Antihyperglycemic effect of extra virgin sacha inchi oil in type 2 diabetic rats: Mechanisms involved in pancreatic β-cell function and apoptosis

Abstract

Background and aim

The purpose of the study was to investigate the anti-hyperglycemic effect of extra virgin sacha inchi oil (EVSIO) and its possible mechanisms and actions against pancreatic β-cell death and dysfunction in type 2 diabetic (T2D) rats.

Experimental procedure

T2D rats were induced with a high-fat diet and low-dose of streptozotocin. The rats were then treated for 5 weeks with EVSIO (0.5, 1, and 2 ml/kg), or pioglitazone. Biochemical and histopathological studies, oxidative and inflammatory markers, and expression of apoptotic-related proteins were then evaluated.

Results

EVSIO treatment exhibited a dose-dependent reduction of fasting blood glucose, area under the curve of glucose, total cholesterol, and triglyceride levels in the diabetic rats, while improved pancreatic β-function was demonstrated by increasing pancreatic and serum insulin levels. EVSIO treatment effectively lowered atrophic pancreatic islets and reduced the level of serum and pancreatic MDA in the diabetic rats. In addition to serum and pancreatic GPx activities in the diabetic rats, EVSIO also augmented serum SOD. Increased levels of NF-κB, TNF-α and IL-6 present in the diabetic rats were greatly reduced by EVSIO treatment. Furthermore, EVSIO revealed an anti-apoptotic effect on the diabetic rat pancreas by upregulating Bcl-2, and downregulating Bax and cleaved caspase-3 protein expression.

Conclusion

The overall study results demonstrated the potential anti-hyperglycemic effect of EVSIO in the diabetic rats. The beneficial effects of EVSIO may be attributed to its ability to improve pancreatic β-cell function and ameliorate β-cell apoptosis by inhibiting oxidative stress and inflammatory cytokines.

Graphical abstract

Highlights

• •Extra virgin sacha inchi oil triggered antidiabetic activity in diabetic rats.
• •Extra virgin sacha inchi oil ameliorated hyperglycemia in diabetic rats by improving pancreatic β-cell function.
• •Extra virgin sacha inchi oil protected pancreatic β-cells from apoptosis in diabetic rats.
• •Extra virgin sacha inchi oil possessed antioxidative and anti-inflammatory properties in diabetic rats.

List of abbreviations

ALA

α-linolenic acid

AUC_BG

Area under the curve of blood glucose

BCA

Bicinchoninic acid

Bcl-2

B-cell lymphoma 2

BSA

Bovine serum albumin

b.w.

Body weight

CAT

Catalase

DHA

Docosahexaenoic acid

DM

Diabetes mellitus

ELISA

Enzyme-linked immunosorbent assay

EPA

Eicosapentaenoic acid

EVSIO

Extra virgin sacha inchi oil

FBG

Fasting blood glucose

FFAs

Free fatty acids

GPx

Glutathione peroxidase

GSH

Reduced glutathione

GSSG

Oxidized glutathione

HDL-C

High-density lipoprotein cholesterol

HFD

High-fat diet

HNF-4α

Hepatocyte nuclear factor- 4 alpha

HRP

Horseradish peroxidase

H&E

Hematoxylin and Eosin

LDL-C

Low-density lipoprotein cholesterol

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

MDA

Malondialdehyde

MTTP

Microsomal triglyceride transfer protein

NADPH

Nicotinamide adenine dinucleotide phosphate

NF-κB

Nuclear factor kappa-B

OGTT

Oral glucose tolerance test

ω-3 PUFAs

Omega-3 polyunsaturated fatty acids

ω-6 PUFAs

Omega-6 polyunsaturated fatty acids

PBS

Phosphate buffered saline

PVDF

Polyvinylidene fluoride

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

SDS

Sodium dodecyl sulfate

SOD

Superoxide dismutase

STZ

Streptozotocin

TBARS

Thiobarbituric acid reactive substances

TC

Total cholesterol

TG

Triglycerides

TNF-α

Tumor necrosis factor-alpha

T2D

Type 2 diabetes

VLDL-C

Very low-density lipoprotein cholesterol

Taxonomy (classification by EVISE)

Metabolic Disorder; Diabetes mellitus; Natural Products; Biochemical analysis and Histopathology.

1. Introduction

Diabetes mellitus is a complex metabolic disorder that has emerged as a major healthcare problem worldwide. Over 90% of diabetic cases are type 2 diabetes (T2D), which is mainly attributable to impaired insulin secretion and peripheral insulin resistance.^1,2 Insulin produced by pancreatic β-cells is the most important hormone for the regulation of glucose homeostasis. When β-cells are unable to release adequate insulin to compensate for insulin insensitivity, hyperglycemia is induced.³ Pancreatic β-cell death and dysfunction are core defects in the development of T2D. β-cell damage in diabetes has traditionally been associated with obesity, an excess of free fatty acids (FFAs), and hyperglycemia.^3,4 Under these conditions, inflammatory stress, oxidative stress, and endoplasmic reticulum (ER) stress, are activated, which ultimately leads to pancreatic β-cell apoptosis and dysfunction with reduced insulin synthesis and secretion as a consequence.³ Pancreatic β-cells are noted to have low antioxidant defense and are highly susceptible to a condition that leads to oxidative damage.^5,6 Excessive reactive oxygen species (ROS) production causes a depletion of Ca²⁺ mobilization and insulin secretion, and induces pro-apoptotic signaling.^1,6 ROS generation also enhances inflammatory molecules such as the tumor necrosis factor-alpha (TNF-α), interleukin-1β and −6 (IL-1β and IL-6) molecules that recruit macrophages and activate pancreatic islet inflammation.6, 7, 8 In turn, these inflammatory cytokines can induce oxidative stress and the two processes synergistically interact to amplify the activation of apoptotic pathways, thereby causing β-cell apoptosis.⁷ This notion has been proven in many animal models of T2D that demonstrated a reduction in β-cell mass with increased β-cell apoptosis, linking the elevated oxidative stress markers and inflammatory mediators.^9,10 Excessive production of ROS and inflammatory cytokines are known to inhibit anti-apoptotic Bcl-2 family proteins but also to induce the pro-apoptotic Bcl-2 proteins-mediated mitochondria apoptotic pathway. This eventually leads to β-cell apoptosis.^7,9

