Sacha Inchi (Plukenetia volubilis L.) Oil Improves Hepatic Insulin Sensitivity and Glucose Metabolism through Insulin Signaling Pathway in a Rat Model of Type 2 Diabetes

Abstract

This study aimed to evaluate the role of sacha inchi oil (SI) in alleviating hepatic insulin resistance and improving glucose metabolism by inhibiting oxidative stress and inflammation in a rat model of type 2 diabetes. This model was established by providing a high-fat diet and streptozotocin to the rats, thereby inducing diabetes. The diabetic rats were treated orally with 0.5, 1, and 2 mL/kg body weight (b.w.) of SI or 30 mg/kg b.w. of pioglitazone daily for 5 weeks. Blood and hepatic tissues were used for insulin sensitivity, carbohydrate metabolism, oxidative stress, and inflammatory status assessment. Treatment with SI attenuated hyperglycemia and insulin resistance indices, and improved hepatic histopathological alterations in the diabetic rats in a dose-dependent manner, which is correlated with the decreased serum levels of the liver enzymes, alanine transaminase and aspartate transaminase. SI significantly diminished the hepatic oxidative status of the diabetic rats by inhibiting malondialdehyde and enhancing the antioxidant superoxide dismutase, catalase, and glutathione peroxidase activities. Moreover, pro-inflammatory cytokine levels, including tumor necrosis factor-α and interleukin-6, in the liver of the diabetic rats were significantly decreased by the SI. Furthermore, SI treatment enhanced the hepatic insulin sensitivity of the diabetic rats, as shown by the increased insulin receptor substrate-1 and p-Akt protein expression, decreased phosphoenolpyruvate carboxykinase-1 and glucose-6-phospatase protein expression, and increased hepatic glycogen content. Overall, these findings suggest that SI exerts a potential hepatic insulin-sensitizing effect and an improvement in glucose metabolism in the type 2 diabetic rats, at least in part through enhancing insulin signaling, antioxidant defense, and inhibiting inflammation.

INTRODUCTION

Type 2 diabetes (T2D) is one of the most common metabolic disorders, affecting millions of people worldwide, and it is a disease that leads to poor health outcomes (Magliano and Boyko, 2021). This disease is presented as hyperglycemia resulting from peripheral insulin resistance and relative insulin deficiency (Galicia-Garcia et al., 2020). Disturbances in carbohydrate, fat, and protein metabolisms occur because of an impairment of insulin action on target tissues, particularly the muscle, adipose tissue, and liver (Wilcox, 2005). By regulating glycolysis, gluconeogenesis, and glycogenolysis, the liver is a major target organ for insulin action (Postic et al., 2004; Titchenell et al., 2017). A defect in the hepatic insulin signaling pathway leads to increased gluconeogenesis and glycogenolysis, thereby inducing glucose production and release by the liver, ultimately resulting in excessive hyperglycemia (Guo, 2014; Jiang et al., 2020). Therefore, to maintain euglycemia, improving the hepatic insulin signaling-mediated carbohydrate metabolism is one of the important factors in the treatment of T2D.

Several mechanisms are postulated to explain the impaired insulin effect on the liver (Petersen and Shulman, 2018; Yaribeygi et al., 2019). Previous studies suggested that both oxidative stress and inflammation are the main causes contributing to hepatic insulin resistance and impaired glucose metabolism in T2D (Keane et al., 2015; Bin-Jumah, 2019). In the persistent hyperglycemia state, reactive oxygen species (ROS) overproduction along with decreasing antioxidant enzyme protection, and coupled with increasing pro-inflammatory mediators, have been reported to interfere with the insulin signaling pathway by the inhibition of insulin receptor substrate (IRS)/phosphoinositide 3-kinase (PI3K)/Akt pathway activation, thereby leading to enhanced hepatic insulin insensitivity and altered glucose metabolism, ultimately promoting severely increased blood glucose levels (Rains and Jain, 2011; Rehman and Akash, 2016; Huang et al., 2018). Therefore, suppressing oxidative/inflammatory stress activation in diabetes is a significant objective in ameliorating insulin resistance for T2D treatment.

A high-fat diet (HFD) and low-dose streptozotocin (STZ) combination has been widely used to develop an animal model of T2D, which closely mimics the pathogenesis and natural history of human T2D (Srinivasan et al., 2005; Gheibi et al., 2017). HFD causes obesity with an increased visceral fat accumulation, contributing to whole-body insulin resistance by suppressing the IRS/PI3K/Akt pathway activation (Gheibi et al., 2017). By inducing ROS generation and DNA fragmentation, STZ selectively destroys insulin-producing pancreatic β-cells (Eleazu et al., 2013). Low-dose STZ has been known to produce partial β-cell damage, causing relative insulin deficiency (Zhang et al., 2008; Eleazu et al., 2013). Therefore, an animal model of T2D caused by HFD combined with low-dose STZ provides similar characteristics to human T2D, such as dysfunctional pancreatic-β cells, insulin resistance, and impaired glucose tolerance (Srinivasan et al., 2005; Gheibi et al., 2017). This makes it an appropriate model for studying the beneficial actions of new therapeutic agents for T2D.

