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Journal of Diabetes and Metabolic Disorders logoLink to Journal of Diabetes and Metabolic Disorders
. 2021 Oct 21;20(2):1685–1696. doi: 10.1007/s40200-021-00924-x

Acetate supplementation restores testicular function by modulating Nrf2/PPAR-γ in high fat diet-induced obesity in Wistar rats

Kehinde S Olaniyi 1,2,✉, Christopher O Akintayo 1, Adesola A Oniyide 1, Adams O Omoaghe 1, Mosunmola B Oyeleke 1, Adedamola A Fafure 2
PMCID: PMC8630149  PMID: 34900819

Abstract

Purpose

Several studies have established impaired testicular function in obese male population, including the young males with childhood obesity, contributing to increased male infertility, which is a universal trend in the last few decades. Short chain fatty acids (SCFAs) have been recently demonstrated to inhibit progression to metabolic comorbidities. The present study therefore hypothesized that SCFAs, acetate attenuates testicular dysfunction in high fat diet (HFD)-induced obese rat model, possibly by modulating Nrf2/PPAR-γ.

Methods

Adult male Wistar rats weighing 160–190 g were randomly allotted into three groups (n = 6/group): The groups received vehicle (distilled water), 40% HFD and sodium acetate (200 mg/kg) plus 40% HFD respectively. The administration lasted for 12 weeks.

Results

HFD caused obesity, which is characterized with increased body weight and visceral adiposity and insulin resistance/hyperinsulinemia. In addition, it increased testicular lipid deposition, malondialdehyde, pro-inflammatory mediators, lactate/pyruvate ratio, γ-Glutamyl transferase, and circulating leptin as well as decreased testicular glutathione, nitric oxide, Nrf2, PPAR-γ and circulating follicle stimulating hormone and testosterone without a significant change in testicular lactate dehydrogenase, blood glucose and luteinizing hormone when compared to the control group. Nevertheless, administration of acetate reversed the HFD-induced alterations.

Conclusion

The present results demonstrates that HFD causes obesity-driven testicular dysfunction, associated with testicular lipid deposition, oxidative stress, and inflammation. The study in addition suggests the restoration of testicular function in obese animals by acetate, an effect that is accompanied by elevated Nrf2/PPAR-γ.

Keywords: Acetate; Inflammation; Lipid, Nrf2; Obesity; PPAR- γ

Introduction

Despite the awareness and strategies for prevention and management of obesity, the rising prevalence of obesity represents an important public health issue [1]. This exponential increase is attributable to accelerated nutritional transition and globalization, which have involved developing countries [2, 3]. Epidemiological report showed that obesity affects over 650 million of adult population globally, and over 340 million children and adolescents are overweight or obese worldwide [1]. With persistence in this secular trend, it is estimated that over 20% of the world’s adult population would be obese by 2030 [4, 5], supporting the possible transition of obesity from epidemic to pandemic proportions. It is generally characterized by excessive fat mass with or without insulin resistance, hyperglycemia and overweight [6–8]. Several studies have associated the risk of developing common chronic diseases such as diabetes mellitus, hypertension, heart disease, stroke, neurodegeneration, reproductive dysfunction and certain cancers with overweight and obesity in both men and women [4, 9, 10]. Similarly, accumulating evidence shows that male obesity impacts negatively on male reproductive potential not only reducing sperm quality, but in particular altering the physical and molecular structure of testicular cells, including Leydig and Germ cells with subsequent disruption of steroidogenic and spermatogenic functions as well as eventual reduction in male fertility [11–13].

In addition, obesity has been well associated with multiple adverse reproductive outcomes, including testicular dysfunction and impaired fertility [14–16], but the mechanisms involved are largely under elucidation. Although previous studies have demonstrated the influence of lipotoxity on reproductive tissues in obesity or metabolic-related syndrome [17–19]. This process is characterized by increased lipolysis that triggers the excessive circulation of long-chain saturated fatty acids which are produced by adipocytes, and when the adipocytes can no longer store these fatty acids, other non-adipose cell types, including testicular cells begin to store fat, leading to increased production of reactive oxygen species (ROS) and subsequent mitochondrial dysfunction and endoplasmic reticulum stress as well as cell death [18, 20]. Similarly, adipokines are signaling molecules produced by adipose cells and their production vary with adipose mass. Dysregulation of adipokines such as leptin, tumor necrosis factor-alpha (TNFα), interleukin 6 (IL-6) among others has been demonstrated in models of obesity, causing inflammation and abnormal cell signaling with consequent cellular metabolic dysfunction [21–23]. This has been recently validated with a report that hyperleptinemia induced by diet in obese mouse model causes central leptin resistance and hypogonadism with corresponding reduction in testicular function [24]. However, further investigation of the pathogenic link between obesity and testicular dysfunction might unravel a better therapeutic target.

