influence of combined high-fat-high-carbohydrate … · hfhcd for 16 weeks, the control groups -...

IOSR Journal of Dental and Medical Sciences (IOSR-JDMS)

e-ISSN: 2279-0853, p-ISSN: 2279-0861.Volume 16, Issue 10 Ver. XIII (Oct. 2017), PP 01-12

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DOI: 10.9790/0853-1610130112 www.iosrjournals.org 1 | Page

Influence Of Combined High-Fat-High-Carbohydrate Diet On

Ghrelin In Female And Male Rats

Peter Ivanov Hrischev¹,*Nadya Ivanova Penkova², Katerina Nikolova

Georgieva¹, Dora Dimitrova Terzieva³, Pepa Koseva Atanassova² ¹ Department of Physiology, Faculty of Medicine, Medical University-Plovdiv, 15a Vasil Aprilov st., Plovdiv,

Bulgaria

² Department of Anatomy, Histology and Embryology,Faculty of Medicine, Medical University – Plovdiv, 15a

Vasil Aprilov st.,Plovdiv, Bulgaria

³ Department of Clinical Laboratory, Faculty of Pharmacy, Medical University – Plovdiv, 15a Vasil Aprilov st.,

Plovdiv ,Bulgaria

Corresponding Author: * Nadya Ivanova Penkova

Abstract : Ghrelin is secreted from stomach and plays a key role in appetite and glucose regulation.

Aim: The aim of our work was to study the influence of high-fat-high-carbohydrate diet (HFHCD) on ghrelin

expression in female and male rats.

Material and methods: Two-month-old Wistar rats were divided into four groups: control - female (FC) and

male (MC), and dietary-manipulated - female (FD) and male (MD). The dietary-manipulated animals were fed

HFHCD for 16 weeks, the control groups - standard rat chew. Weight, BMI and waist circumference were

measured in the course and at the end of the experiment. After the sacrifice of the animals, serum

concentrations of glucose, insulin and ghrelin were measured. The stomach of each rat was examined

immunohistochemically for ghrelin expression.

Results: FD and MD compared with the controls had higher values of body weight, BMI, waist circumferences,

glucose, insulin and HOMA-IR and developed insulin resistance. Their serum ghrelin concentrations were

lower, with gender differences – lower in FD than MD. Stronger immunohistochemical reaction was detected in

the stomach of the dietary-manipulated rats.

Conclusion: HFHCD induces changes in ghrelin expression and makes it a risk factor for the development of

obesity and metabolic syndrome.

Keywords: high-fat-carbohydrate-diet, ghrelin, rats

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Date of Submission: 04 -10-2017 Date of acceptance: 28-10-2017

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I. Introduction Ghrelin is a gastrointestinal hormone secreted mainly by gastric mucosa. [1] It is the basic peripheral

humoral signal to the central nervous system during hunger and is referred to as „hunger hormone‟. [2] Changes

in ghrelin secretion are adaptive reactions which stimulate or suppress appetite depending on the current energy

imbalance. [3] Ghrelin regulates the exocrine pancreatic function and is related to insulin secretion. [4] It

influences glucose and lipid metabolism, and energy homeostasis. [5] The various metabolic effects of ghrelin

make it a potential risk factor for atherosclerosis, hypertension, diabetes type 2, obesity. [6,7] Serum ghrelin

levels in obesity fall below normal values. [8,9]

The chief factors which regulate the secretory activity of ghrelin-producing cells and ghrelin blood

level respectively are the contents of the food. All nutrient types given orally are capable of inhibiting ghrelin

secretion.[10] The high protein content induces long-term reduction of appetite and body weight despite the

compensatory changes in the daily plasma concentrations of ghrelin. [11] In patients with diabetes type 2 a five-

week high-protein diet does not influence the ghrelin level in blood. [12] The high fructose level of food in the

form of corn syrup increases ghrelin secretion. [13] Peroral and intravenous glucose administration decreases

plasma ghrelin concentration in rodents [14,15] and man [16,17] Fat-rich diet in rats results in an increase in

body weight and a significant reduction in ghrelin levels. [18,19,20] Despite the numerous researches, molecular

mechanisms of the effect of basic nutrients on ghrelin-producing cells and ghrelin release remain unclear. In

available literature there aren‟t any studies on ghrelin expression in the course of dietary induction of obesity

and metabolic syndrome with a high-fat-high-carbohydrate diet.