Accumulating evidence has revealed that several functional foods demonstrate an anti-hyperglycemic activity by improving the function of β-cells and ameliorating inflammatory and oxidative status. The presence of nutrients and bioactive ingredients in extracts from plants, such as omega-3 polyunsaturated fatty acids (ω-3 PUFAs), polyphenols, flavonoids, and glycosides, are well documented to have an anti-diabetic activity.^11,12 Sacha inchi oil extracted from the seeds of sacha inchi (Plukenetia volubilis L., Euphorbiaceae family) is an excellent source of polyunsaturated fatty acids (PUFAs), especially, α-linolenic acid (ALA, 18:3 n-3; 45.20–50.41%), a kind of ω-3 PUFAs, and linoleic acid (LA, 18:2 n-6; 32.66–36.80%), a kind of ω-6 PUFAs.¹³ ALA serves as a precursor of eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) synthesis, and provides beneficial effects against diabetes, hypercholesterolemia, hypertension, cardiovascular disease, cancer, and inflammatory disorders.¹⁴ Additionally, sacha inchi oil contains high contents of tocopherols, phytosterols and polyphenolic compounds, which exhibit strong antioxidant activity.¹³ Studies have also shown that sacha inchi oil alleviates total cholesterol and triglycerides levels and increases the high-density lipoprotein cholesterol (HDL-C) level in rats.^15,16 Similarly, treatment with sacha inchi oil of patients with hypercholesterolemia was shown to lead to decreased total cholesterol and non-esterified fatty acids, and augmented HDL-C and insulin levels.¹⁷ Obese rats treated with sacha inchi oil showed a decrease in hyperlipidemia, oxidative damage and inflammatory markers, and an increase in serum adiponectin.¹⁶ Furthermore, sacha inchi oil supplementation improved insulin sensitivity in humans with less healthy metabolic status and higher glycemic responses to fat load.¹⁸ Although many pharmacological activities of sacha inchi oil have been identified in previous studies to exert anti-oxidative, anti-inflammatory, anti-hypercholesterolemia, and anti-bacterial properties,¹⁹ the anti-diabetic action of sacha inchi oil remains unclear. Thus, this research study was undertaken to investigate the anti-hyperglycemic and pancreatic protective activities of extra virgin sacha inchi oil (EVSIO) in a T2D rat model, and to evaluate the possible mechanisms of this oil.

2. Materials and methods

2.1. Chemicals

Extra virgin sacha inchi oil (EVSIO) was locally purchased from the company in Thailand. The commercial EVSIO was produced by cold-pressed method. Extracting sacha inchi oil through the cold pressing provides a high quality with substantial contents of phenolic compounds, phytosterols, tocopherols, α-linolenic acid and linoleic acid.^13,20 The major fatty acid components in EVSIO used in this study were analyzed and reported by Central Laboratory Co., Ltd., Thailand. The EVSIO consists of 41.29–41.73% α-linolenic acid (ω-3), 40.16–40.54% linoleic acid (ω-6), 8.73–9.16% oleic acid (ω-9), 4.41–4.65% palmitic acid, 3.55–3.62% stearic acid, and ω-6/ω-3 ratio of 0.96–0.98 (Supplementary Table 1). Streptozotocin (STZ) was obtained from Merck Millipore, USA. Pioglitazone hydrochloride was purchased from Berlin Pharmaceutical Industry Co., Ltd., Thailand. All other chemicals were commercial products of analytical grade quality from Sigma-Aldrich Chemicals and Merck Millipore, USA.

2.2. Animals

Male Sprague Dawley rats aged 5–6 weeks with a weight of 160–180 g were procured from Nomura Siam International (Bangkok, Thailand). All rats were kept at the Laboratory Animal Center of Naresuan University, Thailand, under humid conditions in a temperature-controlled room in 12-hr light/dark cycles. The animals were fed a normal commercially available diet for rats and water ad libitum, prior to the dietary manipulation. The animal experiments were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee of Naresuan University, Thailand (Ethic Number: NU-AE620616).

2.3. Induction of T2D and experimental design

T2D was induced in the sample rats as described previously,^21,22 with minor modifications. After seven days of acclimatization, the rats were fed for 2 weeks with a high-fat diet (HFD; 58% kcals from fat, 20% kcals from protein, and 22% kcals from carbohydrates), which was procured from Bangkok Animal Research Center Co., Ltd., Thailand for 2 weeks, followed by intraperitoneal injection of low-dose STZ (40 mg/kg body weight (b.w.), prepared in 0.1 M sodium citrate buffer, pH 4.5). The control rats were given a normal chow diet and vehicle citrate buffer. Three days after injection, the rats were assessed for fasting blood glucose (FBG) of 200 mg/dl or above to confirm the diabetes condition. The fasting blood glucose (FBG) levels were measured from tail blood using a digital glucometer (Roche Diagnostics GmbH, Germany). The schematic diagram of the experimental protocol is shown in Fig. 1.

Fig. 1.

The experimental procedure of the study. The T2D rats were established by feeding 2 weeks of high-fat diet (HFD), followed by intraperitoneal injection of 40 mg/kg streptozotocin (STZ), and the control rats were fed with a normal diet. The T2D rats were orally treated with 0.5, 1, and 2 ml/kg EVSIO or 30 mg/kg pioglitazone (PG) for 5 weeks. C: Control rats, DM: Diabetic rats, DM + EVSIO 0.5: Diabetic rats treated with extra virgin sacha inchi oil at a dose of 0.5 ml/kg, DM + EVSIO 1: Diabetic rats treated with extra virgin sacha inchi oil at a dose of 1 ml/kg, DM + EVSIO 2: Diabetic rats treated with extra virgin sacha inchi oil at a dose of 2 ml/kg, DM + PG 30: Diabetic rats treated with pioglitazone at a dose of 30 mg/kg, DW: Distilled water, STZ: Streptozotocin.

Thirty six experimental rats were randomly divided into 6 groups (n = 6 for each group), as follows: Group I (control group); normal rats received 1 ml/kg of distilled water, Group II (diabetic group); diabetic rats received 1 ml/kg of distilled water, Groups III, IV and V; diabetic rats received EVSIO at the three different-doses of 0.5, 1, and 2 ml/kg b.w., respectively, and Group VI (positive control group); diabetic rats received 30 mg/kg b.w. of pioglitazone. All treatments were orally administered, once per day, for 5 weeks. A range of doses of EVISO used in this study was based on the findings of a previous study, which reported that sacha inchi oil at doses of 0.5, 1, and 1.5 ml/kg b.w./day dose-dependently improved lipid metabolism and reduced hepatic steatosis and inflammation in HFD-fed rats.²³ The fatty acid composition in each dosage of EVSIO has been calculated on the basis of the fatty acid profiles of the sample and is shown in Supplementary Table 2.

During the treatment period, food and water intake were checked daily. Alterations of FBG and body weight were determined weekly throughout the study period. At the end of the 5-week study period, all rats were anesthetized with thiopental sodium (100 mg/kg b.w., i.p.), and blood and pancreas were collected for further analysis.

