High fructose-enriched diet synergistically exacerbates endocrine but not metabolic changes in letrozole-induced polycystic ovarian syndrome in Wistar rats.

BACKGROUND: Polycystic Ovarian Syndrome (PCOS) is a multifactorial endocrine-metabolic disorder that highly contributes to the prevalence of infertility globally. The increased consumption of refined carbohydrate, particularly fructose has been associated with pandemic metabolic disorders, including in women of reproductive age. However, the effects of high fructose consumption (FRD) on endocrine and metabolic disorders associated with PCOS are not clear. Therefore, this study investigated the effects of FRD on endocrine/metabolic changes in letrozole-induced PCOS in Wistar rats.
MATERIALS AND METHODS: Twenty-eight adult female Wistar rats were randomly allotted into 4 groups and treated with vehicle, letrozole (LET; 0.5 mg/kg), FRD (D-fructose chow pellet mixture) and LET + FRD. The treatment lasted for 21days.
RESULTS: Data showed a significant increase in ovarian weight, liver weight, luteinising hormone (LH), testosterone and decrease in follicle stimulating hormone as well as moderate histopathological changes in the fallopian tube, uterus and liver of animals with PCOS. FRD-treated group showed a significant increase in ovarian weight and liver weight but no significant alteration in hormonal profile or histopathological changes in uterus and fallopian tube. However, FRD significantly altered hormonal profile with consequent histopathological changes in fallopian tube and uterus but FRD did not alter ovarian/liver weight or blood glucose in animals with PCOS when compared with animals without PCOS.
CONCLUSION: The present results demonstrate that FRD synergistically aggravates endocrine but not metabolic changes in PCOS, suggesting that FRD might deteriorate endocrine-related phenotypes in PCOS.

Introduction

Polycystic ovarian syndrome (PCOS) is a multifactorial reproductive disorder that is characterized by polycystic ovaries, endocrine and metabolic phenotypes (Witchel, 2006). Several studies have established a strong correlation between enlarged polycystic ovaries and obesity, hirsutism and amenorrhea/oligomenorrhea which are critical features of PCOS (Trofimova et al., 2017; Liu et al., 2017; Mykhalchenko et al., 2017). In addition, women with hirsutism and oligomenorrhea have been characterized with hyperplasia of theca and stromal cells, elevated testosterone and luteinising hormone, low estradiol and follicle stimulating hormone (Trofimova et al., 2017). The consequences of PCOS vary throughout a woman's lifespan and often lead to infertility and other life-threatening diseases such as cardiovascular diseases (Xita et al., 2002) and metabolic/reproductive-related syndrome (McCartney et al., 2006; Franks et al., 2006). Six-ten percent (6–10%) of women within the reproductive age are affected by PCOS (Mastorakos et al., 2006). Similarly, studies in women between the ages of 17 and 45 showed 6.6% prevalence of PCOS and ranging percent on the symptoms particularly menstrual irregularities and hirsutism (Jodie and Braunack-Mayer, 2007). Previous studies have also reported ethnic and racial variations in its prevalence and symptoms (Jakubowski, 2005).

In addition to genetic and environmental factors, sedentary lifestyle, unhealthy diets and infectious mediators contribute in large proportional to the onset of phenotypic traits in PCOS (Goodarzi, 2011). Weight gain contributes to insulin resistance, hyperinsulinemia and atherogenic lipids which are the features of metabolic dysfunction. These affect androgen level resulting in reproductive disorder, particularly anovulation (Diamanti-Kandarakis and Legro, 2006). The candidate genes which involves in PCOS affect neuroendocrine, biochemical, metabolic and reproductive pathway in women (Joseph et al., 2015). Although the etiology of PCOS is unknown, the pathogenesis of metabolic, endocrine and reproductive dysfunctions is strongly linked with PCOS (Allahbadiaa and Merchant, 2010).