Medicinal plants and foods possessing antioxidant and anti-inflammatory properties have received attention as potential new targets in diabetes management (Necyk and Zubach-Cassano, 2017; Xu et al., 2018). Plukenetia volubilis L., commonly known as sacha inchi, is a perennial plant of the family Euphorbiaceae, which produces oil- and protein-rich seeds (Goyal et al., 2022). Sacha inchi oil (SI) is reported to have essential active ingredients, including phenolic acids, tocopherols, flavonoids, and phytosterol; these active compounds demonstrate strong antioxidant activity that inhibits oxidative stress (Cárdenas et al., 2021). Additionally, omega-3 fatty acids, found in quantity in SI, are known to possess antioxidant, anti-inflammatory, and anti-hyperglycemic properties (Adeyemi and Olayaki, 2018; Eraky et al., 2018). Several studies have demonstrated the beneficial properties of SI in moderating inflammation, controlling cholesterol levels, and decreasing cardiovascular diseases (Alayón et al., 2019; Cárdenas et al., 2021; Goyal et al., 2022). In a clinical study, SI improves the lipid profile and insulin level of individuals with dyslipidemia (Garmendia et al., 2011). Similar studies have reported that SI increases high-density lipoprotein cholesterol levels and decreases low-density lipoprotein cholesterol levels as well as arterial blood pressure in adult human participants (Gonzales and Gonzales, 2014). In another study, SI has also shown anti-lipidemic and anti-inflammatory properties in metabolically unhealthy patients (Alayón et al., 2019). In the obesity-induced animal model, SI exhibits a positive effect on the lipid profile and reduces oxidative stress by increasing antioxidant activities, as well as pro-inflammatory factors in the rats induced to obesity (Ambulay et al., 2020). Recently, by inhibiting hepatic triglyceride accumulation, de novo lipogenesis, and inflammation, as well as enhancing fatty acid β-oxidation, SI treatment has been shown to alleviate lipid dysmetabolism in the liver of HFD-fed rats (Li et al., 2020).

Considering these previously reported findings, we hypothesized that the presence of these active compounds in SI may have a beneficial effect on improving insulin sensitivity in the diabetes state. Therefore, we investigated the role of SI in alleviating hepatic insulin resistance and improving glucose metabolism by inhibiting oxidative stress and inflammation in the experimental animal model of T2D.

MATERIALS AND METHODS

Reagents

SI was obtained from a local company in Thailand; its composition is shown in Table 1. Pioglitazone hydrochloride (PZ) was acquired from Berlin Pharmaceutical Industry Co., Ltd.. Primary antibodies, including insulin receptor-β (IR-β), IRS-1, phosphoenolpyruvate carboxykinase-1 (PEPCK/PCK-1), p-Akt (Ser 473), and Akt, and a secondary antibody were procured from Cell Signaling Technology, Inc.. Primary antibody against glucose 6-phosphatase (G-6-Pase) was obtained from Abcam. STZ and all chemicals were purchased from Sigma-Aldrich Co. and Merck Millipore.

Table 1.

Main composition of the sacha inchi oil administered to rats

Fatty acids	Molecular formula	Percentage
Myristic acid	C14:0	0.02
Palmitic acid	C16:0	4.41
Heptadecanoic acid	C17:0	0.11
Stearic acid	C18:0	3.62
Arachidic acid	C20:0	0.11
Behenic acid	C22:0	0.03
Tricosanoic acid	C23:0	0.01
Lignoceric acid	C24:0	0.01
Saturated fat	−	8.32
Palmitoleic acid	C16:1n7	0.04
Cis-10-heptadecenoic acid	C17:1n10	0.05
Trans-9-elaidic acid	C18:1n9t	0.05
Cis-9-oleic acid	C18:1n9c	9.16
Cis-11-eicosenoic acid	C20:1n11	0.27
Monounsaturated fatty acid	−	9.58
Cis-9,12-linoleic acid	C18:2n6	40.54
γ-Linolenic acid	C18:3n6	0.20
α-Linolenic acid	C18:3n3	41.29
Cis-11,14-eicosadienoic acid	C20:2	0.05
Cis-11,14,17-eicosatrienoic acid	C20:3n3	0.01
Total polyunsaturated fatty acid		82.10
Total unsaturated fat		91.68
Total omega-3		41.30
Total omega-6		40.75
Total omega-9		9.16

Animals

Male Sprague-Dawley rats [approximately 160∼180 g body weight (b.w.)] purchased from the Nomura Siam International were maintained in the Center for Animal Research Naresuan University under temperature at 22±1°C with a 12/12-h light-dark cycle. All animals were acclimated for 1 week before starting the experiment. Animal procedures in this study were approved by the Animal Ethics Committee of Naresuan University (no. NU-AE620616).

Induction of diabetes and experimental design

Rat diabetes was induced with an HFD (58%, 20%, and 22% kcal from fat, protein, and carbohydrate, respectively, Bangkok Animal Research Center Co., Ltd.), which was fed to the rats for 2 weeks, followed by intraperitoneal injection of STZ (40 mg/kg b.w.). In the control group, the rats were fed with a normal diet and intraperitoneally injected with citrate buffer (0.1 M; pH 4.5). After 3 days of STZ injection, blood was collected from the rats’ tail veins for fasting blood glucose (FBG) level measurement. The animals with FBG levels of more than 11.1 mmol/L were defined as diabetic and used for further experiments.

