Caloric Restriction

Adult zebrafish (AB line, ZIRC, Eugene, OR, USA) were kept at 28°C under a 14-h light:10-h dark cycle, and water conditions of environmental quality were maintained according to an established protocol [68 ]. Zebrafish at 3.5 mpf were assigned into two dietary groups (overfeeding and maintenance groups) with approximately five fish per 2-L tank. Zebrafish in the overfeeding group were fed three times per day with freshly hatched Artemia (corresponding to 60 mg cysts/fish/day). Zebrafish in the maintenance group were fed freshly hatched Artemia (corresponding to 5 mg cysts/fish/day) once per day. For calorie restriction, the zebrafish were fed freshly hatched Artemia (corresponding to 2.5 mg cysts/fish/day) for 2 weeks after being overfed for 8 weeks. Commercial flake food (Hikari Tropical Fancy Guppy, Kyorin, Hyogo, Japan) consists of 9% fat, 17% carbohydrate and 59% protein, whereas Artemia nauplii consist of 22% fat, 16% carbohydrate and 44% protein (dry weight basis).
The percentage of consumed Artemia was estimated by counting Artemia before and after feeding. Briefly, 1 mL of water was collected from 1700 mL of water containing freshly hatched Artemia corresponding to 100 or 8.5 mg cysts. The numbers of hatched Artemia were counted three times to determine a mean count. After counting, the samples were returned to the 2-L tank. Then, five fish were transferred to the tank for feeding. After 2 hours, the number of Artemia was counted as before feeding. Zebrafish fed 5 or 60 mg of Artemia per day consumed about 80 or 50% of the provided Artemia, respectively. Zebrafish fed 2.5 mg of Artemia per day consumed almost all of the provided Artemia.

This prospective cohort study is based on data collected during the first 2 years of the PREDIMED-Plus (PREvención con DIeta MEDiterránea Plus) study. Briefly, the PREDIMED-Plus study is an ongoing randomized, parallel-group, 6-year multicenter, controlled trial designed to assess the effect of lifestyle interventions on the primary prevention of cardiovascular disease. The primary aim of the trial is to assess the effects of an intensive weight loss intervention based on an energy-reduced Mediterranean diet (MedDiet), physical activity promotion, and behavioral support (intervention group) compared to usual care and dietary counseling only with an energy-unrestricted MedDiet (control group) on the prevention of cardiovascular events. Details of the design and methods of PREDIMED-Plus have been previously described [35 (link), 36 (link)] and are available at https://www.predimedplus.com/.

From January to March 2021, the study was carried out at the Buraydah Central Hospital. This was a repeated-measurement uncontrolled intervention research. The prospective participants listened to a presentation on a very low carbohydrate or ketogenic diet, which provided details of the diet, the significance of nutritional composition, recommended eating times each day, the benefits of the diet, and the high-fat diet. All potential participants were asked to consume a KD for eight weeks while maintaining their normal physical activity levels. The study protocol and potential risks were fully explained to the participants before their written consent was obtained. Participants were asked to attend six sessions during the intervention period, at baseline (pre-intervention), weeks 1, 2, and 4 (mid-intervention), week 6, and week 8 (post-intervention) for an assessment of their body weight, height, BMI, waist circumference, and BHB levels.
The session was held at the Nutrition Clinic of the Buraydah Central Hospital. A very low carbohydrate or ketogenic diet with caloric restriction was used as the experimental intervention. At the start of the intervention, all participants attended a two-hour lecture about a KD, nutritional composition, the proper times to eat, and the beneficial effects of a KD, as well as an educational session about their prescribed diet plan. The participants were given a list of very low-carbohydrate foods to choose from in order to consume 20 g of carbohydrates per day. They were provided with a typical plan and a menu for all main dishes, snacks, and drinks allowed for seven days, with instructions to limit their carbohydrate intake to 5–10% of energy intake (EI) and to derive at least 75% of it from fat, incorporate a protein intake of 20%, and follow a very low-energy diet with a deliberate caloric deficit (1200–1500 kcal). Unlimited quantities of meat, poultry, fish, shellfish, and eggs were permitted, as were two cups of salad and vegetables per day, one cup of low-carbohydrate vegetables per day, four ounces of hard cheese, and limited amounts of cream, avocado, olives, and lemon juice. Transformed fats were prohibited, but fats and oils were not. Detailed explanations of how to apply the KD principles in practice and appropriate tracking and monitoring procedures were provided. The participants were instructed not to use drugs or antioxidants to eliminate the possibility of confounding effects. BHB levels were measured weekly to assess adherence to the dietary regimen.