Fructose is a monosaccharide found in plants such as honey, fruits, flowers, berries, sugar cane, sugar beets, corn and most root vegetables. It exists in foods as either free monosaccharide or unit of a sucrose. It is absorbed by the intestine via the GLUT 5 transporter, transmitted from the enterocytes to the blood vessel leading to the liver by the GLUT 2 transporter where it enters the process of glycolysis and is used as a substrate for gluconeogenesis, glucogenolysis and lipid synthesis (Rajasekar and Anuradha, 2007). The excessive consumption of fructose is promoted through its endemic use as sweetener on a commercial scale, resulting in lipogenesis that has been linked with metabolic syndrome as well as cardiovascular disease (György, 2018). A number of previous studies suggest that fructose consumption greater than 50 g is associated with metabolic syndrome, which is usually characterized by obesity, insulin resistance, dyslipidemia, high blood pressure, prediabetes and diabetes as well as inflammation and thrombosis. In addition, studies in animal models showed that high fructose intake led to insulin resistance, impaired glucose tolerance and high blood pressure (Celep et al. 2015; Dornas et al. 2015). Increased fructose intake leads to hyperglycemia, stimulating the pancreatic beta cells to increase insulin secretion, which results in stimulation of theca cells, increasing androgen production, which possibly manifests as hirsutism and acne (Brown et al., 2005).

Letrozole, a non-steroidal inhibitor suppresses the action of aromatase enzymes from converting adrenal androgens to estrogen. Estrogen is responsible for the maturation and growth of ovarian follicles and its deficiency impairs ovarian function. Letrozole reduces the biosynthesis of estrogen in all tissues by competitively binding to the haem of cytochrome P450 subunit of aromatase enzyme (Yager and Davidson, 2006), which reduces the negative feedback effect of estrogen at the hypothalamic pituitary axis, leading to alteration of gonadotropin secretion and follicular development. It is well documented that administration of letrozole induces PCOS in animal models (Yager and Davidson, 2006; Fauser et al., 2012).

In all these models, endocrine disturbances similar to those in human PCOS were observed but the metabolic changes of the syndrome were not well characterized (Crosignani and Nicolosi, 2001). The heterogeneity of the syndrome is reflected in many animal models of PCOS. However, few of these models have focused on the analysis of predisposing conditions that increase the risk of PCOS, particularly genetic background and environmental factors, such as endocrine disruptors and lifestyle like high fructose consumption (Strauss, 2003). Despite several attempts, PCOS is still reaching worldwide epidemic proportions and increasing epidemic metabolic-related complications. It is therefore imperative better understand the metabolic/endocrine dysfunctions associated with PCOS. This study was therefore designed to investigate the effects of high fructose consumption on metabolic/endocrine disorders associated with letrozole-induced PCOS in Wistar rats.

Materials and methods

Animals and treatment

All experimental protocols were conducted in line with the guidelines of the National Institutes of Health Guide (NIH) for the Care and Use of Laboratory Animals and it was approved by the Institutional Ethical Review Committee, Afe Babalola University, Ado-Ekiti, Nigeria. Every effort was made to minimize both the number of animals used and their suffering. Twenty-eight (28) female Wistar rats which weighed between 147 and 193 g were used for the study. Rats had unrestricted access to standard rat chow and water. They were maintained under standard environmental conditions of temperature, relative humidity, and 12-h dark/light cycle. After acclimatizing the animals for one week, rats were randomly assigned to 4 groups of n = 7/group; control, letrozole group (LET), D-fructose group (FRD) and letrozole + D-fructose group (LET + FRD).

By oral gavage control received vehicle and LET-treated group received 0.5 mg/kg of letrozole, FRD-treated group received D-fructose chow pellet mixture and LET + FRD group received 0.5 mg/kg of letrozole and D-fructose pellet. The administration lasted for 21 days.