The rats were randomly assigned into six groups (n=6/group): 1) normal control group; 2) diabetic group; 3) SI (0.5 mL/kg b.w.)-treated diabetic group; 4) SI (1 mL/kg b.w.)-treated diabetic group; 5) SI (2 mL/kg b.w.)-treated diabetic group; and 6) antidiabetic drug, PZ (30 mg/kg b.w.)-treated diabetic group. The chosen doses of SI were based on a previous study that demonstrated the beneficial roles of SI in ameliorating HFD-induced hyperlipidemia and hepatic inflammation and steatosis in rats (Li et al., 2020). The three different doses of SI (0.5, 1, and 2 mL/kg b.w./d) used in this study are equivalent to dietary supplements of approximately 5, 10, and 20 mL/d of SI for a 60-kg human, respectively, as calculated using the rat-to-human translation method described by Nair and Jacob (2016). A clinical study has reported that consumption of 10 and 15 mL/d of SI for 16 weeks is safe (Gonzales and Gonzales, 2014). Another clinical trial has shown that SI supplementation at a dose of 15 mL/d can increase postprandial insulin sensitivity in healthy humans (Alayón et al., 2018).

After 5 weeks of orally administered treatment, the rats’ FBG levels were determined using an Advanced Accucheck glucometer (Roche Diagnostics GmbH). Furthermore, to evaluate glucose tolerance, the oral glucose tolerance test (OGTT) was performed. After glucose load (2 g/kg b.w.) to the fasting rats, blood samples were obtained from the rats’ tail veins at 0, 0.5, 1, 1.5, and 2 h for glucose level measurement. The rats were subsequently euthanized by injection with 100 mg/kg b.w. of sodium thiopental, and blood and liver tissue samples were collected.

Insulin sensitivity determination

Homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) are generally accessible for insulin resistance assessment (Li and Pan, 1993; Singh and Saxena, 2010). HOMA-IR and ISI were calculated using the following equations (Li et al., 2019):

$$\begin{array}{l}\text{HOMA-IR=}\frac{\text{fasting insulin (}\mu \text{U/mL)}\times \text{fasting glucose (mmol/L)}}{22.5}\\ \text{ISI=ln}\frac{1}{\text{fasting insulin (}\mu \text{U/mL)}\times \text{fasting glucose (mmol/L)}}\end{array}$$

Measurement of serum liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST)

Blood samples were collected from the rats in each group by cardiac puncture. The samples were centrifuged at 14,000 g for 30 min at 4°C to obtain the serum samples, which were sent to Biolab Medical Clinic for analysis. The serum ALT and AST levels were determined using the automated biochemical analyzer (Cobas, Roche Diagnostics GmbH).

Histopathological analysis of the liver tissue

To study the alterations of liver histopathology, the hepatic tissues were stained with hematoxylin and eosin (H&E). In brief, the liver tissues that were obtained from the experimental rats were washed with cold phosphate-buffered saline (PBS) and fixed in 10% neutral-buffered formalin for 2 days. The tissues were dehydrated by the graded ethanol and isopropanol series and infiltrated by paraffin. The embedding tissues in paraffin blocks were cut into 5-μm-thick sections and subsequently deparaffinized and stained with H&E to assess the liver histopathological changes.

Hepatic antioxidant status and oxidative stress marker evaluation

Assay of superoxide dismutase (SOD) activity: The SOD activity was examined using the pyrogallol method, following the procedures described by Marklund and Marklund (1974). The liver tissue was homogenized with cold PBS and centrifuged at 14,000 g for 15 min at 4°C. Subsequently, the supernatant was obtained and mixed with Tris-ethylene-diamine-tetraacetic acid (EDTA) buffer (23.5 mM Tris and 2.98 mM EDTA) and 0.2 mM pyrogallol. The change in absorbance was kinetically measured for 10 min on a spectrophotometer at 420 nm. The SOD activity in the liver tissue was calculated on the basis of the ability of one unit of enzyme activity to inhibit pyrogallol autooxidation by 50% and expressed as U/mg protein.

Assay of catalase (CAT) activity: The CAT activity was determined as described previously with modifications (Prasartthong et al., 2022). The liver homogenate was prepared with cold PBS with a pH of 7.4. To determine the CAT activity, the supernatant was obtained by centrifugation at 14,000 g for 15 min at 4°C. The sample was added to the reaction buffer consisting of sodium phosphate buffer, 0.017% hydrogen peroxide (H₂O₂), and 2.54 M sulfuric acid (H₂SO₄). The reaction was started by adding 0.1 mM potassium permanganate (KMnO₄). The rate of absorbance change was recorded spectrophotometrically at 525 nm. The hepatic CAT activity was expressed as U/mg protein.

Assay of glutathione peroxidase (GPx) activity: The GPx activity was assessed by modifying the method of Paglia and Valentine (1967). The liver tissues were extracted with GPx assay buffer. After centrifugation at 10,000 g for 30 min at 4°C, the supernatant was collected and added to the reaction mixture containing 1 mM reduced glutathi-one, one unit of glutathione reductase, 0.2 mM β-nicotin-amide adenine dinucleotide phosphate (NADPH), and 1.96 mM H₂O₂ and subsequently incubated for 15 min at room temperature. The decreased absorbance of reduced NADPH was read at 340 nm using a spectrophotometer; the value was expressed as U/mg protein.

Assay of the level of malondialdehyde (MDA) as a lipid peroxidation marker: The MDA content in the liver was examined using the thiobarbituric acid (TBA) reactive substance method (Parveen et al., 2010). Briefly, the liver tissues were homogenized using cold PBS with a pH of 7.4. The mixture solution containing 8.1% sodium dodecyl sulfate (SDS), 0.8% TBA, and 20% acetic acid solution (pH, 3.5) was added to the sample or MDA standard. The reaction mixture was incubated at 95°C for 60 min, kept on ice for 5 min, and subsequently centrifuged at 4,000 rpm for 10 min at room temperature. The supernatant of the MDA-TBA product was measured using a microplate reader at 532 nm. The MDA level was expressed as nmol/g protein.