2.4. Evaluation of oral glucose tolerance test (OGTT)

Glucose tolerance was investigated using OGTT. After overnight fasting, blood samples were collected from the lateral tail vein of the rats to determine the blood glucose at the baseline (0 min). Glucose solution (2 mg/kg) was then orally administered to the rats and blood samples were obtained at 30, 60, 90, 120, and 180 min for measurement of blood glucose. The area under the curve for blood glucose (AUC_BG) was calculated using the following equation:

AUC_BG (mg • h/dl): (BG0 + BG30)/2 x 0.5 + (BG30 + BG60)/2 x 0.5 + (BG60 + BG90)/2 x 0.5 + (BG90 + BG120)/2 x 0.5 + (BG120+BG180)/2 x 1

2.5. Evaluation of serum and pancreatic insulin levels

Blood samples were centrifuged at 4,000 g for 30 min to collect blood serum. The serum insulin level was measured using a rat insulin enzyme-linked immunosorbent assay (ELISA) kit, following the manufacturer's instructions (Cat. Number # EZRMI-13 K, Merck Millipore, USA).

For determination of pancreatic insulin content, pancreatic tissues were washed with phosphate buffered saline (PBS) and homogenized with acid ethanol (0.18 M HCL in 70% ethanol), overnight at −20 °C. The homogenates were centrifuged at 14,000 g, 4 °C for 30 min to obtain supernatant for determining the insulin content. A rat insulin ELISA kit was used, following the manufacturer's instructions (Cat. Number # RAB0904, Sigma Aldrich, USA).

2.6. Determination of lipid profiles

The serum lipid profiles, consisting of total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), were assessed using Roche Cobas C501 Chemistry analyzer, as analyzed by the Biolab Medical Clinic, Phitsanulok, Thailand.

2.7. Pancreatic histopathology

Pancreatic tissues were fixed in 10% neutral buffered, dehydrated, and embedded in paraffin. The sections were then stained with hematoxylin and eosin (H&E). The slides were examined under a light microscope (Olympus BX53F2, Japan) and images of the histological sections were obtained using an Olympus DP74 digital camera at 10X magnification. The area of the pancreatic islet (μm²) was analyzed by Image J version 1.53g software.

2.8. Determination of pro-inflammatory cytokine TNF-α and IL-6 levels in serum and pancreatic tissues

To obtain protein samples, pancreatic tissues were homogenized with cold radioimmunoprecipitation assay (RIPA) buffer (Cat. Number # 89900, Thermo Scientific, USA) supplemented with 1% halt protease inhibitor, and centrifuged at 14,000 g, 4 °C for 15 min. Protein concentration was measured using a micro bicinchoninic acid (BCA) protein assay kit (Cat. Number # 71285, Merck Millipore, USA). TNF-α levels in the serum and pancreatic tissues were analyzed using rat TNF-α ELISA kit (Cat. Number # 900-M73) provided by PeproTech Asia, Israel. For IL-6 detection, IL-6 levels in the serum and pancreatic tissues were assessed using a rat IL-6 ELISA kit (Cat. Number # 900-M86, PeproTech Asia, Israel). All procedures were performed according to the manufacturer's instructions.

2.9. Measurement of serum and pancreatic malondialdehyde (MDA) contents

Malonaldehyde (MDA) is used as a lipid peroxidation marker. Excessive endogenous ROS production causes an increase in the generation of lipid peroxidation.²⁴ In this study, MDA was measured in both the serum and pancreatic tissues by the thiobarbituric acid reactive substances (TBARS) method. The samples were mixed with 50 μl of 8.1% sodium dodecyl sulfate (SDS), 375 μl of 0.8% TBA, and 375 μl of 20% acetic acid solution (pH 3.5), then heated at 95 °C for 60 min then cooled. After centrifugation at 4,000 rpm for 10 min, the supernatants were collected to determine the TBARS levels at an absorbance of 532 nm using a spectrophotometer (Molecular Devices, California, USA). The concentrations of the MDA in serum (nmol/ml) and pancreatic tissue (nmol/g protein) were calculated using a calibration curve with 1,1,3,3-tetramethoxypropane (TMP).

2.10. Determination of enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in serum and pancreatic tissues

Serum and pancreatic SOD activities were evaluated by the ability to inhibit the autoxidation of pyrogallol. The procedure was carried out according to a previous protocol with modifications.²⁵ The absorbance decrease of pyrogallol was detected at 420 nm. Serum SOD activity was calculated as units per ml and pancreatic SOD activity was expressed as units per mg protein, where one unit of SOD activity was defined as the amount of enzyme required for inhibition of pyrogallol autoxidation by 50% per min.

Serum and pancreatic GPx activities were performed following the method of Paglia and Valentine,²⁶ with modifications. This assay is based on the oxidation of reduced glutathione (GSH) into oxidized glutathione (GSSG) catalyzed by GPx. GSSG is then reduced by glutathione reductase in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), to become GSH and NADP⁺. The decrease in NADPH absorbance was read spectrophotometrically at 340 nm. Serum and pancreatic GPx activities were expressed as nmol/min/ml and nmol/min/mg protein, respectively.

The modified method of Goth²⁷ was employed to measure the activities of catalase (CAT) in serum and pancreatic tissues. The CAT activity was assessed by adding the enzyme sample to the substrate containing, 65 mM H₂O₂ in 60 mM sodium phosphate buffer, pH 7.4, at 37 °C for 1 min. The reaction was stopped with ammonium molybdate, which produces a yellow complex. The absorbance of a complex reaction between H₂O₂ and ammonium molybdate was then detected at 405 nm. CAT activities in the serum and pancreatic tissue were expressed as units per ml and units per mg protein, respectively.

2.11. Western blot analysis

Pancreatic proteins (80 μg) were extracted by RIPA buffer and then separated on 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, USA). The membranes were blocked in 5% (w/v) bovine serum albumin (BSA) dissolved in 1xTBST (Tris-buffered saline, 0.1% Tween 20) and incubated overnight at 4 °C with each primary antibody; anti–NF–κB (1:500, Cell Signaling Technology, USA), anti-cleaved caspase-3 (1:500, Cell Signaling Technology, USA), anti-Bax (1:500, Cell Signaling Technology, USA), anti-Bcl-2 (1:500, ABclonal, USA), or anti-GADPH (1:2500, Cell Signaling Technology, USA) antibodies. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) (Cell Signaling Technology, USA) at room temperature for 1 h. Protein bands were detected by the enhanced chemiluminescence system and analyzed using Image Lab software (Bio-Rad, USA).