Sample collection and handling

The animals were sacrificed by anaesthetizing with sodium pentobarbital (50 mg/kg; ip) after overnight fasting and measurement of blood glucose. Blood was collected by cardiac puncture into non-heparinised sample bottles. The blood was centrifuged at 3,000 rpm for 15 min to get clear serum samples, which were subsequently kept frozen at -20 °C until needed for biochemical assay. The liver, fallopian tube and uterus were removed, weighed and prepared for histological analysis.

Measurement of hormonal profile

The serum hormonal concentration (FSH, LH, and testosterone) were carried out using ELIZA kits obtained from Inteco Limited (UK).

Histopathological assessment of liver, fallopian tube and uterus

For hematoxylin and eosin (H & E) stains, a section of liver, fallopian tube and uterus were fixed in 10% formolsaline overnight and thereafter dehydrated, embedded in paraffin and sectioned at 5-μm thickness. The slides were prepared and examined using light microscopy.

Statistical analysis

All experimental data were analysed using Graphpad Prism 5 software and expressed as means ± S.E.M. One-way analysis of variance (ANOVA) was used for the comparison of the mean values of variables among the groups. Bonferroni's test was used to identify the significance of pairwise comparison of mean values among the groups. Statistically significant differences were accepted at p < 0.05.

Result

Effect of fructose on body weight in letrozole-induced PCOS Wistar rats

There was no significant change in body weight of PCOS animals with or without fructose-enriched diet compared with control animals (Figure 1).

Figure 1

Effects of fructose-enriched diet on body weight in letrozole-induced PCOS in Wistar rats. Data are expressed as mean ± S.E.M. n = 7 and analyzed by one-way ANOVA followed by Bonferroni post hoc test. (∗p < 0.05 vs. CTL; #p < 0.05).

Effect of fructose on ovarian and liver weight in letrozole-induced PCOS Wistar rats

Ovarian and liver weight significantly increased in PCOS animals with or without fructose-enriched diet when compared with control group. However, there was no significant change in LET + FRD group when compared with LET-treated group (Figure 2).

Figure 2

Effects of fructose-enriched diet on ovarian and liver weight in letrozole-induced PCOS in Wistar rats. Data are expressed as mean ± S.E.M. n = 7 and analyzed by one-way ANOVA followed by Bonferroni post hoc test. (∗p < 0.05 vs. CTL; #p < 0.05).

Effect of fructose on blood glucose in letrozole-induced PCOS Wistar rats

There was no significant change in blood glucose of PCOS animals with or without fructose-enriched diet compared with control animals (Figure 3).

Figure 3

Effects of fructose-enriched diet on blood glucose in letrozole-induced PCOS in Wistar rats. Data are expressed as mean ± S.E.M. n = 7 and analyzed by one-way ANOVA followed by Bonferroni post hoc test. (∗p < 0.05 vs. CTL; #p < 0.05).

Effect of fructose on hormonal profile in letrozole-induced PCOS Wistar rats

There was a significant decrease in FSH and increase in LH and testosterone in LET-treated group compared with control group. Whereas fructose-enriched diet alone did not alter hormonal profile when compared with control group. However, FSH decreased and LH and testosterone significantly increased in LET + FRD-treated group compared to the control group. In addition, fructose-enriched diet significantly increased LH and testosterone in PCOS animals compared with PCOS without fructose-enriched diet (Table 1).

Table 1

Effects of fructose-enriched diet on hormonal profile in letrozole-induced PCOS in Wistar rats.

Animal Group	FSH (μg/ml)	LH (μg/ml)	Testosterone (μg/ml)
Control	10.39±1.79	46.31±0.86	10.31±0.15
Letrozol	6.52±1.73∗	64.64±2.82∗	20.18±0.09∗
Fructose	8.22±1.76	47.01±0.60	10.11±0.08
Letrozol+ Fructose	5.72±1.83∗#	87.46±0.43∗#	35.40±0.10∗#

Data are expressed as mean ± S.E.M. n = 7 and analyzed by one-way ANOVA followed by Bonferroni post hoc test. (∗p < 0.05 vs. CTL; #p < 0.05).