Evaluation of tumor necrosis factor-αlpha (TNF-α) and interleukin-6 (IL-6) levels in the liver tissue

The hepatic TNF-α and IL-6 levels were determined using enzyme-linked immunosorbent assay kits (PeproTech Asia) following the manufacturer’s instructions. Briefly, the liver proteins were bound against anti-rat TNF-α and IL-6 antibody in pre-coated 96-well plates. After washing, the samples were incubated with rat TNF-α and IL-6 detection antibody, followed by horseradish peroxidase avidin D (HRP Avidin D) solution. The peroxidation reaction products were subsequently created by adding a substrate and stop solution. The absorbance of the samples was measured using a microplate reader at 420 nm.

Hepatic glycogen content evaluation

The glycogen content in the liver was detected using the anthrone reagent according to the method of Hayanga et al. (2016) with modifications. The hepatic tissue was extracted by it boiling with 30% potassium hydroxide solution. The glycogen was then precipitated from the extract by 95% ethanol and centrifuged at 14,000 g for 30 min at 4°C. The glycogen products were created by adding a 0.2% anthrone reagent. Using a spectrophotometer, the absorbance at 620 nm was read. The result was expressed as the ratio of mg of glycogen per g of tissue.

Western blotting analysis

Proteins were extracted from the liver tissues using a radioimmunoprecipitation assay buffer containing 1% halt protease inhibitor. Protein concentrations were measured using a micro bicinchoninic acid protein assay kit from Merck Millipore. Subsequently, the liver proteins (40 μg) were separated on 8∼10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane using a Trans-Blot^Ⓡ wet electrophoretic transfer system (Bio-Rad Laboratories). The membranes were blocked for nonspecific protein with 5% bovine serum albumin in 1× Tris-buffered saline Tween for 1 h. After washing, the membranes were incubated with the primary antibodies recognized as anti-IR-β (1:1,000), anti-IRS-1 (1:1,000), anti-PCK-1 (1:1,000), anti-G-6-Pase (1:1,000), anti-p-Akt (Ser 473) (1:500), anti-Akt (1:500), or anti-β-actin (1:2,500) overnight at 4°C and again incubated with HRP-conjugated secondary antibodies (1:3,000) for 1 h at room temperature. After washing, the protein bands were detected using the enhanced chemiluminescence reagent kit, and the intensities of protein expressions were quantified using the Amersham ImageQuant^TM 800 system (Cytiva Corp.).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad). Data were expressed as means±standard error of the mean. Statistical significance was analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s methods for multiple comparisons. Two-way ANOVA followed by Bonferroni’s post-hoc test was used to analyze the blood glucose data. P-value <0.05 was considered statistically significant.

RESULTS

SI decreases the increased FBG levels, glucose intolerance, and insulin resistance in the diabetic rats

Data on FBG levels are presented in Fig. 1A. Results showed that following diabetes induction with HFD and STZ, the diabetic rats had higher FBG levels than the control rats. However, after treatment with SI at different doses of 0.5, 1, and 2 mL/kg b.w., the FBG levels of the diabetic rats were significantly dose-dependently decreased compared with those of the untreated diabetic rats. Similarly, 30 mg/kg b.w. of PZ treatment caused a significant decrease in the FBG levels in the diabetic rats.

Fig. 1.

Effects of sacha inchi oil (SI) on the levels of fasting blood glucose (FBG) (A), homeostatic model assessment for insulin resistance (HOMA-IR) (B), insulin sensitivity index (ISI) (C), glucose tolerance (D), serum alanine transaminase (ALT) (E), and aspartate transaminase (AST) (F) in the diabetic rats. The FBG level is measured using a digital glucometer before and after treatment with SI or pioglitazone. To estimate insulin sensitivity, HOMA-IR and ISI are calculated. Glucose levels are determined at different time points during the oral glucose tolerance test. The serum ALT and AST levels are assessed using an automated biochemistry analyzer. Data are expressed as mean±standard error of the mean (n=approximately 5~6). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 vs. the control group; ^#P<0.05, ^##P<0.01, and ^###P<0.001 vs. the diabetic group. Tt, treatment; DM, diabetic rats; PZ, pioglitazone.

Additionally, HOM-IR and ISI were used for surrogate measures of insulin resistance to determine the effect of SI on insulin sensitivity in the diabetic rats. As illustrated in Fig. 1B and 1C, compared with the control rats, the diabetic rats show a significant increment of HOMA-IR and a decrement of ISI, indicating the presence of insulin resistance in the diabetic rats. However, HOMA-IR was attenuated, and ISI was enhanced in the SI (0.5, 1, and 2 mL/kg b.w.)-treated diabetic rats, of which those in the 1 and 2 mL/kg b.w. of the SI-treated groups significantly improved. A similar outcome, demonstrated by reduced HOMA-IR and induced ISI, was observed in the diabetic rats treated with 30 mg/kg b.w. of PZ.

The measurement of the rate of glucose disposal during the OGTT can provide changes in insulin sensitivity (Chen et al., 2018). As shown in Fig. 1D, the blood glucose level of the diabetic rats reach its peak values at 1 h after oral glucose loading; compared with those in the control rats, this value was significantly maintained at a high level and reduced at the slow rate during 2-h observation periods of OGTT, reflecting impaired insulin action to promote glucose disposal in the diabetic rats. However, compared with the untreated diabetic rats, treatment of the diabetic rats with 2 mL/kg b.w. of SI can accelerate the decrease in the blood glucose levels and significantly decrease the 2-h blood glucose level. Conversely, treatment with 0.5 and 1 mL/kg b.w. of SI in the diabetic rats gradually decreased the blood glucose levels. In the PZ-treated diabetic rats, a clear decreasing trend was observed in the blood glucose levels during the OGTT. Overall, these findings suggest that SI could effectively ameliorate hyperglycemia and enhance insulin sensitivity and glucose tolerance in the HFD- and STZ-induced diabetic rats.