Moreover, fat oxidation in the mitochondria yields abundant ROS and free radicals, which normally stimulate cellular antioxidants response to prevent oxidative damage [25]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays regulatory role in the production of antioxidant proteins. Nuclear factor erythroid 2-related factor 2 is mainly localized in the cytoplasm but is translocated to the nucleus upon exposure to oxidative and inflammatory signals, where it binds with enhancer, antioxidant response element (ARE), which in turn mediates the activation of genes coding for antioxidant proteins especially the glutathione (GSH) [26]. Beside its role in ROS clearance, Nrf2 was also proposed to play a second role in regulating lipid metabolism [27] Similarly, peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors crucial in regulating many physiological processes such as energy homeostasis, glucose/lipid metabolism, inflammation, as well as cell proliferation and differentiation [28]. Peroxisome proliferator-activated receptors isoforms, particularly PPAR-γ are well expressed in reproductive tissues, including testicular cells such as Leydig cells, Sertoli cells and germ cells, where lipid metabolism, specifically β-oxidation of fatty acids are important for testicular functions and in addition plays essential role in spermatogenesis [29, 30]. Earlier studies have associated defects in PPAR-γ with metabolic-related syndrome [31, 32]. However, PPAR-γ agonist, pioglitazone has been shown to improve testicular function and semen quality in diabetic rabbits [33].

Nevertheless, recent study also demonstrated that Obesity alters testicular microbiota composition in Zebrafish, causing testicular inflammation/dysfunction with consequent decrease in sperm quality [34]. Therefore, supplementation with SCFAs, particularly acetate might be a potential intervention for obesity-associated testicular dysfunction. Besides, acetate, the most abundant form of SCFAs produced by fiber-fermenting bacteria in the distal gut are associated with improved metabolic profile by exerting antioxidant, anti-inflammatory and glucoregulatory effects [35–37]. Our recent study also showed that acetate improves hepatic lipid dysregulation and its accompanied injury in diabetic rodents via suppression of histone deacetylase [38]. Therefore, the present study attempted to test the hypothesis that acetate attenuates testicular dysfunction associated with high fat diet (HFD)-induced obese rat model, possibly by modulating Nrf2/PPAR-γ.

Materials and methods

Ethical approval and experimental design

This study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ethical approval was obtained from the institutional Ethical Review Board of Afe Babalola University, Ado-Ekiti, Nigeria (ABUADERC/07/2020). Effort was made to minimize the number of animals used and their suffering. Twenty-four male Wistar rats weighing 160–190 g were obtained in bred from the institution and the animals were giving access to water and standard rat chow ad libitum. After acclimatizing the animals for two weeks, they were randomly divided into three groups with n = 6 per group. The groups include Control (CTL), HFD, and acetate-treated (SACE) plus HFD groups. The maintenance of animals was ensured under standard environmental conditions of 22-260C temperature, 50–60% relative humidity and 12-h dark/light cycle. The experimental induction of obesity was performed by exposing the animals to 40% HFD ad libitum as previously described [39] and this exposure lasted for 12 weeks.

Chemicals and reagents

All chemicals and reagents used were of analytical grade and they include: sodium acetate (Sigma-Aldrich, St Louis, MI), sodium pentobarbital (Apro Pharmacy, Ado-Ekiti, Nigeria) and assay kits/reagents (Calbiotech Inc; Cordell Ct., El Cajon, CA 92,020, USA, Elabscience Biotechnology Inc; Wuhan, Hubei, P.R.C., China; Fortress Diagnostics Ltd; Antrim, UK, Randox Laboratory Ltd; Co. Antrim, UK and Oxford Biomedical Research Inc; Oxford, USA).