Effect of fructose on the histology of liver, fallopian tube and uterus in letrozole-induced PCOS Wistar rats

The histological assessment of liver showed a section of hepatic tissue with normal architecture, central venule and sinusoids without cellular infiltration in control group, relatively normal architecture, central venules and sinusoids with mild cellular infiltration as well as focal area of fluid accumulation in LET-treated group, hepatic tissue with poor architecture, focal areas of moderate lymphocytes aggregate, there is mild perivascular infiltration, the sinusoids appear mildly dilatated with mild infiltration of inflammatory cells in FRD-treated group and normal architecture with mild disruption of central venules and sinusoids, without infiltration of inflammatory cells in LET + FRD-treated group (Figure 4).

Figure 4

Photomicrographs showing a section of hepatic tissue in control (A), LET-treated (B), FRD-treated and LET + FRD-treated groups. (Haematoxylin and Eosin Stain; x100, Longitudinal section).

Histology of fallopian tube showed a section of fallopian tube with normal slender plicae or folds resting on the normal muscularis layer, the tubular epithelial cells appear normal and there is no pathological lesion in control group, altered slender plicae or folds resting on the muscularis layer and altered tubular epithelial cells in LET-treated group, normal slender plicae or folds resting on the normal muscularis layer and tubular epithelial cells in FRD-treated group and altered slender plicae or folds resting on the muscularis layer and disrupted tubular epithelial cells in LET + FRD-treated group (Figure 5).

Figure 5

Photomicrographs showing a section of fallopian tissue in control (A), LET-treated (B), FRD-treated and LET + FRD-treated groups. (Haematoxylin and Eosin Stain; x100, Longitudinal section).

In addition, histological assessment of uterus revealed a section of uterine lining with normal endometrial layer, epithelial layer and endometrial cell glands without infiltration of inflammatory cells as well as normal myometrium in control group, mildly altered endometrial layer, epithelial layer and endometrial cell glands without infiltration of inflammatory cells and altered myometrium in LET-treated group, normal endometrial layer, myometrial layer and endometrial layer with normal epithelial lining, there are normal endometrial cell glands without infiltration of inflammatory cells in FRD-treated group and altered endometrial layer, myometrial layer and endometrial layer with normal epithelial lining, there are moderately altered endometrial cells glands with cellular infiltration in LET + FRD-treated group (Figure 6).

Figure 6

Photomicrographs showing a section of uterine tissue in control (A), LET-treated (B), FRD-treated and LET + FRD-treated groups. (Haematoxylin and Eosin Stain; x100, Longitudinal section).

Evaluation of synergy using Bliss theory

The table below showed that the administration of letrozole and fructose expressed a synergistic effect on body weight change, blood glucose liver weight and endocrine profile but not ovarian weight. Table 2.

Table 2

Interaction between letrozole and fructose administration using Bliss independence method.

Parameters	Bliss prediction	Inference
B.W change	1.06±0.75	synergistic
Blood glucose	3.32±1.08	synergistic
Ovarian weight	-2.13±0.70	antagonistic
Liver weight	0.74±0.23	synergistic
FSH	0.50±0.24	synergistic
LH	1.05±0.61	synergistic
Testosterone	0.73±0.44	synergistic

The interaction between letrozole and fructose using Bliss independence method (Bliss prediction; lnYa + lnYb-lnYab = 0, which indicates that the two agents (Ya and Yb) act independently. lnYa + lnYb-lnYab>1 indicates synergy and lnYa + lnYb-lnYab<1 indicates antagonism (Demidenko et al., 2017; Demidenko and Miller, 2019) Ya (Letrozole), Yb (Fructose), Yab (Letrozole + Fructose), B.W (Body weight), FSH (Follicle stimulating hormone), LH (Luteinising hormone).