SI ameliorates the increment in serum ALT and AST levels in the diabetic rats

To assess liver injury, the levels of hepatic enzymes, ALT and AST, were measured. The levels of these enzymes are presented in Fig. 1E and 1F. Compared with the normal rats, the diabetic rats showed a noticeable increase in the induction of serum ALT and AST levels, indicating liver damage. After SI (0.5, 1, and 2 mL/kg b.w.) administration to the diabetic rats, the increased ALT and AST levels in the diabetic rats were markedly suppressed in a dose-dependent manner compared with those in the untreated diabetic rats. However, treatment with 30 mg/kg b.w. of PZ in the diabetic rats showed a decreasing trend in the AST level; a significant inhibition of the ALT level increment was noted in the diabetic rats. Therefore, these results indicate that SI may have a protective effect on diabetes-induced liver damage.

SI improves the hepatic histopathological changes in the diabetic rats

As illustrated in Fig. 2A and 2B, the liver of diabetic rats shows an evident hepatic abnormal morphology with a loss of normal arrangement of hepatocytes, sinusoidal dilatation, cytoplasmic vacuolation diffusion, and hepatic central vein degeneration. Quantified analysis revealed that the diabetic rats had a significant increase in the number of hepatic vacuole droplets compared with the control rats. However, these alterations in the liver histopathology of the diabetic rats were significantly reversed to closely normal conditions by treatment with SI (0.5, 1, and 2 mL/kg b.w.) in a dose-dependent manner. A similar improvement was observed in the PZ-treated diabetic rats.

Fig. 2.

Effects of sacha inchi oil (SI) on hepatic histopathology in the diabetic rats. (A) Photomicrographs showing the histopathological alterations of the liver tissues in different experimental groups measured by hematoxylin and eosin (H&E) staining. Upper images, magnification 20×; lower images, magnification 10×; scale bar=50 μm. (B) Bar graph presents the number of vacuole droplets per area (mm²) in the liver tissues of each group. Data are expressed as mean±standard error of the mean (n=6). ****P<0.0001 vs. the control group; ^####P<0.0001 vs. the diabetic group. DM, diabetic rats; PZ, pioglitazone; HC, hepatocytes; V, cytoplasmic vacuolation; SI, sinusoid; CV, central vein.

SI abolishes the increased TNF-α and IL-6 levels in the liver of the diabetic rats

Pro-inflammatory cytokines are widely accepted to be involved in insulin resistance pathogenesis. Thus, the hepatic TNF-α and IL-6 levels were determined in this study. As shown in Fig. 3A and 3B, in the diabetic group, the levels of pro-inflammatory cytokines, TNF-α and IL-6, are significantly increased in the liver compared with those in the control rats. Inversely, the increased hepatic TNF-α and IL-6 levels in the diabetic rats were significantly inhibited by treatment with 2 mL/kg b.w. of SI or PZ. However, the increased TNF-α and IL-6 levels in the liver of the diabetic rats were unaffected by treatment with SI at doses of 0.5 and 1 mL/kg b.w.

Fig. 3.

Effects of sacha inchi oil (SI) on the hepatic tumor necrosis factor-αlpha (TNF-α) (A) and interleukin-6 (IL-6) (B) levels in the diabetic rats. The hepatic TNF-α and IL-6 levels are measured using enzyme-linked immunosorbent assay kits. Data are expressed as mean±standard error of the mean (n=approximately 4~5). *P<0.05 vs. the control group; ^##P<0.01 and ^###P<0.001 vs. the diabetic group. DM, diabetic rats; PZ, pioglitazone.

SI enhances antioxidant enzyme activities and mitigates lipid peroxidation in the liver of the diabetic rats

As presented in Fig. 4A∼4C, compared with those in the control rats, the activities of antioxidant enzymes SOD, CAT, and GPx are significantly decreased in the liver of the diabetic rats. Compared with the non-treated diabetic rats, treatment with SI significantly increased the SOD, CAT, and GPx activities in the liver of diabetic rats in a dose-dependent manner. In the PZ-treated diabetic group, a significant restoration in the activities of hepatic SOD and CAT levels was observed; however, no significant improvement in the GPx activity was noted.

Fig. 4.

Effects of sacha inchi oil (SI) on the activities of antioxidant enzymes, superoxide dismutase (SOD) (A), catalase (CAT) (B), glutathi-one peroxidase (GPx) (C), and malondialdehyde (MDA) (D) level in the liver of the diabetic rats. Data are expressed as mean±standard error of the mean (n=approximately 4~5). **P<0.01 vs. the control group; ^#P<0.05, ^##P<0.01, and ^###P<0.001 vs. the diabetic group. DM, diabetic rats; PZ, pioglitazone.

Moreover, compared with the control group, the diabetic rats demonstrated a significant enhancement in the hepatic MDA content owing to their diabetic conditions. Conversely, the increased hepatic MDA content in the diabetic rats was markedly suppressed by treatment with SI in a dose-dependent manner, whereas PZ treatment slightly decreased the hepatic MDA level of the diabetic rats (Fig. 4D).