2.12. Statistical analysis

Data were presented as mean ± standard error of the mean (S.E.M.). Data from the initial and final treatment were calculated by using two-way ANOVA with Bonferroni's method. The significant differences between the groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Differences with p-value <0.05 were defined as statistically significant. All statistical analysis was performed using Prism for Windows, version 9.00 (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Effects of EVSIO on FBG, body weight, food and water intake in the diabetic rats

As shown in Table 1, the initial and final FBG levels were evaluated in the experimental rats. The diabetic rats showed a significant increase in the FBG level through the experimental period as compared to the control group. At the end of 5 weeks of experimentation, treatment with EVSIO (0.5, 1, and 2 ml/kg) dose-dependently decreased the FBG level of the diabetic rats. Similarly, the diabetic rats treated with pioglitazone (30 mg/kg) revealed a significant decline in the FBG level compared with that of the diabetic group.

Table 1.

Effects of extra virgin sacha inchi oil (EVSIO) on the initial and final levels of fasting blood glucose (FBG), body weight, food intake, and water intake in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic rats.

Group	FBG (mg/dl)		Body weight (g)		Food intake (g/day)		Water intake (ml/day)
	Initial	Final	Initial	Final	Initial	Final	Initial	Final
Control	110.17 ± 2.76	86.50 ± 4.41	372.32 ± 9.25	542.50 ± 13.44	27.69 ± 0.93	31.27 ± 0.77	39.64 ± 2.55	44.94 ± 5.10
DM	371.17 ± 20.00∗∗∗∗	387.00 ± 26.42∗∗∗∗	315.47 ± 5.84	369.33 ± 20.88∗∗∗∗	25.26 ± 0.75	35.32 ± 1.78	128.37 ± 4.57	206.41 ± 34.48∗∗∗∗
DM + EVSIO 0.5 ml/kg	386.17 ± 14.79∗∗∗∗	343.33 ± 67.68	326.20 ± 7.18	390.00 ± 29.14	25.20 ± 1.06	31.15 ± 3.31	125.00 ± 7.39	199.52 ± 26.76
DM + EVSIO 1 ml/kg	352.50 ± 19.57∗∗∗∗	220 ± 25.24#	309.13 ± 9.33	401.33 ± 18.16	24.52 ± 0.77	30.05 ± 1.34	115.83 ± 10.39	150.54 ± 23.68
DM + EVSIO 2 ml/kg	338.00 ± 37.97∗∗∗∗	213.67 ± 49.51#	320.80 ± 3.19	421.17 ± 26.19	24.54 ± 1.49	31.78 ± 3.27	101.43 ± 20.13	161.36 ± 44.81
DM + PG 30 mg/kg	334.67 ± 33.50∗∗∗∗	219.00 ± 39.04#	311.03 ± 4.15	421.67 ± 39.99	25.98 ± 0.81	34.82 ± 1.20	105.24 ± 11.14	173.12 ± 25.31

Values are expressed as mean ± S.E.M., for six rats in each group. ∗∗∗∗p < 0.0001; compared to control rats, ^#p < 0.05; compared to diabetic rats. PG: pioglitazone.

The diabetic rats showed a significant attenuation of body weight as compared to that of the control group by the end of the 5-week experiment. However, the diabetic rats’ body weight tended to improve when treated with either 0.5, 1, and 2 ml/kg EVSIO or with pioglitazone. Before and after treatment, the food intake did not show any significant differences among the groups. In the diabetic rats, a significant induction of the final water intake was also observed. After treatment with EVSIO (0.5, 1, and 2 ml/kg) or pioglitazone for 5 weeks, the increased final water intake of the diabetic rats was slightly reduced compared with the untreated diabetic rats.

3.2. Effect of EVSIO on serum lipid alterations in the diabetic rats

As demonstrated in Table 2, the diabetic rats showed a significant elevation in the serum levels of TC and TG as compared to the control rats, while for the diabetic rats treated with 0.5, 1, and 2 ml/kg EVSIO or pioglitazone, those indices were significantly decreased in comparison with the untreated diabetic rats. However, the levels of HDL-C did not differ among any groups.

Table 2.

Effect of extra virgin sacha inchi oil (EVSIO) on lipid profiles in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic rats.

Group	Total cholesterol (mg/dl)	Triglyceride (mg/dl)	HDL-C (mg/dl)
Control	51.40 ± 2.60	89.40 ± 9.81	37.80 ± 2.25
DM	161.80 ± 22.89∗∗∗∗	250.20 ± 49.55∗∗	45.20 ± 3.50
DM + EVSIO 0.5 ml/kg	84.80 ± 2.96###	121.00 ± 28.49#	41.40 ± 2.79
DM + EVSIO 1 ml/kg	87.40 ± 11.72##	111.60 ± 22.58#	42.40 ± 2.89
DM + EVSIO 2 ml/kg	57.40 ± 2.84####	93.20 ± 11.30##	40.80 ± 5.49
DM + PG 30 mg/kg	84.80 ± 9.40###	92.80 ± 32.14##	43.20 ± 4.51

Values are expressed as mean ± S.E.M., for five rats in each group.

∗∗p < 0.01, ∗∗∗∗p < 0.0001; compared to control rats.

^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001; compared to diabetic rats. PG: pioglitazone.

3.3. Effect of EVSIO on glucose tolerance in the diabetic rats

The glucose AUG (AUC_BG) of OGTT is shown in Fig. 2A. The diabetic rats showed a significant augmentation in the AUC_BG as compared to that of the control rats. The diabetic rats treated with EVSIO at doses of 0.5, 1, and 2 ml/kg attenuated the AUC_BG in a dose-dependent manner compared with the diabetic rats. Also, the diabetic rats treated with pioglitazone demonstrated a trend in the reduction of the AUC_BG. These results indicate that EVSIO improves glucose tolerance in rat diabetes.

Fig. 2.

Effect of extra virgin sacha inchi oil (EVSIO) on glucose tolerance, and serum and pancreatic insulin levels in the diabetic rats. (A) The AUC_BG for oral OGTT; glucose tolerance of the experimental rats was measured using OGTT method, at the end of a 5-week treatment. (B) Serum insulin and (C) pancreatic insulin contents were determined by the ELISA technique. Values are expressed as mean ± S.E.M., for five to six rats in each group. ∗∗∗∗p < 0.0001versus the control rats, ^#p < 0.05 versus the diabetic rats. OGTT: oral glucose tolerance test.

3.4. Effect of EVSIO on serum and pancreatic insulin levels in the diabetic rats

Fig. 2B and C illustrates the insulin levels in serum and pancreas. The untreated diabetic rats revealed a significant reduction in serum and pancreatic insulin levels as compared to the normal control rats. Importantly, 2 ml/kg EVSIO administration markedly induced the levels of serum and pancreatic insulin in the diabetic rats, suggesting its effect on the improvement of pancreatic β-cell function. However, the diabetic rats treated with the antidiabetic drug pioglitazone demonstrated a significant increment in serum insulin level in comparison with the untreated diabetic rats.

