Front Endocrinol (Lausanne) 10: 942

PMC: PMC7002433

PMID: 32082253

Evening Primrose Oil Ameliorates Hyperleptinemia and Reproductive Hormone Disturbances in Obese Female Rats: Impact on Estrus Cyclicity

Diet and Treatments

The high fat diet (HFD) is often utilized for induction of obesity in female rats. It was prepared by mixing the standard chow diet (87.7% w/w) with the following fatty components: 10% w/w pork fat, 2% w/w cholesterol and 0.3% w/w bile salts (28, 29). Each gelatin capsule of Primaleve (GlaxoSmithKline) contained 1,000 mg of EPR oil (including 9–10% γ-LA). The oily content of the capsule was withdrawn with a sharp needle of a 3-ml syringe.

Design of the Experiment

All procedures for animal handling were in accord with the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 80-23, revised in 1978). All experimental procedures were approved by the Institutional Research Ethics Committee at Faculty of Pharmacy, Suez Canal University [approval number 201605RA2].

Twenty four female albino Wistar rats were purchased from Moustafa Rashed Company in Saqqarah (Giza, Egypt). The rats aged 8–9 weeks at the beginning of the experiment. Female rats were kept in plastic cages at room temperature (27± 5°C) and normal light-dark phases with unhindered access to food and water. Rats had initial body weights equal 110–142 g. Six rats received standard diet for 14 weeks (WKs), received oral doses of distilled water (10 ml/kg, weeks 8–14) and categorized as a normal group; other rats received HFD for 14 weeks to begin a model of dietary obesity. Rats receiving HFD were divided equally into three groups. The first one received oral doses of distilled water (10 ml/kg, weeks 8–14) and considered the obese control group while the other two groups received oral treatment with EPR oil (5 or 10 g/kg/day, p.o.) (30, 31). Treatment with distilled water or EPR oil started from the beginning of week 8 and continued along with HFD until the end of week 14 (a 7-week therapeutic period). The time-course of the study is shown in Figure 1.

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Figure 1

A scheme describes the course of the experiment.

Detection of Disturbances in Estrus Cyclicity

The percentage of animals with irregular cycles was determined by vaginal smears. The cytology samples were collected daily in the early morning (8:30–9:30 a.m.) for minimum 2 successive cycles (32) by applying a water-moistened cotton bud to be inserted 1 cm intra-vaginally to ensure loading sufficient number of cells on the swab. The removed aqueous fluids were then spread on slides to prepare smears and left at room temperature until completely dried. The prepared smears were immediately immersed in a jar containing 70% alcohol for 1 or 2 min for fixation followed by rapid staining with 0.5% Loba chemie methylene blue solution (Mumbai, India) for 3 min. This was followed by washing with tap water and examined by a light microscope (33, 34). The distinguishing phases of the cycle can be classified in the following as estrus (E), metestrus (M), diestrus (D), and proestrus (P). A typical regular cycle lasts for 4 days. If the of estrus cycle is <4 or >5 days hence, the cycle is marked as not regular (35).

Blood Glucose Measurement and Collection of Samples

At the end of week 14, body weights of the animals were measured and body weight gain % was calculated relative to the baseline value. After an overnight, fasting blood sugar (FBS) levels were determined using blood samples attained from the tail tip using a blood glucometer. Then, rats were anesthetized and sacrificed by dislocation at the cervical vertebra. Blood samples were taken from the orbital sinus, kept for 20 min at room temperature and centrifuged at 1,600 × g for 8 min. Then, sera were separated and stored at −20°C till the time of analyses. Moreover, the total abdominal white adipose tissues were collected from mesenteric, parametrial, and retroperitoneal tissues and weighed. After that, the adipose tissue index was determined as the percentage of the ratio between adipose tissue weight (g) and whole body weight (g) × 100.