Discussion

Polycystic ovarian syndrome (PCOS) is a complex disorder of both metabolic and endocrine origin; however, its etiology has not been fully elucidated, hence if not treated could result in infertility in women of reproductive age. The main finding of the present study is that high fructose consumption aggravates endocrine disturbances in letrozole-induced PCOS. The present data showed a significant increase in ovarian weight, liver weight, luteinising hormone (LH), testosterone and decrease in follicle stimulating hormone as well as moderate histopathological changes in the fallopian tube, uterus and liver of animals with PCOS. FRD-treated group showed a significant increase in ovarian weight and liver weight but no significant alteration in hormonal profile or histopathological changes in uterus and fallopian tube. However, FRD significantly altered hormonal profile with consequent histopathological changes in fallopian tube and uterus but FRD did not alter ovarian/liver weight or blood glucose in animals with PCOS when compared with animals without PCOS.

The present data showed no significant alteration in body weight and body glucose level in the letrozole + fructose group compared to the control group. One of the crucial effects of obesity is the development of insulin resistance (IR), which the prevalence is rapidly increasing worldwide. Insulin resistance is a central feature of PCOS and is additionally associated with the components of metabolic syndrome, such as hypertension, cardiomyopathy, dyslipidemia, type 2 diabetes and other cardiovascular events. Anovulatory women with PCOS are relatively more insulin resistant and hyperinsulinemic than weight-matched control subjects (Legro et al. 2004). Earlier studies reported that 50–70% of PCOS individuals have some degree of insulin resistance, and this hormone insensitivity possibly contributes to the hyperandrogenism that characterized PCOS (Vignesh and Mohan, 2007). Similarly, study by Dogan and Guleki showed that the association between increased insulin resistance (IR) and PCOS is a consistent finding in all ethnic groups (Dogan and Gulekli, 2006).

Previous studies showed that the prevalence of metabolic syndrome in PCOS women is nearly 2-fold higher than in age-matched women in the general population. Likewise, Apridonidze et al., reported higher significant BMI values with hirsutism and Acanthosis nigricans in women with metabolic syndrome than metabolically disrupted women with normal BMI (Apridonidze et al., 2005). The present results in addition showed a significant decrease in FSH and increase in LH and testosterone in LET-treated group compared with control group. Whereas fructose-enriched diet alone did not alter hormonal profile when compared with control group. However, FSH decreased and LH and testosterone significantly increased in LET + FRD-treated group compared to the control group. Besides, fructose-enriched diet significantly increased LH and testosterone in PCOS animals compared with PCOS without fructose-enriched diet. In PCOS, decreased sensitivity of the GnRH to feedback inhibition by ovarian steroids results in a rapid GnRH pulse frequency and perturbations in gonadotropin secretion, especially LH hypersecretion, a hallmark of PCOS disorder (McCartney et al., 2006) and a cause of hyperandrogenism (Abdallah and Johnny, 2006).

Hypersecretion of Luteinizing hormone has been positively correlated with elevated serum testosterone concentration, a critical feature of PCOS (Barontini et al., 2001) and high endogenous LH levels might detrimentally affect oocyte maturation/fertilization and pregnancy (Allahbadiaa and Merchant, 2010). Hyperandrogenism in PCOS might possibly be caused by the increased synthesis of testosterone precursors owing to a dysregulation of theca cell androgen production intrinsic to the ovary, and aberrant P450c17a, the rate-limiting enzyme that regulates androgen (Allahbadiaa and Merchant, 2010). This may be the primary factor that is responsible for enhanced testosterone secretion in PCOS with a phenotypic manifestation of hirsutism and acne in genetically predisposed individuals (Allahbadiaa and Merchant, 2010). The present study showed a significant decrease in the FSH level in the letrozole and the LET + FRD group when compared with control, which is possibly due to an increase in GnRH pulse frequency with a resultant decrease in FSH production, causing defective follicular growth with multiple small follicles. The ovarian and liver weight significantly increased in PCOS animals with or without fructose-enriched diet when compared with control group. However, there was no significant change in LET + FRD group when compared with LET-treated group.

