Effects of dietary high fructose corn syrup on regulation of energy intake and leptin gene expression in rats

1 Universidad Autónoma del Estado de Hidalgo, Instituto de Ciencias de la Salud, Centro Interdisciplinario de Investigación en Ciencias de la Salud. Carretera Actopan-Tilcuautla, s/n., 42162, San Agustín Talxiaca, Hidalgo, México. Correspondência para/Correspondence to: G LÓPEZ-RODRÍGUEZ. E-mail: . Effects of dietary high fructose corn syrup on regulation of energy intake and leptin gene expression in rats


I N T R O D U C T I O N
In Mexico the use of High Fructose Corn Syrup (HFCS) as a sweetener has increased in the last 20 years from 0.09 kg per capita in 1990 to 5.3 kg in 2000 and 12.5 kg in 2013 1 .Fruit juices, nectars, soft drinks, and cookies are foods that compose the diet of Mexican children sweetened with HFCS.High intake of these foods is associated with the nutritional transition toward ever growing prevalences of overweight and obesity in children 2 , and obesity and diabetes in adults 3 .Associations between sugar intake from beverages and weight gain 4 , type 2 diabetes [5][6][7] , cardiovascular disease, and metabolic syndrome have been reported 8,9 .
High fructose corn syrup was initially regarded as a sweetener suitable for diabetic patients because it has low glycemic index compared with glucose 10 .However, HFCS intake may promote the development of chronic diseases, such as diabetes and cardiovascular disease 11 , which have been related to hypertriglyceridemia 12 and insulin resistance 13 in patients with prolonged HFCS intake.
Direct fructose administration into the hypothalamic arcuate nucleus increases food intake in rats and regulates the expression of some genes, such as orexigenic neuropeptide Y and agouti protein 14 .Glucose but not fructose changes blood flow in brain regions associated with appetite and reward pathways, affecting hunger, fullness, and satiety 15 .Hence, fructose may be associated with lower satiety and thus favor weight gain.The objective of this study was to evaluate the effects of chronic fructose intake on weight gain, on leptin gene expression in adipose tissue and the brain, and on metabolic indicators.

M E T H O D S
Male rats aged 21 days were housed in standard polycarbonate shoebox cages under a 12 hour light and 12 hour dark regimen at 24±2ºC and 55±10% humidity.The control rats received water ad libitum and were fed a standard diet (Formulab Chow 5008, Purina ® , Framinghn, Massachusetts, United States).The other groups received defined diets: High-Fructose diet (HFr, standard diet containing 3.5 kcal/g and HFCS 55 solution in deionized water at 15% concentration) and High-Fat diet (HFat, diet containing 6.5 kcal/g with 40% calories from margarine) for 14 weeks.
Body weight, water intake, and food intake were measured weekly.Blood samples were obtained at 45 and 98 days of life to quantify plasma levels of triglycerides, total cholesterol, High-Density Lipoprotein (HDL) cholesterol, and glucose, using commercial enzymatic colorimetric kits (Winer Lab ® ).All procedures were approved by the Universidad Autónoma del Estado de Hidalgo Animal Research Committee.

Gene expression
At 14 weeks of life the rats were killed in the morning after fasting for 8 hours.After dissection, brain tissue and mesenteric adipose tissue were washed in dulbecco's phosphatebuffered saline to eliminate blood contamination and immediately frozen in liquid nitrogen.Total Ribonucleic Acid (RNA) was extracted with TRIzol ® reagent (Invitrogen ® , Carlsbad, California, United States) supplemented with Ribonuclease (RNAse)free Deoxyribonuclease (DNAse), and cleaned with the Qiagen RNeasy ® mini kit according to the manufacturer's instructions.RNA integrity was assessed by gel electrophoresis.Complementary DNA (cDNA) was synthesized at 42°C for 60 minutes using 2 µg of total RNA and 0.5 µg of Oligo (dT) 18 primers (Invitrogen ® ) denatured at 72°C for 10 minutes.Then, strand buffer (1X), 0.5 µM dithiothreitol, 500 µM of each deoxyribose-containing Nucleotide Triphosphate (dNTP), and 200 U of moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) were added (Invitrogen ® ).Subsequently, MMLV-RT was inactivated at 72°C for 10 minutes.

