrose, HFC more fat, and LPC less palatable food than animals belonging to the other clusters. i Comparison of weight gain between the chow group and the 3 palatable food intake-based clusters. Weight gain was increased in the 3 clusters compared to the chow group. j Proportion of OP/OR rats belonging to the 3 palatable intake-based clusters. The same proportions of OP and OR animals were observed in the 3 clusters. In bar graphs, white dots represent individual data. *p < .005; **p < .001; ***p < .0001, n = 5–23 per group. fcHFHS (n = 25); OP obesity prone (n = 10); OR obesity resistant (n = 15); BMI body mass index; LPC low palatable consumers (n = 13); HSC high sucrose consumers (n = 5); HFC high fat consumers (n = 7)

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