3.5. Effect of EVSIO on histopathological changes in the pancreas of the diabetic rats

As shown in Fig. 3A–F, pancreatic histopathological assessment displayed islet degeneration and atrophy, and a decrease in the number of islets in the diabetic rats. However, the dose-dependent treatment of the diabetic rats with EVSIO ameliorated these changes, as well as the diabetic rats treated with pioglitazone, which showed a considerable improvement in the histological structure of pancreatic islets of the diabetic rats in both treated groups with mild degree injuries. Also, the islet area analysis revealed a significant reduction in the islet area of the diabetic rats as compared to the control rats. Remarkably, 2 ml/kg EVISO and pioglitazone treatment significantly increased the islet area in the diabetic rats (Fig. 3G). These results reflect the beneficial role of EVSIO in the prevention of pancreatic islet damage in the diabetic rats.

Fig. 3.

Effect of extra virgin sacha inchi oil (EVSIO) on pancreatic histopathology in the diabetic rats. The pancreatic tissues were stained with hematoxylin and eosin (H&E) to observe the islet morphology and examined under a light microscope ( × 100; scale bar = 50 μm). The arrows show the pancreatic islets. (A) control rats, (B) diabetic rats, (C–E) treatment of diabetic rats with EVSIO at doses of 0.5, 1, and 2 ml/kg, respectively, (F) diabetic rats treated with pioglitazone 30 mg/kg, (G) the bar graph of the islet area. Values are expressed as mean ± S.E.M., for six rats in each group. ∗∗∗∗p < 0.0001 versus the control rats, ^#p < 0.05, ^##p < 0.01 versus the diabetic rats.

3.6. Effect of EVSIO on oxidative stress parameters in serum and pancreas of the diabetic rats

We measured the oxidative stress marker MDA, which is the end product of lipid peroxidation, and the antioxidant enzyme activities in the serum and pancreas. The diabetic rats showed an increase in the levels of serum and pancreatic MDA and a decrease in levels of the activities of the antioxidant enzymes, SOD, GPx and CAT in the serum. Inversely, increased levels of serum and pancreatic MDA were obviously blocked by the dose-dependent treatment with both EVISO and pioglitazone. Also, the reduced enzymatic antioxidant SOD and GPx activities in the serum of the diabetic rats were markedly restored in a dose-dependent manner after EVISO treatment as well as supplementation treatment with pioglitazone. However, no significant changes in serum antioxidant CAT activity were documented in either the EVISO or the pioglitazone-treated diabetic rats compared with the untreated diabetic rats (Fig. 4A–E).

Fig. 4.

Effect of extra virgin sacha inchi oil (EVSIO) on oxidative stress parameters in serum and pancreas of the diabetic rats. The bar graph shows oxidative status in the serum of the experimental rats; (A) MDA, (B) GPx, (C) SOD, and (D) catalase. The bar graph depicts oxidative stress parameters in the rats' pancreas; (E) MDA, (F) GPx, (G) SOD, and (H) catalase. Values are expressed as mean ± S.E.M., for five to six rats in each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus the control rats, ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001 versus the diabetic rats. MDA: malondialdehyde, GPx: glutathione peroxidase, SOD: superoxide dismutase.

Additionally, as illustrated in Fig. 4F, the pancreatic GPx activity in the diabetic rats was significantly lower than that of the non-diabetic control rats. However, this alteration was improved by only 2 ml/kg of EVSIO treatment. Surprisingly, the antioxidant SOD and CAT activities were found to increase in the pancreas of the diabetic rats in comparison with the non-diabetic rats. On the other hand, administration of EVSIO or pioglitazone to the diabetic rats significantly reduced pancreatic SOD activity compared to the untreated diabetic rats. Meanwhile, both the EVSIO- and pioglitazone-treated diabetic groups revealed no significant changes in pancreatic CAT activity (Fig. 4G and H).

3.7. Effect of EVSIO on inflammatory mediators in serum and pancreas of the diabetic rats

To investigate the anti-inflammatory action of EVSIO under diabetic conditions, serum and pancreatic pro-inflammatory cytokine TNF-α and IL-6 levels were determined. The diabetic rats exhibited a significant enhancement in serum and pancreatic TNF-α and IL-6 levels. Treatment with 2 ml/kg EVSIO and pioglitazone effectively attenuated serum and pancreatic IL-6 levels, as well as the pancreatic TNF-α in the diabetic rats. However, the elevated level of serum TNF-α in the diabetic rats was not alleviated by EVSIO or pioglitazone treatment (Fig. 5A and B).

Fig. 5.

Effect of extra virgin sacha inchi oil (EVSIO) on the inflammatory mediators in the serum and pancreas of the diabetic rats. The bar graphs show serum IL-6 (A) and TNF-α (B), and pancreatic IL-6 (C) and TNF-α (D) levels, measured by ELISA technique. (E) Top; a representative Western blot analysis of the NF-κB and GAPDH protein expression, bottom; the bar graph of the relative expression of NF-κB normalized to GAPDH protein. Values are expressed as mean ± S.E.M., for four rats in each group. ∗p < 0.05, ∗∗p < 0.01 versus the control rats, ^#p < 0.05, ^##p < 0.01 versus the diabetic rats. IL-6: interleukin-6, TNF-α: tumor necrosis factor-alpha.

In addition, the transcription factor NF-κB, which activates the expression of inflammatory cytokines, was determined in the pancreatic tissue. As illustrated in Fig. 5E, there was a significant elevation in pancreatic NF-κB protein expression in the diabetic rats as compared to the control rats. However, the dose-dependent treatment with EVSIO and the treatment with pioglitazone exhibited a significant reduction in pancreatic NF-κB protein in the diabetic rats as compared to the untreated diabetic rats.

3.8. Effect of EVSIO on the protein expression of molecules involved in pancreatic apoptosis of the diabetic rats

Alteration of the Bcl-2 family molecules and activation of the caspase pathway are the crucial players in the regulation of the apoptotic cells.⁹ To ascertain the role of EVSIO against pancreatic apoptosis, we measured the expression proteins of the anti- and pro-apoptotic Bcl-2 and cleaved caspase-3. Our data showed a diminution in anti-apoptotic Bcl-2 protein and an increment in pro-apoptotic Bax and cleaved caspase-3 proteins that were markedly detected in the diabetic rats. However, the dose-dependent treatment with EVSIO and with pioglitazone effectively restored the alterations of the anti-apoptotic Bcl-2 and reduced the increment in Bax and cleaved caspase-3 protein expression in the diabetic rats (Fig. 6A–D). These findings collectively suggest that EVSIO has an inhibitory property on the apoptosis of the pancreas in the diabetic rats.