R E S U L T S Intake regulation and weight gain
After 11 weeks of treatment, the mean weights of the groups differed significantly (Figure 1); however, calorie intake per kilogram (kcal/g weight) at 77 days of treatment was similar in all study groups because only the distribution of energy nutrients varied between the groups (Table 1).Rats fed the HFat diet were heavier by the end of the treatment (429.3±25.6 g).Rats fed the HFr diet presented the same pattern of weight gain as the control rats.This similar pattern of weight gain occurred despite different protein intakes (27% in the standard diet and 18% in the HFr diet).Rats fed the HFat diet consumed 10% of their energy as protein in week 14 of life.
Intake of a 15.0%HFCS solution ad libitum for 11 weeks did not cause the rats to lose their energy intake regulation (kcal/g weight), the same was observed when they were fed with HFat diet (Table 1).The animals in the HFr group increased their carbohydrate consumption by consuming a larger volume of the HFCS solution (1.2 mL/g weight/week versus 0.6 mL/g weight/week in the control group, p<0.001Student's t test).The 15.0% HFCS solution in week 14 of life accounted for 33.5±4.1% of the total calorie intake of animals in the HFr diet.Animals fed 15.0% HFCS solution preferred the sweet solution, while water intake was similar in animals fed the standard and HFat diets (Table 1).

Biochemical indicators
Table 2 shows the circulating levels of glucose, total cholesterol, HDL-cholesterol and triglycerides of the three groups.In the HFr group the effect of consuming the 15% HFCS solution on the metabolic indicators was present at 98 days (14 weeks) of treatment as serum triglycerides increased and HDL-cholesterol decreased.Animals in the HFat group had higher levels of glucose than the control group (p<0.01,Student's t test).
Both treatment groups had higher plasma triglyceride levels than the control group (p<0.01,one-way Anova) but as expected, the HFr group had the highest levels.HDL-cholesterol levels decreased in both treatment groups (Table 2).

Expression of the leptin and FAS genes
Levels of gene expression are shown in Figure 2. The HFat diet had higher leptin gene expression in adipose tissue than the control diet (p<0.05,Student's t test) or the HFr diet, by 1.5 times in the latter.Leptin expression in the adipose tissue of rats fed the HFat diet contrasted expression in the brain, which was the lowest of the study groups (p<0.01,one-way Anova).Animals fed the HFr diet had lower leptin expression in adipose tissue and brain than the control group (p<0.01,Student's t test).
Leptin expression was lowest in the brains of the HFat and HFr groups with the increased expression of FAS in the brain.Compared with the control group, they presented 2.7-and 3.1-fold changes, respectively (p<0.01, one-way Anova).