Treatment

Control group received vehicle (Distilled water) by oral gavage, HFD group received 40% HFD and HFD + SACE-treated group received 40% HFD plus 200 mg/kg body weight of sodium acetate (oral gavage). The administration was carried out for 12 weeks. Initial and final body weights were monitored, and the percentage body weight gain was determined.

Fasting blood glucose and insulin resistance

After the last day of administration, the fasting blood glucose was determined with a hand-held glucometer (ONETOUCH®-LifeScan, Inc., Milpitas, CA, USA) following 12-h overnight fasting of the animals. Insulin resistance was determined using the homeostatic model assessment for insulin resistance (HOMA-IR) = fasting glucose (mmol/l) * fasting insulin (µU/l)/22.5) as previously described [38].

Animal sacrifice and sample preparation

The animals were anesthetized by intraperitoneal injection of 50 mg/kg b.w. of sodium pentobarbital. Cardiac puncture was used for the collection of blood into the heparinized tube and blood was centrifuged at room temperature for 5 min at 3000 rpm. Plasma was decanted and stored frozen until when it was needed for the biochemical analysis. In addition, testicular tissue homogenates were prepared after weighing the testes, 100 mg section of each tissue was carefully removed and homogenized in phosphate buffer solution with a glass homogenizer and centrifuged at 10,000 rpm for 10 min at 4 °C. The homogenates were decanted and stored frozen until when it was needed for biochemical assays.

Determination of visceral adiposity and testicular weight

After dissection, visceral fat mass was determined as previously described [40, 41]. In addition, testicular weight was determined by weighing the testes and corrected to tibial length to eliminate variability.

Measurement of biochemical parameters

Plasma insulin

Insulin Rat ELISA kits obtained from Calbiotech Inc. (Cordell Ct., El Cajon, CA 92,020, USA) was used to determine the plasma insulin concentration. The method is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule.

Tumor necrosis- α and interleukin-6 concentration

The concentration of TNF-α and IL-6 was determined from the testicular tissue homogenates by quantitative standard sandwich ELISA technique using monoclonal antibody specific for these parameters with Rat kits obtained from Elabscience Biotechnology Inc. (Wuhan, Hubei, P.R.C., China).

Nuclear factor erythroid 2-related factor 2 and peroxisome proliferator-activated receptor-γ

The testicular tissue concentration of Nrf2 and PPAR-γ was determined with Rat ELISA kits obtained from Elabscience Biotechnology Inc. (Wuhan, Hubei, P.R.C., China) and in compliance with the manufacturer’s assay procedures.

Endocrine profile

Plasma level of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone was determined using Rat ELIZA kits from Calbiotech Inc. (El Cajon, USA), while plasma leptin concentration was determined with Rat ELISA kit obtained from Elabscience Biotechnology Inc. (Wuhan, Hubei, P.R.C., China).

Lipid profile

Testicular concentration of triglycerides (TG), total cholesterol (TC) and high-density lipoprotein (HDLc) was determined by standardized colorimetric methods using assay kits obtained from Fortress Diagnostics Ltd. (Antrim, UK).

Malondialdehyde (MDA) and glutathione (GSH)

Malondialdehyde was determined from the testicular tissue homogenates by standard non-enzymatic spectrophotometric method using assay kits from Randox Laboratory Ltd. (Co. Antrim, UK). This method involves the reaction of MDA in the sample with thiobarbituric acid (TBA) to generate a MDA-TBA adduct, which was quantified spectrophotometrically. In addition, GSH was determined using a non-enzymatic spectrophotometric method with assay kits from Oxford Biomedical Research Inc. (Oxford, USA).

Lactate, pyruvate and lactate dehydrogenase (LDH) activity

The testicular concentration of lactate was determined by standardized non-enzymatic colorimetric method using assay kit obtained from Randox Laboratory Ltd. (Co. Antrim, UK). Whereas testicular activity of LDH was measured by standardized enzymatic colorimetric method using assay kit obtained from Randox Laboratory Ltd. (Co. Antrim, UK). Pyruvate concentration was estimated by non-enzymatic colorimetric method using assay kits from Randox Laboratory Ltd. (Co. Antrim, UK). Besides, ratio of lactate to pyruvate was also estimated.