Influence Of Combined High-Fat-High-Carbohydrate Diet On Ghrelin In Female And Male Rats


II. Aim The aim of our work was to study the influence of a combined high-fat-high-carbohydrate diet

(HFHCD) on the ghrelin serum levels and its stomach immunohistochemical expression in female and male rats.

III. Material And Methods In order to follow the recommendations of the National Institute of Health (NIH, 1993) to conduct

parallel investigations in both sexes, male and female animals were used in this experiment. An equal number of

female (n=16) and male (n=16) Wistar rats were used, with an initial body mass of 160-180 g. They were kept

in individual metabolic cages at 20 ± 2 oC, controlled humidity and 12:12 h light-dark period. The experiment

was approved by the Commission on Ethical Treatment of Animals of the Bulgarian Food Safety Agency (№

119, 18.06.2015). In the preparatory period all rats received a standard rat chew for two weeks while adapting to

the conditions in metabolic cages. At the end of the preparatory period, the rats were divided into four groups: 2

control groups – male (MC) and female (FC) and 2 dietary-manipulated – male (MD) and female (FD). The rats

from MD and FD groups were subjected to HFHCD and that from MC and FC received standard rat chew for 16

weeks. The energy content of the food of MC and FC groups was 2908 kcal.kg

-1 and of groups MD and FD -

4298 kcal.kg

-1. HFHCD was prepared by adding lard and sucrose to standard pellets.

Body weight of each animal was measured once a week. An hour before, the food was temporarily

deprived and returned to the metabolic cages after determining the body mass. BMI was calculated using the

formula:

BMI = body weight (g) / length (cm)

2.

Experimental animals' waist circumference was measured with a meter in its widest part once a month.

At the end of the trial, the experimental animals were decapitated under narcosis with Thiopental 30 mg.kg

-1 i.p.

and mixed blood was collected. Then it was centrifuged in order to measure the serum levels of ghrelin, glucose

and insulin in blood serum. Serum concentration of glucose was assayed by analyzer Konelab 60 i

(Thermophisher Scientific, USA). Ghrelin and insulin serum levels were measured by the sandwich ELISA

method with Ghrelin Rat ELISA Kit (BioVendor Inc.) and Rat Insulin HS ELISA Kit (BioVendor Inc.),

respectively. Doubled samples were elaborated on Sirio S microplate reader (SEAC, Italy) and the average of

the two measurements was used as a representative for each rat. The homeostatic model for insulin resistance

(HOMA-IR), based on measured insulin and fasting blood glucose, was determined. HOMA-IR was calculated

by the formula:

Glucose (mmol/L) x Insulin (mU/L) / 22.5

Stomach specimens were taken from each animal after the decapitation, fixed in 10% formalin and

after paraffin inclusion were studied immunohistochemically with a primary antibody for ghrelin (goat

polyclonal ghrelin antibody: sc-10368 - Santa Cruz Biotechnology USA) by the ABC method with ABC

Staining System (Santa Cruz Biotechnology, USA). The ghrelin antibody was diluted in PBS in 1:100 ratio.

Positive reaction for ghrelin was reported in the presence of black granules in the cell cytoplasm. The specificity

of immunohistochemical reactions was confirmed by negative controls in which the specific antibodies were

substituted with a buffer (PBS) or normal non-immune serum. There was no product of the respective reaction

in them. Observation and photo documentation of the microscopic preparations were performed with digital

photo microscopic camera of a light microscope “Olympus BX51”.