The histological assessment of liver also showed a relatively normal architecture, central venules and sinusoids with mild cellular infiltration as well as focal area of fluid accumulation in LET-treated group compared to section of hepatic tissue with normal architecture, central venule and sinusoids without cellular infiltration in control group. Likewise, hepatic tissue with poor architecture, focal areas of moderate lymphocytes aggregate and mild perivascular infiltration, sinusoids appear mildly dilatated with mild infiltration of inflammatory cells in FRD-treated group. Extreme increase in the intake of fructose (up to 80 g/day) moderately reduces the sensitivity of the liver to insulin, but this does not affect blood sugar levels, it increases fat accumulation in the liver and, as a consequence, the insulin resistance of liver cells (Genet, 2016; Busko, 2015). Several experiments on rodents demonstrated insulin resistance and weight gain caused by long-term consumption of significant amounts, and similar consequences of drinks sweetened with fructose and large amounts of dietary fructose (Celep et al., 2015). However, a normal hepatic tissue architecture with mild disruption of central venules and sinusoids, without infiltration of inflammatory cells was observed in LET + FRD-treated group.

There was a significant increase in the ovarian weight of the LET + FRD group compared with control. When compared to control group, ovaries from LET + FRD group showed high incidence of subscapular ovarian cyst and capsular thickening together with incomplete luteinisation and decreased number of corpora luteal. Women with PCOS frequently present with reproductive dysfunction. Ovarian function might be disturbed, with resultant abnormal folliculogenesis and steroidogenesis (Van der Spuy and Dyer, 2004). Perturbations in gonadotropin secretion in PCOS, such as decreased FSH levels and LH hypersecretion, owing to a dysregulation of the GnRH pulse regulator, may result in abnormal follicular dynamics culminating in anovulatory infertility Barnes (1998); Franks et al. (2000). In addition, histology of fallopian tube showed a section of fallopian tube with normal slender plicae or folds resting on the normal muscularis layer, the tubular epithelial cells appear normal and there was no pathological lesion in control group, altered slender plicae or folds resting on the muscularis layer and altered tubular epithelial cells in LET-treated group, normal slender plicae or folds resting on the normal muscularis layer and tubular epithelial cells in FRD-treated group and altered slender plicae or folds resting on the muscularis layer and disrupted tubular epithelial cells in LET + FRD-treated group.

Furthermore, histological analysis of uterus revealed a section of uterine lining with normal endometrial layer, epithelial layer and endometrial cell glands without infiltration of inflammatory cells as well as normal myometrium in control group, mildly altered endometrial layer, epithelial layer and endometrial cell glands without infiltration of inflammatory cells and altered myometrium in LET-treated group, normal endometrial layer, myometrial layer and endometrial layer with normal epithelial lining, there are normal endometrial cell glands without infiltration of inflammatory cells in FRD-treated group and altered endometrial layer, myometrial layer and endometrial layer with normal epithelial lining, there are moderately altered endometrial cells glands with cellular infiltration in LET + FRD-treated group. Previous studies have suggested that LH and insulin hypersecretion probably play a secondary role in PCOS by exacerbating the preexisting ovarian dysregulation (Jodie and Braunack-Mayer, 2007). LH hypersecretion may result in follicular growth arrest either directly by causing premature granulosa cell maturation and luteinization or indirectly by LH-induced hyperandrogenism.

Conclusion

The present result demonstrates that FRD synergistically aggravates endocrine but not metabolic changes in PCOS, suggesting that FRD might deteriorate endocrine-related phenotypes in PCOS.

Declarations

Author contribution statement

C.O. Akintayo, A.D. Johnson: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials and analysis tools or data.

K.S. Olaniyi, O.C. Badejogbin: Analyzed and interpreted the data; Contributed reagents, materials and analysis tools or data; Wrote the paper.

A.B. Kayode, O.I. Adeyomoye, A.O. Ojewale, I.O. Ajadi, A.A. Oniyide: Analyzed and interpreted the data; Contributed reagents, materials and analysis tools or data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.