Nitric oxide concentration and γ-Glutamyl transferase activity (GGT)

Testicular concentration of nitric oxide was assayed spectrophotometrically by measuring the accumulation of its stable degradation products, nitrate and nitrite using kits from Oxford Biomedical Research Inc., (Oxford, UK). This kit employs Griess reagent, thus providing for accurate determination of total NO production. Whereas testicular activity of GGT was quantified by standardized enzymatic colorimetric method using assay kit obtained from Randox Laboratory Ltd. (Co. Antrim, UK).

Data and statistical analysis

Shapiro–Wilk test was used to determine the distribution of the data and all data were normally distributed. Data are presented as means ± SD. Statistical group analysis was performed using the Graphpad prism 5. One-way ANOVA was used to compare the mean values of variables and post hoc analysis was performed with Bonferroni’s test. Statistically significance was considered at p < 0.05.

Results

Impact of acetate on body weight, visceral adiposity and testicular weight in male Wistar rats fed with high fat diet

High fat diet significantly increased (p < 0.05) body weight and visceral adiposity but not testicular weight when compared with control group. However, treatment with acetate significantly reduced (p < 0.05) these alterations (body weight and visceral adiposity) in HFD + SACE group compared with untreated HFD group (Fig. 1).

Fig. 1.

Fig. 1

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Impact of acetate on body weight (a), visceral adiposity (b) and testicular weight (c) in Wistar rats fed with high fat diet. HFD increases body weight and visceral adiposity, which are reversed by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE) and High fat diet (HFD)

Impact of acetate on metabolic indices in male Wistar rats fed with high fat diet

There was a significant increase (p < 0.05) in circulating insulin concentration in animals fed with high fat diet compared with control group, which was attenuated when treated with acetate in HFD + SACE group. In addition, insulin resistance was demonstrated in animals fed with high fat diet with significant increase in HOMA-IR compared to the control group. However, treatment with acetate reversed insulin resistance with significant reduction (p < 0.05) in HOMA-IR in HFD + SACE group compared with untreated HFD group. Nevertheless, blood glucose level did not change in animals fed with high fat diet compared to the control group (Fig. 2).

Fig. 2.

Fig. 2

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Impact of acetate on fasting blood glucose (a), fasting insulin (b) and HOMA-IR (c) in Wistar rats fed with high fat diet. HFD increases fasting insulin and causes insulin resistance, which are attenuated when treated with acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD) and Homeostatic model of assessment of insulin resistance (HOMA-IR)

Impact of acetate on testicular lipid profile in male Wistar rats fed with high fat diet

There was a significant increase (p < 0.05) in testicular TG and TC with corresponding decrease in HDLc in animals fed with high fat diet compared to the control group, resulting in testicular lipid accumulation, which was attenuated when treated with acetate in HFD + SACE group compared with untreated HFD group (Fig. 3).

Fig. 3.

Fig. 3

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Impact of acetate on testicular triglyceride (a), total cholesterol (b) and high-density lipoprotein cholesterol (c) in Wistar rats fed with high fat diet. HFD increases TG, TC and decreases HDLc, which are attenuated by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD), High-density lipoprotein cholesterol (HDLc); Triglyceride (TG) and Total cholesterol (TC)

Impact of acetate on testicular lipid peroxidation and redox status in male Wistar rats fed with high fat diet

High fat diet significantly increased (p < 0.05) testicular MDA with a significant decrease in GSH and nitric oxide concentration compared to the control group. However, treatment with acetate reduced the testicular MDA with corresponding increase in GSH and nitric oxide in HFD + SACE group compared with untreated HFD group (Fig. 4).

Fig. 4.

Fig. 4

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Impact of acetate on testicular malondialdehyde (a), glutathione (b) and nitric oxide (c) in Wistar rats fed with high fat diet. HFD increases MDA and decreases glutathione and nitric oxide, which are attenuated by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD); Malondialdehyde (MDA)

Impact of acetate on testicular Nrf2, TNF-α and IL-6 in male Wistar rats fed with high fat diet

There was a significant decrease (p < 0.05) in testicular Nrf2 with corresponding increase in TNF-α and IL-6 in animals fed with high fat diet compared to the control group. Nonetheless, treatment with acetate significantly increased Nrf2 and decreased TNF-α and IL-6 in HFD + SACE group compared with untreated HFD group (Fig. 5).