Fig. 6.

Effect of extra virgin sacha inchi oil (EVSIO) on the apoptosis-related protein expression in the pancreas of the diabetic rats. (A) A representative Western blot analysis of cleaved caspase-3, Bcl-2, and GAPDH protein expression in the rats' pancreas. (B–C) The bar graphs show the relative expression of cleaved caspase-3 and Bcl-2 proteins normalized to GAPDH, respectively. (D) A representative Western blot band and bar graph depicting Bax protein expression. Values are expressed as mean ± S.E.M., for four to five rats in each group. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus the control rats, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus the diabetic rats. Bcl-2: B-cell lymphoma 2.

4. Discussion

T2D is characterized by defective insulin secretion from pancreatic β-cells, coupled with insulin resistance.¹ It is well known that β-cell failure to compensate for insulin resistance contributes to persistent hyperglycemia.^1,3,4 Therefore, exploring the therapeutic agents for the protection of pancreatic β-cells and the improvement of their function to synthesize and release insulin is important in the prevention and control of diabetes. Several researchers have reported that the active ingredients in functional foods demonstrate an anti-diabetic effect and potential biological activity in the protection of β-cells.²⁸ Among these active compounds, tocopherols, phytosterols, polyphenols, and ω-3 PUFAs that are present in sacha inchi oil exert strong antioxidant and anti-inflammatory activities, which are associated with positive effects in the control of hyperglycemia.^{11,12,14,19,29,30} In our study, we scientifically evaluated the anti-diabetic activity of extra virgin sacha inchi oil (EVSIO) and its mechanism of action on the protection of pancreatic β-cells in the HFD/STZ-induced diabetic model. These findings demonstrated for the first time that EVSIO has a hypoglycemic effect by improving pancreatic β-cell function and preserving β-cells from apoptotic death in the rat model of diabetes. The beneficial activities of EVSIO on diabetic rats also proceed via enhancing its antioxidant and anti-inflammatory properties.

The combination of HFD and STZ to induce diabetes in a rat model is widely accepted in studies evaluating the antidiabetic actions of medicines and functional foods. The HFD/STZ-treated rats present the metabolic characteristic of T2D, indicated by the elevated blood glucose, impaired glucose tolerance, insulin resistance, and reduced insulin hormone.^10,22 HFD causes peripheral insulin resistance and glucose intolerance, while low-doses of STZ cause mild destruction of pancreatic β-cells, which leads to diminished insulin release and action.²¹ This further reduces peripheral glucose utilization and enhances hepatic glucose production, thereby ultimately inducing a hyperglycemia state.²² In our study, increased FBG, along with decreased serum insulin and impaired glucose tolerance, were found in the diabetic rats induced by a combination of HFD and STZ. The diabetic rats exhibited a reduction in body weight, which is due to insulin insufficiency to prevent fat and protein breakdown.³¹ Also, the HFD/STZ-induced diabetic rats demonstrated excessive water intake, due to a glucosuria-mediated loss of urine, and subsequent stimulation of excessive thirst.³² However, food intake was not altered in the diabetic rats; probably due to satiation from the consumption of HFD.³² Importantly, we found that the dose-dependent treatment of the diabetic rats with EVSIO (0.5, 1, and 2 ml/kg b.w.) significantly ameliorated FBG and tended to decrease body weight loss and water intake, suggesting its anti-diabetic potential (Table 1). Furthermore, our results demonstrated that the glucose AUG was significantly elevated in the diabetic rats during OGTT. This observation indicates dysfunction of β-cells and insulin insensitivity. On the other hand, EVSIO treatment was able to dose-dependently reduce the AUG of glucose in the diabetic rats (Fig. 2A.). The decrease in the glucose AUG level may be due to improved β-cell function releasing insulin, along with increased glucose uptake and utilization by peripheral tissues. This is consistent with prior clinical study that reported sacha inchi oil reduced high blood glucose levels and glucose intolerance in healthy humans.¹⁸ Sacha inchi oil also contains high contents of ω-3 PUFAs, tocopherol, and polyphenolic compounds, which provide beneficial effects in the prevention of diabetes.^12,19,29 We found that EVSIO consists of 41.29–41.73% ω-3, 40.16–40.54% ω-6, 8.73–9.16% ω-9, 8.32–8.54% total saturated fatty acids, and ω-6/ω-3 ratio of 0.96–0.98 (Supplementary Table 1). According to previous studies, an increment in ω-3 PUFAs intake and a decrement in the ω-6/ω-3 PUFAs ratio are likely associated with reducing the risk of metabolic disorders such as obesity, insulin resistance, and diabetes.³³ ω-3 PUFAs therapy exhibited beneficial actions in lowering blood glucose and improving insulin sensitivity in T2D rats.³⁴ Dietary flaxseed oil rich in ω-3 PUFAs supplementation was also shown to reduce FBG in diabetic rats.³⁵ In addition, dietary polyphenols have been reported to possess antidiabetic properties in several animal models.^12,36 Therefore, it is postulated that EVSIO has these compounds which were responsible for the anti-hyperglycemic effect in the diabetic rats.