D I S C U S S I O N
Growing obesity rates in Mexico have been linked to changes in eating habits and physical activity, along with higher intake of sweetened beverages, beginning in the first years of life 16 .HFCS is widely used by the food industry to sweeten a large variety of foods consumed mainly by children and adolescents 17 .Chronic HFCS and excess fat intakes by humans 4,18 and rodents 19,20 promotes weight gain, further altering blood metabolic indicators, such as triglycerides and glucose 21 .
To determine whether the changes in weight are explained by loss of energy intake regulation or by nutrient distribution, we used an animal model that allowed intake of a 15% HFCS ad libitum.The energy intake of rats fed a high-fat diet, high-fructose diet, or standard chow was quantified and changes in body weight were measured.
The results indicate that consumption of 15% HFCS did not affect energy intake regulation, only the animals fed the HFat diet gained weight after 11 weeks of treatment.This weight difference was not solely related to dietary protein intake because protein intake was lower in the HFat diet group.The data suggest that male rats fed with a 15% HFCS solution and a standard diet after weaning can maintain a body weight similar to that of control rats.
In several studies there is controversy regarding the amount of fructose that promotes weight gain.Intake of 70% fructose, but not glucose, negatively affects weight gain in Wistar rats fed the same amount of protein 22 ; however, weight gain in female rats fed a solution of 13% HFCS ad libitum for 8 weeks has also been reported 20 .HFCS may have little effect on females at the 30 th week of life but still favor significant weight gain in males (8% HFCS) 19 .The data suggest that weight gain or loss is related to the proportion of HFCS consumed.Weight changes are not explained solely by protein intake; total glucose intake can also affect weight gain.The amount of glucose in the diet of animals consuming fructose should not be less than 23% of total energy intake 22 .In our study, the glucose proportions in the control and HFr diets were 36 and 37%, respectively, thus same glucose intake could explain the equal weight gain of the animals.
As expected, the animals that consumed HFCS had hypertriglyceridemia. De novo lipogenesis promoted by high fructose intake is a primary mechanism in the development of hepatic steatosis 23 .Several animal studies indicate that Sterol Regulatory Element-Binding Protein 1 (SREBP1c) is a major mediator of lipogenesis in the liver when high-carbohydrate diets are consumed 24,25 .In rats' liver, hypertriglyceridemia from fructose is related to activation of the transcription factor SREBP-1c, which activates AT: Adipose Tissue; mRNA: Messenger Ribonucleic Acid.
http://dx.doi.org/10.1590/1415-52732015000600003transcription of genes required for fatty acid synthesis, such as acetyl coenzyme A, carboxylase, fatty acid synthase, and stearoyl coenzyme-A desaturase-1 26 .Sirtuin 1 (SIRT1) is a deacetylase protein involved in the cellular metabolism of lipids and glucose 27 .Downregulation of SIRT1 expression resulted in a significant increase of triglyceride accumulation in 3T3-L1 cells 28 .In humans' and rats' liver cells, fatty acid oxidation is inhibited by fructose by reducing peroxisome proliferator-activated receptor alpha expression and activity, through a mechanism involving SIRT1 downregulation 29 .This evidence suggests that high triglycerides in the serum of animals fed the HFr diet is mediated by SREBP-1 and SIRT1.
Elevated triglyceride levels may inhibit the transport of leptin across the blood-brain barrier of rodents 30 , and high-fructose diets (60%) induced hepatic triglycerides synthesis 31 and hypothalamic leptin resistance 32 , so there is a relation between dietary fructose and leptin.The study animals fed the HFr or HFat diet had hypertriglyceridemia and low levels of leptin expression in the brain.These results suggest that, in the brain, the leptin gene is regulated by fat and fructose availability.Diet-induced obesity is associated with lower levels of leptin receptor expression in the hypothalamus yet high serum leptin 33 .Leptin mRNA levels in the hypothalamus, cortex, and pituitary were undetectable in fasted rats 34 , which may have had fasting-induced hypertriglyceridemia. Haring & Harris 35 described peripheral leptin resistance in rats fed fructose (40%).High fat diets also induced central leptin resistance 36 .Lower leptin gene expression by HFCS intake also may be associated with leptin resistance.
Lower leptin expression compared with controls was associated with higher FAS expression in rats' brains.Fructose intake (10%) increases the expression of the FAS gene up to six times from the seventh day of intake 29 , and fructose directly inhibits the oxidation of fatty acids in rats' livers 37 .This process is mediated by an increase in xylulose-5-phosphate, which activates protein phosphatase 2A, which affects leptin signaling, ultimately producing a fatty acid oxidation deficit in the liver 38 .These data suggest that the effect of fructose on fatty acid oxidation is mediated by leptin and that resistance to it and/or low leptin signaling may be associated with a higher level of fatty acid synthesis.

C O N C L U S I O N
In conclusion, 15% HFCS did not affect weight gain in Wistar rats, possibly because the animals in the HFCS and control groups had the same glucose intake.The two experimental diets reduced the expression of leptin in the brain.This lower expression may be associated with hepatic synthesis of fatty acids, thereby producing the hypertriglyceridemia observed in the animals.Furthermore, the 15% HFCS in the HFr diet related to lower leptin expression in adipose tissue.The metabolic basis of low leptin expression in rats fed high-fructose diets has not been determined.It would be interesting if future studies determined the effect of HFCS intake on the expression of other genes associated with the regulation of hunger and satiety.

R E F E R E N C E S
1. Corporacion Nacional de Consumidores y Usuarios.

Figure 1 .
Figure 1.Body weight gain of weaned (21 days of life) male Wistar rats fed High-Fat Diet (HFat), High-Fructose Diet (HFr), or standard diets.The results are presented as mean, standard deviation with six animals per group.Note: Differences are in relation to the standard diet, * p<0.05, ** p<0.01;one-way analysis of variance, Fisher's least significant difference post hoc test.

A
C K N O W L E D G M E N T S This study was supported by Programa del Mejoramiento del Profesorado (UAEH-EXP-090) and Consejo Nacional de Ciencia y Tecnología, Project nº 216092.C O N T R I B U T O R S G LÓPEZ-RODRÍGUEZ designed the study, performed the data analysis, and wrote the study report.SK OSUNA and T SUÁREZ DIEGUEZ conducted the experimental analysis.M GALVÁN GARCÍA contributed to the writing and editing of the manuscript.

Table 1 .
Comparison of effects of High Fructose Corn Syrup Solution (HFCS) and vegetable fat on food energy intake, liquid intake and energetic nutrient distribution in male rats.