The results are expressed as means ± SEM. In order to assess the two main effects (gender and diet)

and their interaction, the results of all tests, which were repeated several times during the experiment (body

weight, BMI and waist circumference), were assessed by two-way ANOVA for repeated measures. The results

obtained only at the end of the experiment (serum concentration of ghrelin, insulin, glucose, HOMA-IR) were

analyzed with two-way ANOVA. The level of significance was set at P<0.05.

IV. Results 1. Body weight

At the beginning of the experiment the body weights of the experimental animals in the four

experimental groups were almost equal - male controls (182.13 ± 9.64 g), female controls (172.00 ± 16.49 g),

male dietary-manipulated (184.75 ± 13.66 g), female dietary-manipulated (176.25 ± 14.39 g) rats (P>0.05).

Until the 11th

week HFHCD diet had no effect on the weight of the test animals (P>0.05). From the 12th

week

until the end of the experiment the administered HFHCD had a significant main effect on the body weight, as

dietary-manipulated animals had higher weight than the controls (P<0.05). At the end of the experiment the

dietary-manipulated rats had higher weight compared with the controls (P<0.05) (Fig. 1.).



Fig. 1. Body weight (g) of dietary-manipulated and control rats from 12th

week until the end of the experiment.

*P<0.05 dietary-manipulated vs control rats.

The gender had a significant main effect on the body mass from the 2nd

week until the end of the

experiment - male animals had higher body weight than female (P<0.05) (Fig. 2.).

Fig. 2. Body weight (g) of female and male rats during the experiment (*P<0.05).

At the end of the experiment, male rats increased their body weight with 53.17%, female rats - with

44.95%. Analyzing the obtained data of changes in the body mass during the experiment, we did not detect

statistically significant two-way interaction (P>0.05).

Body Mass Index (BMI)

At the beginning of the study there was no difference in the BMI of the experimental rats - male

controls (0.45 ± 0.05 g·cm-2

), female controls (0.50 ± 0.05 g·cm-2

), male dietary-manipulated (0.46 ± 0.05 g·cm-

2), female dietary-manipulated (0.47 ± 0.04 g·cm

-2) rats (P>0.05). A significant main effect of HFHCD on BMI

was found at the end of the experiment (16th

week), as dietary-manipulated rats had higher body mass index

compared to the controls (P<0.01) (Fig. 3.).

Fig. 3. Body mass index (g·cm-2

) of dietary-manipulated and control animals in the course of the experiment.

*P<0.01 dietary-manipulated vs control rats.



At the end of the 8th

week we found a significant main effect of the gender on BMI (P<0.01). The male

rats had higher BMI than female and this effect was maintained until the end of the experiment (P<0.01) (Fig.

4.). No significant interaction between the gender and HFHCD on BMI values of the experimental animals was

found (P>0.05).

Fig. 4. Body mass index (g·cm-2

) of female and male rats in the course of the experiment. *P<0.01 male vs

female rats.

Waist circumferences

From the 12th week until the end of the experiment, HFHCD had a significant main effect on the

values of the waist circumferences of the experimental animals. At the end of the experiment dietary-

manipulated rats had larger waists compared with the controls (P<0.001) (Fig. 5.).

Fig. 5. Waist circumferences (cm) of dietary-manipulated and control animals in the course of the experiment.

*P<0.001, dietary-manipulated vs control rats.

The gender had a significant main effect on the waist circumference from the 4th

week until the end of

the experiment. At the end of the experiment the male rats had higher values than the female (P<0.001) (Fig. 6.).

No significant two-way interaction on the waist circumference values of the experimental animals was found

(P>0.05).

Fig. 6. Waist circumferences (cm) of male and female animals in the course of the experiment. *P<0.001, male

vs female rats.



Glucose

We found that HFHCD had a significant main effect on serum fasting blood sugar concentration. The

dietary-manipulated animals had higher glucose concentrations compared to the controls (10.10 ± 0.17

mmol·l־ l vs 7.53 ± 0.17 mmol·l־ ,l P<0.001). There was a significant main effect of the gender on the serum

glucose concentration. At the end of the experiment male rats had higher fasting blood glucose concentrations

compared with the female rats (8.45 ± 0.27 mmol·l־ l vs 7.53 ± 0.27 mmol·l־ ,l P<0.05). No significant

interaction between the gender and HFHCD was found on the blood glucose concentration (P> 0.05).