Fig. 5.

Fig. 5

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Impact of acetate on testicular nuclear factor erythroid 2–related factor 2 (a), TNF-α (b) and IL-6 (c) in Wistar rats fed with high fat diet. HFD decreases Nrf2 and increases TNF-α and IL-6, which are attenuated by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD); Nuclear factor erythroid 2–related factor 2 (Nrf2); Tumor necrosis factor- α (TNF- α) and Interleukin-6 (IL-6)

Impact of acetate on testicular tissue injury markers in male Wistar rats fed with high fat diet

High fat diet significantly increased (p < 0.05) testicular lactate/pyruvate ratio and GGT activity but not LDH activity compared to the control group which were attenuated when treated with acetate in HFD + SACE group compared with untreated HFD group (Fig. 6).

Fig. 6.

Fig. 6

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Impact of acetate on testicular lactate/pyruvate ratio (a), LDH activity (b) and GGT activity (c) in Wistar rats fed with high fat diet. HFD increases lactate/pyruvate ratio and GGT activity but not LDH activity and the alterations are reversed by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD); Lactate dehydrogenase (LDH) and γ-Glutamyl transferase (GGT)

Impact of acetate on circulating endocrine profile in male Wistar rats fed with high fat diet

There was a significant decrease (p < 0.05) in circulating FSH and testosterone but not LH in animals fed with high fat diet compared to the control group. However, treatment with acetate significantly increased the circulating levels of FSH and testosterone in HFD + SACE group compared with untreated HFD group (Fig. 7). In addition, plasma leptin concentration significantly increased (p < 0.05) in the group fed with high fat diet and this increase was reversed when treated with acetate as shown in HFD + SACE group compared with untreated HFD group (Fig. 8a).

Fig. 7.

Fig. 7

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Impact of acetate on circulating follicle stimulating hormone (a), Luteinizing hormone (b) and testosterone (c) in Wistar rats fed with high fat diet. HFD decreases FSH and testosterone but not LH and the alterations are attenuated by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD); Follicle stimulating hormone (FSH) and Luteinizing hormone (LH)

Fig. 8.

Fig. 8

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Impact of acetate on circulating leptin (a) and PPAR-γ (b) and testicular PPAR-γ (c) in Wistar rats fed with high fat diet. HFD increases leptin and decreases PPAR-γ, which are attenuated by acetate. Values are represented as mean ± SD. n = 6. (*p < 0.05 vs. CTL; #p < 0.05 vs. HFD). Control (CTL); Sodium acetate (SACE); High fat diet (HFD) and peroxisome proliferator- activated receptor- γ (PPAR-γ)

Impact of acetate on circulating and testicular level of PPAR-γ in male Wistar rats fed with high fat diet

There was a significant decrease (p < 0.05) in the circulating and testicular level of PPAR-γ in animals fed with high fat diet when compared to the control group, and these were attenuated when treated with acetate in HFD + SACE group compared with untreated HFD group (Fig. 8b & c).

Discussion

The key finding from the present study is that SCFAs, acetate ameliorates testicular dysfunction associated with HFD-induced obesity by attenuating excessive testicular lipid deposition, oxidative stress and inflammation, a beneficial effect that is associated with Nrf2/PPAR-γ modulation. The results of the study demonstrate that HFD increased body weight and visceral adiposity and induced insulin resistance/hyperinsulinemia with corresponding increase in testicular lipid (TG and TC)/lipid peroxidation, pro-inflammatory mediators (TNF-α and IL-6), injury biomarkers (GGT and lactate) and leptin concentration. Besides, HFD also decreased testicular antioxidant defense (GSH) and nitric oxide as well as circulating FSH and testosterone with corresponding suppression of Nrf2/PPAR-γ when compared with control group. However, administration of acetate attenuated these alterations in HFD fed animals compared to the untreated HFD group (Fig. 9).

Fig. 9.