SI increases the hepatic glycogen content in the diabetic rats

As presented in Fig. 5, the diabetic rats show a significant reduction in the hepatic glycogen content compared with the control rats. Treatment with SI at 0.5 and 1 mL/kg b.w. doses failed to enhance the glycogen content in the liver of the diabetic rats. Conversely, treatment with 2 mL/kg b.w. of SI or PZ significantly improved the glycogen content in the liver of diabetic rats compared with that in the untreated diabetic rats.

Fig. 5.

Effects of sacha inchi oil (SI) on the hepatic glycogen content in the diabetic rats. The glycogen level is detected using the anthrone method. Data are expressed as mean±standard error of the mean (n=5). *P<0.05 vs. the control group; ^##P<0.01 and ^###P<0.001 vs. the diabetic group. DM, diabetic rats; PZ, pioglitazone.

SI improves the insulin signaling protein expression levels in the liver of the diabetic rats

Fig. 6A∼6C illustrates the IR-β, IRS-1, p-Akt (Ser 473), and Akt protein expressions. The diabetic rats showed IR-β protein expression upregulation; however, compared with the control rats, a significant downregulation in the IRS-1 protein expression and the p-Akt (Ser 473)/Akt ratio in the liver was noted. On SI administration (0.5, 1, and 2 mL/kg b.w.) to the diabetic rats, the hepatic IR-β protein was significantly decreased, and the IRS-1 expression levels and the p-Akt (Ser 473)/Akt protein ratio were dose-dependently increased compared with the untreated diabetic rats. The PZ-treated diabetic rats showed similar results with a significant reduction in the IR-β protein expression and a significant induction in the protein expression of IRS-1 and the p-Akt (Ser 473)/Akt ratio in the liver.

Fig. 6.

Effects of sacha inchi oil (SI) on the expression of proteins associated with the insulin signaling pathway and carbohydrate metabolism in the liver of the diabetic rats. The protein levels of insulin receptor-β (IR-β), IR substrates-1 (IRS-1), p-Akt (Ser 473), protein kinase B (Akt), phosphoenolpyruvate carboxykinase-1 (PCK-1), and glucose 6-phosphatase (G-6-Pase) are detected using western blotting. In each figure, the upper panel shows a representative western blot band, and the lower graph represents the statistical analysis of the relative protein expression of IR-β (A), IRS-1 (B), p-Akt (Ser 473) (C), PCK-1 (D), and G-6-Pase (E) over the control. β-Actin is used for the normalization of IR-β, IRS-1, PCK-1, and G-6-Pase proteins, and p-Akt (Ser 473) is normalized to the total Akt protein. Data are expressed as mean±standard error of the mean (n=approximately 4~5). *P<0.05, ***P<0.001, and ****P<0.0001 vs. the control group; ^#P<0.05, ^##P<0.01, ^###P<0.001, and ^####P<0.0001 vs. the diabetic group. DM, diabetic rats; PZ, pioglitazone.

SI modulates the protein expression of hepatic carbohydrate metabolic enzymes in the diabetic rats

As shown in Fig. 6D and 6E, the PCK-1 and G-6-Pase protein expression levels are significantly enhanced in the liver of the diabetic rats compared with those of the control rats. Compared with the untreated diabetic rats, treatment with SI 2 mL/kg b.w. or PZ in the diabetic rats resulted in a significant alleviation in the PCK-1 and G-6-Pase protein expression in the liver. However, the PCK-1 protein expression levels in the diabetic rats treated with SI (0.5 and 1 mL/kg b.w.) did not show a significant difference compared with those in the untreated diabetic rats.

DISCUSSION

Insulin resistance plays a major role in the pathophysiology of hyperglycemia in diabetic conditions. In this study, we evaluated the beneficial role of SI in improving insulin sensitivity in the liver of rats with HFD- and STZ-induced diabetes. Our findings showed that by inhibiting oxidative stress and inflammatory status, SI has the potential to decrease hyperglycemia in T2D rats, which is strongly related to enhancing hepatic insulin action and carbohydrate metabolism through activating insulin signaling. Moreover, SI has a protective effect against diabetes-induced damage to the liver.

In this study, we observed that rats with HFD- and STZ-induced diabetes showed an obvious increase in FBG levels and impaired insulin sensitivity, as evidenced by decreased ISI and glucose tolerance and increased HOMA-IR, which is consistent with previous reports (Bin-Jumah, 2019; Li et al., 2019). The HFD and STZ combination can develop both insulin resistance and loss of pancreatic β-function, which presents the characterization of the T2D pathophysiology (Gheibi et al., 2017). Importantly, our results demonstrated that the 5-week treatment of diabetic rats with SI (0.5, 1, and 2 mL/kg b.w.) decreased the FBG level and insulin resistance indices in a dose-dependent manner. This effect may be because of the bioactive compounds in SI. This result is consistent with those of earlier studies (Misawa et al., 2008; Hfaiedh et al., 2011; Kim et al., 2013; Alayón et al., 2018; Eraky et al., 2018; Ajebli and Eddouks, 2019), wherein omega-3 fatty acids decreased the FBG level and improved insulin sensitivity in T2D rats (Eraky et al., 2018). Additionally, α-tocopherol was noted to attenuate the blood glucose level in diabetic rats (Hfaiedh et al., 2011) and have a positive effect on insulin sensitivity in HFD-induced obese rats (Kim et al., 2013). Phytosterol has been reported to possess potential ameliorative actions on hyperglycemia and glucose tolerance deterioration in diabetic rats (Misawa et al., 2008). Moreover, flavonoid-enriched extract from Warionia saharae exhibited antihyperglycemic activity in diabetic rats (Ajebli and Eddouks, 2019). Furthermore, in a clinical study, it has been shown that SI supplementation inhibited an increase in plasma glucose levels and improved insulin sensitivity in healthy participants (Alayón et al., 2018).