Determination of Serum Hormone and Cytokine Levels

The insulin level was measured by the SunRed Bio rat insulin ELISA kit (Shanghai, China). Rat estrogen(E) ELISA kit (MBS703614) employs the competitive inhibition enzyme immunoassay technique, a quantitative sandwich rat follicle stimulating hormone (FSH) ELISA kit (MBS017508), a sandwich ELISA kit for rat LH (MBS 2509833), sandwich ELISA for rat prolactin (PRL) (MBS727546) and rat testosterone ELISA kit (MBS262661) were obtained from MyBioSource (San Diego, California, USA). Rat progesterone ELISA kit (CSB-E07282r) employed the competitive enzyme immunoassay technique obtained from CUSABIO (Hubei, China). Serum levels of adiponectin, leptin, interlukin1β (IL1β) and tumor necrosis factor-α (TNF-α) were estimated by CUSABIO rat ELISA kits (8400 Baltimore Avenue, MD, USA) in accordance to the manufacturer's instructions.

Calculation of the Insulin Resistance Index

For calculation of the homeostasis model assessment of insulin resistance [HOMA-IR] index, the next formula was applied: HOMA-IR index = [FBS mM per L x fasting insulin μU per mL]/22.5 (36).

Colorimetric Assays for Liver Enzymes and Lipid Profile

Serum level of liver enzyme activities including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed colorimetrically. Furthermore, serum lipid profile fractions comprising triglycerides (TG), total cholesterol (TC) in addition to low- and high-density lipoprotein–cholesterol (LDL-C, HDL-C) were determined using assay kits from Biodiagnostic (Cairo, Egypt) and an ultraviolet-visible spectrophotometer UNICO 7200 Spectrophotometer (New Jersey, USA).

Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction for Adipokines mRNAs in Adipose Tissue

White adipose tissue expression of leptin, leptin receptor, adiponectin, and visfatin was determined by one step quantitative real-time PCR as described previously (37). GAPDH was measured as a housekeeping gene. Table 1 demonstrates the specific primers and annealing temperatures used for each determined gene. Relative quantification of the gene in each sample was estimated using LIVAK method (38).

Table 1

Primer sequence for the measured genes.

Gene	Forward primer	Reverse primer	Annealing temp
Leptin	5′-GACATTTCACACACGCAGTC−3′	5′-GAGGAGGTCTCGCAGGTT-3′	53°C
Leptin receptor (Ob-Rb)	5′-TGCTCGGAACACTGTTAAT−3′	5′-GAAGAAGAGCAAATATCA-3′	52°C
Adiponectin	5′-AATCCTGCCCAGTCATGAAG-3′	5′- CATCTCCTGGGTCACCCTTA-3′	53°C
Visfatin	5′-CCTTACCTTAGAGTCATTCA−3′	5′-GACATTCTCAATACTCCAC−3′	46°C
GAPDH	5′-ATGACTCTACCCACGGCAAG−3′	5′-GATCTCGCTCCTGGAAGATG-3′	54°C

Histopathology

Retroperitoneal fats were fixed for 48 h in formalin-alcohol, inserted in paraffin, cut into 3-μm thick specimens and finally hematoxylin and eosin (HE) staining was done. Sections were examined by a CX21 microscope (Olympus, Tokyo, Japan) and images were captured at 20 magnification. Adipocyte space diameters were measured in two different microscopic fields and the average was then calculated.

Furthermore, samples were collected from ovarian tissues and fixation was done in 10% paraformaldehyde for 1 day. After that, sections were dipped in water followed by serial dilutions of alcohol. Sections were then inserted in paraffin wax and were sectioned at 4-μ. The cut tissue specimens were stained by HE or Masson's trichrome and inspected by a light microscope (39) to detect pathological changes. A calibrated digital microscope camera (Tucsen ISH1000, Yuscen Photonics Co. Ltd., China) using Olympus® CX21 microscope, with resolution of 10 megapixels (3,656 × 2,740 pixel for every image). All Masson's trichrome stained sections were captured at original magnification 400x (Objective 40x), UIS optical system (Universal Infinity System, Olympus, Tokyo, Japan).