Fig. 9

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Schematic diagram showing the possible effects of acetate and the involvement of Nrf2/PPAR-γ in obesity-associated testicular dysfunction. HFD (High fat diet); Sodium acetate (SACE); Peroxisome proliferator-activated receptor-γ (PPAR-γ); Nuclear factor erythroid 2–related factor 2 (Nrf2); Testicular oxidative stress (TOS) and Insulin resistance (IR)

Accumulating evidence exists that HFD causes obesity in humans and experimental animals [24, 39, 42], which was validated with the results obtained from the present study that revealed increased body weight and visceral adiposity with insulin resistance and hyperinsulinemia, these are well documented features of obesity, indicating that animals fed with HFD were obese regardless of the blood glucose level when compared to the control group. Insulin resistance which is known to be triggered by increased adiposity [8] inhibits adipose function, thus causing lipolysis that results to excess lipid influx in the non-adipose tissue, including testicular tissue as shown by elevated testicular TG, TC and reduced HDLc in obese animals compared to the control group. The present observations show consistency with earlier studies which reported that obesity often leads to ectopic fat accumulation [9, 10, 16] and as extension the present results revealed elevated testicular lipid. The lack of significance difference in blood glucose of obese animals compared to the control animals might be due to compensatory hyperinsulinemia [43], though beneficial to prevent hyperglycemia but known to alter spermatogenesis and usually characterized with higher percentage of sperm with poor DNA chromatin packaging and DNA fragmentation and increased reactive oxygen species in metabolic-related syndrome, including obesity [44, 45].

In addition, excess testicular lipid did not increase testicular weight in obese animals compared to the control group but possibly caused lipotoxicity that increased lipid peroxidation as revealed by elevated MDA and correspondently decreased antioxidant defense (GSH), resulting in testicular oxidative stress. This promotes testicular injury that is biochemically expressed with elevated GGT activity in obese animals compared to the control group. Therefore, the findings that obesity causes testicular oxidative stress with corresponding injury are consistent with previous studies [18, 20]. Besides, the present study showed increase in testicular pro-inflammatory mediators (TNF-α and IL-6) in obese group compared to the control group, which might result from the excessive visceral fat mass, aggravating testicular oxidative stress. This is known to cause cellular metabolic dysfunction as previously reported [21, 22], and revealed by elevated lactate/pyruvate ratio, though without significantly altering LDH activity in obese animals compared to control group in the present study. Lactate/pyruvate ratio has been earlier described as a marker of cytosolic or cellular redox status [46], and its elevation in the testicular tissue is a reflection that the tissue energy requirement outweighed the supply, contributing to ischemic testicular tissue injury. Decrease in testicular nitric oxide in obese animals compared to the control group might also contribute to elevated cellular redox status. All together promoted testicular dysfunction with corresponding decrease in circulating FSH and testosterone without significant alteration in LH in obese animals compared to the control group. These observations are consistent with previous results that demonstrated low level of FSH and testosterone with impaired spermatogenesis in obese rodents and humans [20, 47] as well as other studies, who reported normal level of LH in moderately obese males and attributable to partial suppression of pituitary-gonadotropin release or mild elevated estradiol level, which affects LH pulsatility and bioactivity but not plasma level [48, 49]. In addition, the present result also demonstrated high level of leptin in obese animals compared with control group, which is usually induced by increased visceral fat mass and leptin resistance or reduced leptin clearance and these are critical features of obesity. Besides, hyperleptinemia is known to cause hypogonadism with corresponding testicular disorders [24]. Therefore, the findings herein suggest that obesity causes testicular dysfunction, which is accompanied by testicular oxidative stress and inflammation as well as cellular metabolic dysfunction.