The liver is the principal site for insulin action and glucose homeostasis regulation (Postic et al., 2004; Wilcox, 2005). In the hepatic insulin resistance state, hyperglycemia occurs because of enhanced hepatic glucose production and glycogen breakdown and reduced glucose utilization (Jiang et al., 2020). Key enzymes, including PEPCK and G-6-Pase, play a major role in controlling the hepatic glucose output through the gluconeogenesis pathway (Hatting et al., 2018). PEPCK, as the rate-limiting step enzyme in gluconeogenesis, is essential for the conversion of oxaloacetate to phosphoenolpyruvate, and G-6-Pase catalyzes the dephosphorylation of glucose-6-phosphate to glucose in the final step of both the gluconeogenic and glycogenolytic pathways (Postic et al., 2004; Hatting et al., 2018). An increase in the PEPCK and G-6-Pase activities in the liver promotes the production and release of endogenous glucose into the blood circulation, which results in increased blood glucose levels (Hatting et al., 2018). In diabetes, the decrease in hepatic glycogen content is directly attributed to impaired insulin action and/or secretion owing to the failure of insulin in inhibiting glycogen phosphorylase and G-6-Pase and activating glycogen synthase (Petersen et al., 1998; Jiang et al., 2020). Therefore, in this study, we evaluated the effects of SI on carbohydrate metabolic enzymes and glycogen content in the liver of diabetic rats. Our research results showed enhanced PCK-1 and G-6-Pase protein expression levels and glycogen content depletion in the liver of diabetic rats. These results are consistent with those of previous studies, which demonstrate increased PEPCK and G-6-Pase expressions and decreased glycogen storage in the liver of diabetic rats (Liu et al., 2015; Hayanga et al., 2016). However, we noted that the hepatic PEPCK and G-6-Pase protein upregulation and the decreased glycogen levels in the diabetic rats were reversed by the SI treatment. Our results are consistent with those of previous studies on the properties of omega-3 fatty acids and flavonoids, as bioactive compounds were noted in SI in improving glucose-metabolizing enzymes in the diabetic model (Chiang and Tsai, 1995; Sundaram et al., 2019). Taken together, it is possible that SI suppresses hepatic gluconeogenic enzymes and enhances glycogenesis likely because of improved insulin sensitivity, thereby reducing circulating glucose concentration in diabetes.

It is well established that impaired insulin signaling is a major cause of insulin resistance (Guo, 2014). Insulin maintains glucose metabolism via IRS/PI3K/Akt signaling pathway activation (Titchenell et al., 2017; Petersen and Shulman, 2018). The deletion and dysregulation of IR and its downstream signaling are significant for the development of insulin insensitivity, which leads to promoting hepatic gluconeogenesis and glycogenolysis, ultimately resulting in hyperglycemia in diabetes (Petersen and Shulman, 2018). Therefore, we investigated the effect of SI on the expression of proteins involved in the insulin signaling pathway in the liver of the diabetic rats. Surprisingly, we observed that T2D rats had a significant hepatic IR-β protein expression upregulation, whereas SI treatment reduced the increased hepatic IR-β expression in the diabetic rats. Increased hepatic IR-β protein expression in diabetes may be explained by the compensation of IR upregulation, most likely through changes in decreased circulating insulin levels, which is consistent with the results of a previous study (Sundaram et al., 2019). According to earlier studies (Wei et al., 2017; Liu et al., 2021), the IRS-1 and p-Akt (Ser 473) protein expression downregulation in the liver of the diabetic rats was observed and directly correlated with interrupting glucose metabolism, demonstrating hepatic insulin resistance, as shown in this study. Interestingly, following SI administration to the diabetic rats, a significant increase in the IRS-1 and p-Akt (Ser 473) protein expression in the liver of the diabetic rats was observed, suggesting enhanced insulin sensitivity through the IRS-1/PI3K/Akt signaling pathway. This effect may be because of the high insulin-sensitizing properties of its bioactive compounds, thereby ameliorating insulin resistance and improving glucose metabolism. This notion was supported by previous reports, demonstrating the beneficial effects of omega-3 fatty acids, flavonoids, and β-sitosterols, which are abundant in SI, on insulin sensitivity by activating the IRS/PI3K/Akt signaling pathway in diabetic models (Hu et al., 2014; Jayachandran et al., 2018; Babu et al., 2020).