Insulin

A significant main effect of HFHCD on the insulin levels was found in both male and female rats.

Serum insulin concentration in dietary-manipulated animals was higher than the same in the controls (58.12 ±

17.29 mU·l־ l vs 5.76 ± 17.29 mU·l־ ,l P<0.05). No significant main effect of the gender (P>0.05) and no

significant interaction between the gender and HFHCD on insulin concentration (P>0.05) were found.

HOMA-IR

Statistical analysis of the obtained data showed presence of insulin resistance at the 16th

week of

HFHCD administration in both male and female rats. We found a significant main effect of the diet on HOMA-

IR. In the dietary-manipulated rats it was significantly higher than in the controls (30.79 ± 9.76 vs 1.82 ± 9.76,

P<0.05). No significant main effect of the gender (P> 0.05) and two-way interaction (P>0.05) on HOMA-IR

were found.

Serum ghrelin concentrations

The 16-week HFHCD administration had a significant main effect on the serum ghrelin concentration,

as the dietary-manipulated rats had lower values compared with the controls (3.18 ± 1.09 ng·ml־ˡ vs 16.18 ±

1.09 ng·ml־ˡ, P<0.001). The gender also had a significant main effect, as female rats had lower serum ghrelin

concentrations compared with male animals (7.25 ± 1.09 ng·ml־ˡ vs 12.11 ± 1.09 ng·ml־ˡ, P<0.001). We found

no significant interaction between the gender and HFHCD on the serum ghrelin concentrations of the

experimental animals (P>0.05).

Immunohistochemical expression of ghrelin in the stomach

In the control groups (MC and FC) we established presence of some ghrlein-producing cells scattered

in the body of separate fundus glands of the gastric corpus (Fig. 7. A., B.; Fig. 8. A., B.). The cells with positive

ghrelin immunohistochemical reaction were single, with conical shape. The secretory product was evenly

scattered in the cytoplasm. The intensity of the reaction was moderate. No difference was found in the ghrelin

immunohistochemical expression in male and female rats.

Fig. 7. A male rat from the control group. Single ghrelin-positive cells in the stomach fundus glands. A. Magn. х

200. B. Magn. х 400.



Fig. 8. A female rat from the control group. Single ghrelin-positive cells in the stomach fundus glands. A.

Magn. х 200. B. Magn. х 400.

In the animals fed HFHCD we established a great number of ghrelin-producing cells in extensive areas

of the gastric corpus. They were gathered into cell clusters situated in the body and base of the fundic glands.

The large number of ghrelin positive cells demonstrated strong immunohistochemical reaction. The rats from

the dietary-manipulated groups had more ghrelin positive cells with stronger reaction than that from the control

groups. No difference was found in the ghrelin immunohistochemical expression between the male (MD) (Fig.

9. A., B.) and female (FD) rats (Fig. 10. A., B.).

Fig. 9. A male rat from the HFHCD group. Large number of ghrelin positive cells with strong

immunohistochemical reaction in the stomach fundus glands. A. Magn. х 200. B. Magn. х 400.

Fig. 10. A female rat from the HFHCD group. Large number of ghrelin positive cells with strong

immunohistochemical reaction in the stomach fundus glands. A. Magn. х 200. B. Magn. х 400.



V. Discussion In our work we used HFHCD, which is a combination of high level of carbohydrates and fats

specifically selected to meet the feeding habits of the contemporary man. At the end of the experiment dietary-

manipulated rats had higher values of body weight, BMI, waist circumferences, glucose, insulin and HOMA-IR

and developed insulin resistance. Thus, by inducing obesity and metabolic disorders in both male and female

rats, we mimic what happens in humans. Our study for the first time gives an idea about the changes in ghrelin

production and expression as a result of the 16-week application of HFHCD in both genders and reveals the role

of this hormone in the pathogenesis of metabolic syndrome.