Noteworthily, there was a significant decrease in testicular Nrf2 with corresponding reduction in the circulating and testicular level of PPAR-γ in obese animals compared to the control group. Both correlated to elevated testicular lipid, pro-inflammatory mediators, lipid peroxidation and defective antioxidant defense that characterized the animals fed with HFD (obese animals). Activation of transcription factor Nrf2 has been reported to regulate the redox status and protect against cellular oxidative stress and inflammation (Huang et al. 2010), and its deficiency has been demonstrated to increase body weight and low-density lipoprotein as well as metabolic abnormalities in ovariectomized rats [50]. Similarly, decreased level of Nrf2 has been previously shown to favor oxidative stress and complications found in metabolic-related syndrome, including in obesity, diabetes and atherosclerosis [51]. Recently, shawky et al., reported that activation of Nrf2 attenuates leptin-induced vascular complications associated with obesity [52]. Therefore, the present results that showed a decrease in testicular Nrf2 with corresponding increase in oxidative stress and pro-inflammatory mediators as well as subsequent testicular dysfunction suggests at least in part the critical role of Nrf2 in testicular redox imbalance associated with obese animals. In addition, PPAR-γ level has been earlier shown to be suppressed in metabolic-related syndrome, contributing to lipid/glucose dysmetabolism [31, 32]. The animals fed with HFD demonstrated a decrease in circulating and testicular PPAR-γ, which contributed to impaired spermatogenesis as earlier documented by Gumieniczek et al. in diabetic rabbits [33], indicating the impact of defective PPAR- γ in the progression of testicular dysfunction in obese animals. Therefore, a potent agonist of Nrf2 and PPAR-γ would possibly be an effective therapeutic agent in obesity-associated testicular dysfunction.

Interestingly, supplementation with acetate increased testicular Nrf2 and circulating/testicular PPAR-γ with corresponding decrease in testicular lipid, pro-inflammatory mediators, and lipid peroxidation as well as enhancement of antioxidant defense through increased GSH production in HFD + SACE group compared to untreated HFD group. In addition, acetate significantly decreased the body weight, visceral fat mass, insulin level, insulin resistance and leptin concentration in HFD + SACE group compared to untreated HFD group. Taken together these effects led to attenuation of testicular injury as revealed by decreased GGT activity and lactate/pyruvate ratio with subsequent increase in circulating FSH and testosterone that possibly restored testicular function in HFD + SACE group compared to untreated HFD group. The observation that increase in testicular Nrf2 possibly contributed to decreased testicular oxidative stress/inflammation in HFD + SACE group appears similar to a recent study by Sampath et al., who demonstrated that epigallocatechin 3-gallate-induced activation of Nrf2 in adipose tissue of obese mice improves lipidemic control, decreases oxidative products generation, and reduces body mass as well as insulin level [53]. Similarly, pharmacological activators of PPARs have been shown to lower lipid dysmetabolism and restore energy homeostasis in several disease models, including metabolic disorders [54–56], showing consistency of the present finding that acetate increases circulating/testicular PPAR-γ with consequent attenuation of testicular lipid deposition and oxidative stress/inflammation in HFD + SACE group compared to untreated HFD group. Besides, a number of previous studies, including recent study from our laboratory has demonstrated the beneficial effects of acetate in metabolic disorders and its comorbidities and associate these effects to inhibition of HDACs, antioxidant, anti-inflammatory and insulin sensitizing potentials [35–38]. Therefore, to the best of our knowledge the present study is the first to report the restoration of testicular function in obese animal model by acetate supplementation and this effect is accompanied by elevated testicular levels of Nrf2/PPAR-γ. This study in addition provides insight and justification for future investigation of molecular mechanism and perhaps, clinical relevance for diagnosis and management of infertility associated with testicular dysfunction in obese male individuals.

Conclusion

Taken together, the present results demonstrates that HFD causes obesity-driven testicular dysfunction, accompanied by testicular lipid deposition, oxidative stress, and inflammation as well as cellular metabolic dysfunction. The study in addition suggests that acetate restores testicular function by attenuating testicular oxidative stress and inflammation in obese animals, an effect that is accompanied by elevated Nrf2/PPAR-γ. Therefore, incorporating high fiber diet that trigger acetate production may be a promising intervention for testicular dysfunction and subsequent subfertility associated with obesity.

Acknowledgements

The authors appreciate the financial support of the management of Afe Babalola University, Ado-Ekiti, Nigeria, and the technical support of Bridge-Biotech, GRA, Ilorin, Nigeria.

Authors’ contributions

KSO conceived and designed the research. KSO and COA conducted the experiments. KSO analyzed and interpreted the data. KSO, COA, AAO, AOO, MBO and AAF drafted the manuscript, read, revised and approved the final manuscript.

Declarations

Conflict of interests

The authors have no conflict of interest to declare.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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