Under diabetic conditions, persistent hyperglycemia plays a vital role in the development of hepatocellular injury and liver dysfunction, thereby aggravating metabolic abnormalities (Mohamed et al., 2016). Furthermore, excessive blood glucose promotes increased lipid production and accumulation in the liver, which in turn accelerates the progression of insulin resistance, thereby resulting in disturbances in glucose and lipid metabolisms (Jiang et al., 2020). Growing evidence showed increased hepatic damage in diabetic rat models, as evidenced by the increased serum ALT and AST levels (Jayachandran et al., 2018). The increased liver functional enzyme level is an indicator of liver dysfunction and hepatocyte injury, which allows these enzymes to be released into the blood circulation. The augmented serum transaminases were also involved in hepatic insulin resistance in diabetes (Nannipieri et al., 2005). Consistent with earlier findings (Jayachandran et al., 2018; Yazdi et al., 2019), we noted a significant increase in the serum ALT and AST levels of the diabetic rats. Additionally, the biochemical parameter alterations in the diabetic rats were consistent with the results of a hepatic histopathological study, which showed hepatocyte and central vein degeneration, dilated sinusoids, and increased hepatocellular vacuolization with round border and flattened nucleus similar to a fatty change. Upon insulin resistance progression in diabetes, lipid infiltration in the liver is enhanced by inducing adipocyte lipolysis, hepatic lipogenesis, and lipid deposition (Perry et al., 2014). However, these alterations in the liver of the diabetic rats were improved by the SI treatment, as evidenced by decreased liver marker enzymes and ameliorated hepatic structural abnormalities. Consistent with the results of a previous study, it has been demonstrated that SI treatment decreased serum AST and ALT levels in obese rats (Ambulay et al., 2020). Furthermore, plant flavonoids and omega-3 fatty acids, which are abundantly present in SI, were reported to be efficient in ameliorating liver dysfunction and abnormal structure by inhibiting oxidative and inflammatory status (Rodríguez et al., 2018; El-Gendy et al., 2021). Taken together, it is postulated that SI can protect against diabetes-induced liver damage, possibly through its bioactive constituents that demonstrate potent antioxidant and anti-inflammatory properties.

Growing evidence suggests that the mechanism of diabetes that contributes to liver damage and the negative effect on hepatic insulin sensitivity are crosstalk between oxidative stress and pro-inflammatory mediators (Keane et al., 2015; Mohamed et al., 2016). Increased ROS driven by hyperglycemia activates the transcription factor NF-κB, which translocates to the nucleus and stimulates the expression of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, thereby resulting in inflammation (Lingappan, 2018). In turn, an increase in pro-inflammatory molecules enhances ROS production and reduces antioxidant defense, which leads to oxidative stress and subsequently exacerbates liver damage and the blockade of the insulin signaling pathway (Mohamed et al., 2016; Rehman and Akash, 2016). Thus, a mechanistic study was conducted to investigate the antioxidative and anti-inflammatory properties of SI in a rat T2D model. Our study showed obvious oxidative stress in the liver of the diabetic rats, as demonstrated by the increased MDA level and reduced antioxidant SOD, CAT, and GPx activities. These results are consistent with those of previous studies (Parveen et al., 2010; Bin-Jumah, 2019). However, the SI treatment to the diabetic rats resulted in hepatic lipid peroxidation depletion and increased antioxidant enzyme levels, suggesting that the antioxidant property of SI protects the liver from diabetes-induced oxidative damage. The antioxidant effect of SI may be attributed to its active components with highly potent antioxidant defense, including omega-3 fatty acids, omega-6 fatty acids, tocopherols, phytosterols, and phenolic compounds (Chirinos et al., 2013; Cárdenas et al., 2021). Furthermore, SI treatment was effective in ameliorating hepatic oxidative damage in a rat obesity model through its antioxidant capacity (Ambulay et al., 2020).

TNF-α and IL-6 are key mediators that promote insulin resistance by not only suppressing IR activation and insulin signal transduction but also inducing the damage of insulin target tissues, which lead to reduced insulin sensitivity and abnormal glucose metabolism, thereby accelerating the progression of diabetes (Rehman and Akash, 2016). Earlier studies have exhibited an enhancement of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β, in the diabetic liver, which are strongly associated with diminished insulin response in hepatic tissues (Chen et al., 2013; Bin-Jumah, 2019). Similarly, in this study, we observed increased hepatic TNF-α and IL-6 levels in the diabetic rats. The treatment of the diabetic rats with SI significantly ameliorated these inflammatory cytokines, thereby ameliorating liver inflammation while preserving its functions and enhancing hepatic insulin sensitivity. This hypothesis is supported by previous reports, which demonstrated the anti-inflammatory effect of SI in obese rats by decreasing TNF-α and IL-6 levels and increasing anti-inflammatory cytokine levels, IL-4 and IL-10 (Ambulay et al., 2020). Other active ingredients, such as omega-3 fatty acids, α-tocopherols, and flavonoids, found in SI also exhibited anti-inflammatory properties in the diabetes models, which played a role in improving insulin signal transduction-mediated regulation of glucose metabolism (Jamalan et al., 2015; Ghadge et al., 2016; Othman et al., 2021).

In conclusion, SI has the potential to prevent hypergly-cemia-mediated hepatic damage and improve hepatic insulin sensitivity and carbohydrate metabolism in the diabetic rats. These effects could be because of insulin signaling pathway activation, at least partly through inhibiting oxidative stress and inflammatory process (Fig. 7). In light of these findings, we also suggest that SI could exert beneficial effects in the protection and treatment of diabetes and its complication. However, to determine the detailed mechanisms of action of SI on insulin action, further studies are needed. Moreover, to investigate the efficacy of SI on diabetes, clinical studies are suggested.

Fig. 7.

A possible mechanism mediating the effects of sacha inchi oil on hepatic insulin action and glucose metabolism in type 2 diabetic rats. By oxidative and inflammatory stress inhibition, sacha inchi oil may protect against diabetes-induced liver damage and improve hepatic insulin sensitivity and carbohydrate metabolism via insulin signaling pathway activation. HFD, high-fat diet; STZ, streptozotocin; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; TNF, tumor necrosis factor; IL, interleukin; AST, aspartate transaminase; ALT, alanine transaminase; IR, insulin receptor; IRS, insulin receptor substrates; G-6-Pase, glucose-6-phosphatase; PCK, phosphoenolpyruvate carboxykinase.