Dyslipidemia is characterized by elevated TG, TC and LDL-C, coupled with reduced HDL-C, which are the most common features seen in diabetes.^37,38 Also, it is the major contributor to increased cardiovascular risk in diabetic patients.^37,38 Defects in insulin secretion and actions cause a decrease in lipoprotein lipase activity which affects the clearance of circulating triglycerides, thus promoting diabetic hypertriglyceridemia.^37,38 Hormone-sensitive lipase is regulated by insulin and its actions are enhanced in diabetes, resulting in activating adipocyte lipolysis which ultimately leads to increased triglyceride production and VLDL-C secretion from the liver.^37,38 Additionally, impaired insulin action causes a defect in the clearance of VLDL-C.^37,38 In diabetes, cholesteryl ester transfer protein (CETP)-mediated exchange of VLDL triglyceride for HDL cholesteryl esters is activated, which leads to increased HDL degradation by hepatic lipase enzyme.^37,38 LDL receptors are reduced in diabetes which affects LDL catabolism, causing increased plasma LDL.^37,38 In clinical studies, diabetic patients with insulin deficiency and resistance showed the induction of plasma TG, TC and LDL-C levels, but the reduction of HDL-C levels.³⁸ In our study, HFD/STZ-induced diabetic rats demonstrated a significant increase in serum TG and TC levels, as demonstrated in prior published reports.^31,39 However, the serum HDL-C remained stable in the diabetic rats and this was indicated by the level not being reduced over the 5 weeks of our experiment, even in severe cases of diabetes. Thus, in some conditions of diabetes, the HDL-C level decreases in the long term.³² Interestingly, the different doses of EVSIO significantly reduced the elevated TG and TC levels in the diabetic rats, indicating the hypolipidemic effect of EVSIO (Table 2). The present observations corroborate the earlier findings of decreased TG, TC, and LDL-C levels in obese rats treated with sacha inchi oil.¹⁶ In a previous study of patients with hypercholesterolemia, sacha inchi oil consumption was also shown to result in a marked reduction of TC and LDL-C levels and an induction of the HDL-C.¹⁷ Similar findings have been reported indicating that the consumption of sacha inchi oil can decrease the levels of TC and LDL-C, and increase HDL-C, in healthy people.¹⁸ In addition, ω-3 PUFAs-enriched perilla oil and flaxseed oil exhibited a decrease in hypertriglyceridemia in diabetic mice and rats.^35,40 In an in vitro study of hepatocytes-treated with ω-3 PUFAs, it was found to inhibit VLDL-C secretion and the expression of hepatocyte nuclear factor-4 alpha (HNF-4α) which serves as the activation of microsomal triglyceride transfer protein (MTTP), thereby leading to decreased VLDL-triglyceride export from hepatocytes.⁴¹ Collectively, a decrease in serum TG and TC of the diabetic rats supplemented with EVSIO may be explained by either enhanced insulin action and/or reduced secretion and formation of VLDL-C and LDL-C.

In the progressive stages of diabetes, persistent hyperglycemia accelerates the damage to pancreatic β-cells, thereby aggravating pancreatic β-cell dysfunction and insulin deficiency.^1,3 T2D has also been shown to be closely associated with damaged β-cells of the pancreas.^9,10 Similarly, our study found that pancreatic β-cell defects were observed in HFD/STZ-induced diabetic rats, as shown in a histopathological analysis with islet atrophy and disruption of islet architecture, along with attenuated contents of pancreatic and serum insulin. Conversely, the diabetic rats treated with EVSIO displayed an improvement in pancreatic islet structure as evidenced by increased islet area and decreased islet degeneration (Fig. 3). This correlates with the enhancement of pancreatic β-cell function by a rise in pancreatic and serum insulin levels in EVSIO-treated diabetic rats (Fig. 2B and C), suggesting EVSIO's ability to preserve the pancreatic β-cells from damage, and to restore β-cell function in diabetic rats. Supporting these results, it was reported that the phytoconstituents such as omega-3, β-sitosterol, α-tocopherol, and polyphenol, which are found in sacha inchi oil, show a protective effect on pancreatic β-cells and improvement in the function of β-cells for synthesis and secretion of insulin in diabetic models.^12,42, 43, 44, 45

Extensive studies have reported that oxidative stress plays a vital role in the development of pancreatic β-cell failure.^6,7 In diabetic conditions, excessive glucose causes an increase in ROS production and a decrease in antioxidant defense mechanisms, thereby activating oxidative stress.^3,7 Excess ROS generation has been shown to suppress the pancreatic β-cell function of secreting insulin; and, conversely, a reduction of ROS and increased antioxidant enzyme defenses can protect against pancreatic β-cell damage induced by glucotoxicity and improve their function.5, 6, 7 Several studies have demonstrated the status of oxidative stress in HFD/STZ-induced diabetic rats associated with increased MDA and decreased activities of antioxidant SOD, GPx, and CAT.^31,39 Similarly, we found an induction of the biomarker of oxidative stress-MDA levels in serum and pancreas, and diminution in the activities of serum SOD, GPx and CAT, as well as pancreatic GPx in HFD/STZ-induced diabetic rats (Fig. 4). Surprisingly, our study results showed a significant enhancement in the pancreatic SOD and CAT activities in the diabetic rats (Fig. 4). This could be explained by the fact that SOD and CAT are known to be highly efficient and high-affinity antioxidants that act as the first detoxification enzyme against ROS production.⁴⁶ Increased pancreatic SOD and CAT activities neutralize excessive ROS production in the pancreas. However, this antioxidant response may not be enough to compensate for the scavenging of excess free radicals, thus oxidative stress predominates in the pancreas of diabetic rats. It seems, however, that EVSIO treatment counteracts these alterations where increased serum and pancreatic MDA levels in the diabetic rats were inhibited by EVSIO, while in the EVSIO-treated diabetic rats, the activities of serum SOD, and serum and pancreatic GPx, were restored and antioxidant SOD in the pancreas was decreased (Fig. 4). These results suggest that diabetes promotes increased oxidative stress in both the local pancreas and systemic levels, whereas EVSIO has anti-oxidative properties against diabetes-induced oxidative damage. This finding agrees with a previous study concerning the antioxidant efficacy of sacha inchi oil in rats induced to obesity.¹⁶ Other studies have also demonstrated that ω-3 PUFAs have a potent ability to reduce serum MDA levels, and elevated SOD levels in diabetic rats.^35,47 Certain specific phytochemicals, such as tocopherols and polyphenols were reported to possess a strong antioxidant property as diminished ROS/RNS generations.^12,29 In diabetic animal models, dietary polyphenol supplementation demonstrated anti-oxidative damage in the pancreas by decreasing MDA content and increasing the activities of antioxidant SOD, CAT, and GPx.¹² Furthermore, tocopherol extracted from pumpkin seed oil has been found to inhibit lipid peroxidation and activate endogenous enzymatic and non-enzymatic antioxidant levels in the liver of diabetic rats.⁴⁸ Hence, the presence of these active compounds in EVSIO may mitigate the oxidative damage of the pancreas in diabetes, which leads to improved β-function, further reducing hyperglycemia.