The used by us HFHCD has a negative effect on serum ghrelin level which corresponds with other

works. Previous studies were carried out by loading with only one of the nutrients. Peroral and intravenous

glucose administration decreases plasma ghrelin concentration in rodents. [14,15] The inhibitory effect of

glucose on ghrelin is also observed in man. [16] Serum ghrelin levels were decreased in 5-week-old male Wistar

rats with fructose-induced metabolic syndrome. [21] Significantly reduced plasma levels of ghrelin and

increased body weight were detected in Zucker rats after 8-weeks administration of high-fat diet. [18] Fat-rich

diet for 11-weeks resulted in an increase in body weight and a significant reduction in ghrelin levels. [19]

Decreased ghrelin levels were found in gastrointestinal tract and serum of rats with obesity induced by fat-rich

diet. [20] We found decreased serum ghrelin concentration after the application of HFHCD in both genders,

more pronounced in female rats. In available literature there is no evidence for such a gender comparison

concerning rats. As for humans, there is a report on gender differentiation of the acetylated ghrelin plasma levels

- higher in healthy women than men. [22] We can explain this dissociation in our and other authors‟ results with

the fact that they concern different species.

Our results revealed a correlation between the low serum ghrelin concentrations and the obesity

achieved as a result of the diet. The same negative correlation was proved for ghrelin levels and obesity in

people. [23] Ghrelin levels were reduced in obesity and increased after weight loss. [24] We found low ghrelin

levels to be in association with the other changes as a result of the diet like insulin resistance and abdominal

circumferences. Other works also reported reduced ghrelin levels in correlation with obesity [6,14] and insulin

resistance [25], which are components of metabolic syndrome. Significantly lower ghrelin concentration was

found in metabolic syndrome in association with fasting insulin and insulin resistance. [26] Both forms of

ghrelin were decreased in individuals with metabolic syndrome in connected with insulin resistance. [27] Strong

correlation between ghrelin levels and metabolic syndrom was reported in rats where the decreased ghrelin

levels were considered a compensatory mechanism against elevated insulin and glucose levels. [21] We also

found a negative correlation between serum ghrelin concentration and abdominal circumference which make us

accept the idea that it could predict the development of metabolic syndrome. [25]

In contrast to the serum levels of ghrelin, we established increased immunohistochemical ghrelin

expression in the gastric mucosa in the dietary-manipulated groups. Similar to our results are those concerning

obese patients. Ghrelin producing cells were detected most dense in the proximal stomach of patients with

morbid obesity. [28] More ghrelin positive cells with stronger reaction were found in the stomach fundus glands

of patients with obesity in comparison to healthy people. [29] We accept that ghrelin is involved in the

pathophysiology of obesity [30] and the development of other metabolic disorders, like hyperglycemia, insulin

resistance, dyslipidemia and metabolic syndrome as a whole. Increased ghrelin production is probably an

adaptive reaction which stimulates growth hormone secretion [31], thus interfering indirectly in the regulation of

metabolic processes. Some studies suggest that ghrelin may affect the center of hunger not only by the blood

stream, crossing the blood-brain barrier, but also by activation of intramural vagal afferent fibers. With this

mechanism, there is no need for high plasma levels of ghrelin to activate the feeling of hunger and obesity. [32]

VI. Conclusion This is the first study on the effect of HFHCD on ghrelin expression in female and male rats. HFHCD

leads to obesity and metabolic syndrome, accompanied by proliferation of a large number of ghrelin-producing

cells in the gastric glands and decreased serum ghrelin concentrations. Low blood ghrelin level is an indicator

and risk factor of metabolic syndrome.

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Funding acknowledgements: The study was funded by the Medical University of Plovdiv (Project № SDP 09/2015).



*Nadya Ivanova Penkova. "Influence Of Combined High-Fat-High-Carbohydrate Diet On Ghrelin

In Female And Male Rats ." IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) 16.10

(2017): 01-12

influence of combined high-fat-high-carbohydrate … · hfhcd for 16 weeks, the control groups -...

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