Statistics

SPSS 21 software was utilized to explore the obtained results. Quantitative data were represented as means ± SEM. Assessment was completed by one-way ANOVA followed by Bonferroni's test. Chi square test was applied for qualitative data for % of estrus cycle irregularity. All reported data were two-tailed and P < 0.05 was set as the level of statistical significance.

Dietary Obesity Markers

Rats fed with a HFD for 14 weeks showed approximately 91% rise in body weight vs. 51% in the normal group. Treatment with the large dose of EPR oil produced a significant reduction in the body weight gain vs. the obese control group (Figure 2A). Furthermore, there was a 3-fold increase in the calculated adipose tissue index in the obese control female rats vs. the normal females. Therapeutic doses of EPR oil (5 or 10 g/kg) in obese female rats significantly reduced the adipose tissue index vs. the obese female group (Figure 2B).

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Figure 2

Percent change in body weight and adipose tissue index in the experimental groups. (A) % increase in body weight and (B) Adipose tissue index. Data are mean ± S.E.M. Bonferroni's test was used to determine differences among individual groups. *Different from normal group, ^{different from obese control group at}P < 0.05.

Serum insulin, FBS and hence HOMA-IR index levels in obese control female rats were greater than the levels reported in normal female rats. EPR oil created dose-dependent improvements in fasting insulin and HOMA-IR index vs. the obese control group (Table 2). Further, the serum levels of the adipokines, leptin, and the inflammatory indicators, TNF-α, and IL1β, were elevated in obese female controls compared to the normal females. Treatment with EPR oil significantly decreased these three markers compared to the obese female group. Conversely, serum adiponectin level was significantly lower in obese rats, with a marked increase upon treatment with EPR oil (10 g/kg) (Table 2).

Table 2

Effect of evening primrose oil (5 or 10 g/kg) on serum biochemical parameters in obese female rats.

	Normal	Obese control	Obese + EPR oil (5 g/kg)	Obese + EPR oil (10 g/kg)
FBS (mg/dL)	74.11 ± 1.32	99.33 ± 1.12^*	87.01 ± 0.90^{^#}	80.21 ± 0.89^#
Insulin (μIU/L)	9.14 ± 1.13	42.22 ± 1.23^*	34.02 ± 0.78^{^#}	23.14 ± 1.12^{^#}^$
HOMA-IR index	1.69 ± 0.34	10.33 ± 1.12^*	7.14 ± 1.04^{^#}	4.55 ± 1.30^{^#}^$
TG (mg/dL)	144.12 ± 2.67	224.14 ± 3.34^*	179.12 ± 2.12^{^#}	165.22 ± 2.00^{^#}
TC (mg/dL)	87.21 ± 2.10	130.23 ± 2.99^*	122.23 ± 2.21^*	125.12 ± 2.33^*
LDL-C (mg/dL)	57.22 ± 1.12	71.25 ± 1.23^*	66.13 ± 1.87^*	68.12 ± 1.08^*
HDL-C (mg/dL)	42.12 ± 1.55	29.34 ± 1.67^*	32.12 ± 1.21^*	32.01 ± 1.11^*
ALT (IU/L)	48.22 ± 2.12	80.12 ± 2.33^*	72.34 ± 3.12^*	73.12 ± 3.04^*
AST (IU/L)	68.21 ± 4.12	88.23 ± 5.12^*	84.45 ± 4.22^*	85.12 ± 3.98^*
Leptin(ng/mL)	2.00 ± 0.30	5.50 ± 0.70^*	3.80 ± 0.40	2.70 ± 0.30^#
Adiponectin (ng/mL)	3.20 ± 0.80	1.30 ± 0.20^*	1.90 ± 0.35	2.50 ± 0.50^#
TNF-α (pg/mL)	24.67 ± 3.50	50.50 ± 5.77^*	38.50 ± 3.89	32.30 ± 3.33^#
IL1β (pg/mL)	20.17 ± 3.33	55.17 ± 4.47^*	42.60 ± 4.20^*	30.20 ± 3.90^#

EPR oil, evening primrose oil; FBS, fasting blood sugar; HOMA-IR, homeostasis model assessment of insulin resistance; TG, triglycerides; TC, total cholesterol; LDL-C, low-density lipoprotein–cholesterol; HDL-C, high-density lipoprotein–cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase. Data are represented as mean ± SEM. Comparisons were performed by one-way ANOVA followed by Bonferroni's test for multiple comparisons.

^{Significantly different from Normal group},

^{significantly different from Obese control group},

^{significantly different from obese + EPR oil (5 g/kg) group at p < 0.05}.

Table 2 demonstrates that obese control group showed greater serum TG, TC, LDL-C, and lower HDL-C vs. the normal group. EPR oil (5 or 10 g/kg) equally and significantly lessened the high serum TG level without effect on the other parameters. Serum ALT and AST increased in obese female group vs. the normal female group. EPR oil did not produce decreases in serum levels of these enzymes (Table 2).

Adipose tissue expression of leptin and leptin receptors was increased in obese control rats (≈3.5- and 4-fold increases, respectively) vs. the expression levels in the normal group. A similar increase (≈3.1-fold) was detected in mRNA expression of visfatin; however, a decrease was detected in the expression of adiponectin. Treatment with the large dose of EPR oil ameliorated the aforementioned changes vs. the obese control group. The low dose of EPR oil failed to produce a similar effect (Figure 3).

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Figure 3

Polymerase chain reaction for adipokines in the experimental groups. Data are mean ± S.E.M. Bonferroni's test was used to determine differences among individual groups. *Different from normal group, ^{different from obese control group at}P < 0.05.

Figure 4 shows the adipocyte space diameters in retroperitoneal adipose tissue section which was significantly larger in obese female rats vs. the measured diameters in normal rats. EPR oil (5 or 10 g/kg) equally and significantly reduced the adipocyte diameters.

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Figure 4

Diameter of adipocyte spaces in white adipose tissue. (A) Photographs for adipose tissue specimens stained with hematoxylin and eosin showing different adipocyte spaces in the study group. (B) Column chart showing mean value for adipocyte space diameters. Data are mean ± S.E.M. Bonferroni's test was used to determine differences among individual groups. *Different from normal group, ^{different from obese control group at}P < 0.05.

Estrus Cycles and Sex Hormone Levels

In the vaginal smear, each phase can be primarily identified according to the percentage of types of 3 primary cells present. These types include nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes (Figures 5A,B). Numerous cornified cells were generally observed as clumps and sheets in estrus stage. On the other hand, metestrus stage consists completely of great number of leukocytes and few big non-nucleated epithelial cells. Diestrus stage contains chiefly leukocytes along with large numbers of epithelial cells. Furthermore, proestrus stage is characterized by nucleated epithelial cells. Importantly, obese female rats showed high percentage of estrus irregularity (Figure 5C). A considerable fraction of female rats have 6–7 days cycles. The prolonged cycles were detected in form of existence of a 2nd or 3rd day of a couple of the 4 stages. The commonly prolonged stages were the diestrus and estrus.

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Figure 5

Cell types in vaginal smears and percentages of rats with estrus cycle irregularities. Photos represent the squamous cornified epithelial cells (Thick green arrow) (A), the nucleated epithelial cells (dashed red arrow) (B). The presence or absence of specific cell types indicates the estrus stage estrus, metestrus, diestrus, and proestrus cycle. Methylene blue ×40. (C) Percentage of rats with irregular cycles in the experimental groups. Irregularity of estrus cycle in female rats was tested using vaginal smears for a period of 2 weeks. Data are presented as % of irregular cycles (out of 6 female rats). Chi square test was applied at P < 0.05. EPR oil: evening primrose oil. *Different from normal rats, ^{different from obese control rats.}

Moreover, obese control female rats showed hyperprolactinemia and greater serum testosterone and estrogen levels compared to normal female rats. In contrast, serum progesterone, FSH and LH levels were lower compared to the normal female rats. Treatment with both doses of EPR oil (5 or 10 g/kg) significantly ameliorated the serum levels of these hormones vs. the obese control females (Figure 6).

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Figure 6

Effect of evening primrose oil (5 or 10 g/kg) on serum hormone levels in obese female rats. (A) Prolactin, (B) Testosterone, (C) FSH, (D) Estrogen(E), (E) LH and (F) Progesterone levels. Data are mean ± S.E.M. Bonferroni's test was used to determine differences among individual groups. *Different from normal group, ^{significantly different from obese control group,}^{different from obese + EPR oil (5 g/kg) group at}P < 0.05. EPR oil, evening primrose oil; LH, luteinizing hormone; FSH, follicle stimulating hormone; PRL, prolactin.

Figure 7 shows histopathologic features of HE stained ovarian sections. Ovarian cortices from the normal group contained many mature Graafian follicles with many primordial follicles, stroma in-between formed of spindle cells and vessels. Mature Graafian follicles contained ova in the center surrounded by granulosa cells, antral space, and a nearby primordial follicle (Figures 7a,b). Ovarian cortices from the obese control group showed cysts, atretic follicles and congested dilated vessels. Marked stroma edema with cystification was prominent at high power fields (Figures 7c,d). Ovarian cortices from all obese rats treated with EPR oil (5 g/kg) showed marked improvement, no cysts, multiple numerous primordial follicles detected and secondary follicles. Secondary follicles, primordial follicles and residual edema were shown (Figures 7e,f). Ovarian cortices from obese rats treated with EPR oil (10 g/kg) showed mild to moderate improvement, residual congested vessels, secondary follicles and scattered atretic follicles. Some primordial follicles were shown with residual minimal edema and mild congestion (Figures 7g,h).

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Figure 7

Histopathologic features of ovarian sections stained with hematoxylin and eosin. (a) Low magnification of ovarian cortex of normal group showed many mature Graafian follicles (arrow) with many primordial follicles (arrowhead), stroma in-between formed of spindle cells and vessels. (b) High magnification showed maturing follicle (Antral) with ovum (arrow) in the center surrounded by granulosa cells with antral space (dashed arrow), and a nearby primordial follicle (arrowhead). (c) Low magnification of ovarian cortex of control obese group showed one of the cysts (arrow), atretic follicles (arrowhead), and marked congestion (dashed arrow). (d) High magnification showed marked stromal edema (arrow) with cystification (arrowhead) and scattered vessels showing slight thickening of their walls (V). (e) Low magnification of ovarian cortex of EPR oil (5 g/kg) treated group showed marked improvement, no cysts presented, multiple numerous primordial follicles (arrowhead) detected, and secondary follicles (arrow). (f) High magnification showed secondary follicles (arrow), primordial follicles (arrowhead) and residual mild stroma edema (dashed arrow). (g) Low magnification of ovarian cortex of EPR oil (10 g/kg) treated group showed moderate improvement, residual congested vessels (dashed arrow) secondary follicles (arrow), and scattered atretic follicles (arrowhead). (h) High magnification showed primordial follicles (arrowhead) with residual stromal edema (arrow) and mild congestion (dashed arrow), H&E, 100x, and 400x.

Figure 8 shows Masson's trichrome stained ovarian sections. High degree of fibrosis was shown in ovaries from obese control rats (Figures 8a,b). Treatment with EPR oil (5 or 10 g/kg) decreased the amount of fibrotic tissues shown in ovaries (Figures 8c,d).

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Figure 8

Histopathologic features of ovarian sections stained with Masson's trichrome stain. (a) Normal ovary shows ovarian cellular stroma with scattered small thin vessels (arrow) and no fibrosis. (b) An image from obese control group showing moderate fibrosis and deposition of thick collagen fibers stained green, with more evident perivascular arrangement. (c) An image from obese rats treated with EPR oil (5 g/kg) showing improvement with mild residual fibrosis mainly perivascular. (d) An image from obese rats treated with EPR oil (10 g/kg) showing improvement with cellular ovarian stroma without perivascular fibrosis, with scattered small vessels (arrow), Masson's trichrome stain, 200x.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

^{Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia}

^{Department of Biochemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt}

^{Pharmacology Department, Faculty of Medicine, University of Tabuk, Tabuk, Saudi Arabia}

^{Department of Pharmacology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt}

^{Department of Physiology, Faculty of Medicine, Taibah University, Medina, Saudi Arabia}

^{Department of Physiology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt}

^{Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt}

^{Department of Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt}

^{Clinical Pathology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt}

^{Department of Internal Medicine, Gastroenterology Division, Faculty of Medicine, Al-Azhar University, Cairo, Egypt}

^{Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia}

^{Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt}

Edited by: Oliana Carnevali, Marche Polytechnic University, Italy

Reviewed by: Taisen Iguchi, National Institute for Basic Biology, Japan; Lila Oyama, Federal University of São Paulo, Brazil

*Correspondence: Sawsan A. Zaitone as.ude.tu@enotiazs; ge.ude.zeus.mrahp@nootyaz_naswas

This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology

†ORCID: Sawsan A. Zaitone orcid.org/0000-0002-9688-7683

‡Present address: Sawsan A. Zaitone, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia

Edited by: Oliana Carnevali, Marche Polytechnic University, Italy

Reviewed by: Taisen Iguchi, National Institute for Basic Biology, Japan; Lila Oyama, Federal University of São Paulo, Brazil

Received 2019 Oct 1; Accepted 2019 Dec 31.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

Obesity is a public health burden disturbing all body functions and reproductive hormones. As obesity increases among females, there will be a rising challenge to physicians in care from fertility problems. Evening primrose oil (EPR oil) contains essential fatty acids including omega-6 linoleic acid with strong anti-inflammatory activity. Since EPR oil has utility in alleviating dysmenorrhea, this study aimed to ascertain its modulatory effect on systemic inflammation, reproductive hormones and estrus cycle irregularity in female obese rats. Thirty-two female rats were distributed to 4 groups: (i) normal, (ii) dietary obese-control female rats, and (iii and iv) dietary obese female rats treated with EPR oil (5 or 10 g/kg). Rats were examined for estrus regularity by taking vaginal smears daily during the last 2 weeks of the experiment. Serum level of insulin, leptin, adiponectin, and inflammatory cytokines was measured. In addition, serum lipid profile, and liver enzyme activities were estimated. Adipose tissues were taken for histopathologic examination as well as determination of gene expression for leptin, leptin receptors, adiponectin, and visfatin. Obese rats exhibited significant weight gain (90.69 ± 8.9), irregular prolonged estrus cycles (83.33%), increased serum levels of insulin, leptin, prolactin and testosterone and decreased gonadotropin levels. EPR oil exhibited a curative effect on obesity-related irregularity in estrus cycle and ovarian pathology. The underlying molecular mechanism may be related to reduction of systemic inflammation, alleviating insulin resistance and modulation of adipokine expression. EPR oil may be considered as a promising therapeutic intervention against obesity-related female hormonal disturbances and estrus irregularity.

Keywords: dietary obese female rats, estrus cyclicity, evening primrose oil, hyperleptinemia, reproductive hormone disturbances, insulin resistance

Abstract

Glossary

Abbreviations

ALT	alanine aminotransferase
AST	aspartate aminotransferase
ELISA	enzyme-linked immunosorbent assay
EPR oil	evening-primrose oil
FSH	follicle stimulating hormone
HDL	high-density lipoprotein–cholesterol
HFD	high fat diet
IL1β	interlukin1β
MOMA-IR index	the homeostasis model assessment of insulin resistance index
LA	linoleic acid
γ-LA	γ-Linoleic acid
LDL-C	low-density lipoprotein–cholesterol
LH	luteinizing hormone
PCOS	polycystic ovaries syndrome
PRL	prolactin
WK	week
TG	triglycerides
TC	total cholesterol
TNF-α	tumor necrosis factor-α.

Glossary

Footnotes

Funding. This work was supported by the Deanship of Scientific Research (DSR) at the University of Tabuk, Tabuk, Saudi Arabia [Grant Number S-0140-1439].

Footnotes

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