Inflammation, similar to oxidative stress, is an important process promoting pancreatic β-cell damage.^7,8 Chronic hyperglycemia enhances an increase in the generations of free radicals and pro-inflammatory cytokines, which result in the activation of NF-κB. In turn, transcription factor NF-κB is known to trigger the production of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, which can contribute to local and systemic inflammation.^7,8 Several studies have reported that an increase in NF-κB and pro-inflammatory proteins can induce pancreatic β-cell apoptosis and dysfunction.^7,8 Our study is in agreement with previous findings^49,50 that demonstrated increased levels of both serum and pancreatic TNF-α and IL-6. As well, overexpression of pancreatic NF-κB protein was observed in HFD/STZ-treated diabetic rats. Again, these alterations were significantly abrogated by EVSIO treatment, except that serum TNF-α in the diabetic rats remained unchanged after treatment with EVSIO (Fig. 5), indicating that EVISO might not have the ability to suppress augmented circulating TNF-α levels, caused by the increased proliferation and size of adipocytes in T2D which leads to over-releasing of proinflammatory cytokines, mainly TNF-α, into the serum.⁵¹ Previously, it has been reported that supplementation of sacha inchi oil can inhibit serum IL-6 and hepatic TNF-α in obese rats.¹⁶ In the diabetic rat model, ω-3 PUFAs treatment decreased IL-6 levels in plasma and the pancreas, liver, and kidneys.⁵² As discussed earlier, ω-3 PUFAs in the form of EPA and DHA, demonstrated anti-inflammatory effects, which were found to decrease in the plasma of patients with T1D and T2D.^53,54 Supplementation with dietary ω-3 PUFAs including EPA and DHA also reduced the levels of serum TNF-α, IL-6, and C-reactive protein (CRP) in obese-insulin resistance rats.⁵⁵ Fish oil, a rich source of EPA and DHA, has been reported to alleviate the expression of NF-κB and TNF-α proteins in the hippocampus of diabetic rats.⁵⁶ Furthermore, accumulating evidence has demonstrated that polyphenol-rich diets have a potent anti-inflammatory action by suppressing pro-inflammatory molecules in diabetes models.¹² Overall, it could be suggested that the ω-3 PUFAs and polyphenols with anti-inflammatory effects in EVSIO, may play a role in protecting pancreatic β-cells from inflammation and damage caused under diabetic conditions.

Recent observations have indicated the involvement of hyperglycemia-mediated oxidative and inflammatory stress in the induction of pancreatic β-cell apoptosis.⁷ An increase in ROS and inflammatory mediators inhibits anti-apoptotic Bcl-2 and activate pro-apoptotic Bcl-2 proteins. This promotes the release of cytochrome c and caspase-3 apoptotic cascade, which ultimately leads to β-cell apoptosis.7, 8, 9 Upregulation of cleaved caspase-3 plays a pivotal role in the acceleration of apoptotic caspase cascade in pancreatic β-cells of diabetes.⁹ In this context, the expression of pancreatic Bax and cleaved caspase-3 proteins have been reported to increase in many animal models of diabetes,57, 58, 59, 60 as well as the downregulation of Bcl-2 protein that was observed in the pancreas of diabetic rats.^58,61 In our study, we found the upregulated Bax and cleaved caspase-3 proteins, and downregulated protein expression of Bcl-2 in the pancreas of the HFD/STZ-induced diabetic rats. Importantly, all these protein expressions in the diabetic rats were significantly reversed in a dose-dependent manner by EVSIO treatment (Fig. 6). This effect of EVSIO against β-cells that undergo apoptosis induced by hyperglycemia was probably due to the improved blood glucose and anti-apoptotic Bcl-2 proteins, which play a major role in the regulation of cell apoptosis, and hence induced β-cell survival. According to previous studies, ω-3 PUFA series; EPA and DHA, protected against alloxan-induced death of pancreatic β-cells in vitro,⁴⁵ and also were able to induce cell viability and prevent apoptosis of pancreatic β-cells induced by STZ.⁴⁴ Further, the study of diabetic rats demonstrated that EPA and DHA-enriched fish oil treatment can decrease neuronal apoptosis by reducing caspase-9 protein expression.⁵⁶ Other studies reported that supplementation with ω-3 PUFAs to diabetic rats ameliorated testicular apoptosis, which is related to the upregulation of Bcl-2 and downregulation of caspase-3 expression.⁶² Many research reports on plant phenolic compounds have also reported the ability of these phenolic compounds to inhibit pancreatic β-cell apoptosis in diabetic models by enhancing anti-apoptotic Bcl-2 expression protein and suppressing pro-apoptotic Bcl-2 and cleaved caspase 3 proteins.^12,63 Some studies have also found that γ-tocopherol inhibits proapoptotic Bax in the liver of diabetic mice.⁶⁴ Based on this information, it is reasonable to propose that EVISO, which contains high contents of ω-3 PUFA series, polyphenols, and tocopherol, possess an anti-apoptotic effect, which may play a protective role against diabetes-induced pancreatic β-cell apoptosis.

Sacha inchi oil consumption is regarded as safe in humans. Clinical trials showed that the 16-week supplementation of 5 or 10 ml (2 and 4 g ω-3/day) of sacha inchi oil can decrease serum cholesterol and LDL-C and increase HDL-C in patients with hypercholesterolemia.¹⁷ The consumption of 15 ml/day of sacha inchi oil has a beneficial effect on postprandial insulin sensitivity in healthy adults, which is correlated with increased sirtuin-1 expression.¹⁸ Sirtuin-1 protein plays an important role in the protection of pancreatic β-cells as it improves insulin sensitivity by reducing oxidative stress and inflammation.⁶⁵ In our study, EVSIO at a dose of 2 ml/kg/day showed itself to be much more effective in ameliorating hyperglycemia and pancreatic β-cell dysfunction and death in diabetic rats than other doses (0.5 and 1 ml/kg/day). Using the allometric scaling for calculating and converting doses from rat to human,⁶⁶ the human equivalent dose of 2 ml/kg/day EVSIO (0.84 g ω-3/kg/day; Supplement Table 2) is around 20 ml/day (8.4 g ω-3/day) for a 60 kg person. However, the optimal dose and anti-diabetic effect of EVSIO in clinical studies are needed to further investigate.

5. Conclusion

Based on this study, our study proffers new information on the antidiabetic potential of EVSIO in T2D rats. Overall, the results of the present study provide evidence that the dosage of 2 ml/kg EVSIO treatment is the effective dose for protection against diabetes induced by HFD/STZ in the rat model. EVSIO treatment can reduce hyperglycemia, hyperlipidemia, and improve glucose tolerance in diabetic rats. The beneficial properties of EVSIO on glycemic control are markedly involved in the protection of the pancreatic β-cells against damage and improvement in their function by enhancing insulin production and secretion. Furthermore, EVSIO suppresses oxidative and inflammatory stress, and induces the expression of anti-apoptotic Bcl-2 proteins, thereby causing the inhibition of β-cell apoptosis and improvement in β-cell function, ultimately decreasing hyperglycemia. Therefore, EVSIO is a promising substance for the development of effective anti-diabetic agents. However, further experimental and clinical studies are required to clarify the precise and complete mechanisms of its anti-diabetic action.

Authors’ contributions

WH: supervision, conceptualization, funding acquisition, methodology, investigation, data analysis, data curation, writing-original draft preparation, writing-reviewing and editing. NW: investigation, data analysis, data visualization, data curation, manuscript preparation. WR: investigation. TB: methodology, funding acquisition. All authors read and approved the final manuscript.

Declaration of competing interest

Authors declare that there is no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: