A Citrullus colocynthis fruit extract acutely enhances insulin-induced GLUT4 translocation and glucose uptake in adipocytes by increasing PKB phosphorylation

Highlights

• In traditional medicine, the colocynth is known for its anti-diabetic properties.

• Colocynth fruit extract Pna1 acutely enhances insulin-induced GLUT4 translocation.

• Pna1 acutely increases cellular glucose uptake in insulin-stimulated cells.

• Pna1 enhances insulin action by increasing protein kinase B (PKB) phosphorylation.

• Our study may explain the anti-diabetic properties of the colocynth.

Abstract

Ethnopharmacological relevance

Citrullus colocynthis (L.) Schrad is a common fruit in traditional medicine and used as remedy against various diseases, especially diabetes. Up to now, its anti-diabetic effects have been fully attributed to its enhancement of pancreatic insulin secretion. Whether C. colocynthis also ameliorates insulin action in peripheral tissues has not been investigated.

Aim of the study

In the present study, using 3T3-L1 adipocytes as cell model, we have investigated whether colocynth fruit extracts affect insulin action.

Materials and methods

Various extracts were prepared from the C. colocynthis fruit and screened using a cell-based 96 well plate GLUT4 translocation assay. Promising extracts were further studied for their effects on glucose uptake and cell viability. The effect on insulin signal transduction was determined by Western blot and the molecular composition was established by LC-MS.

Results

The ethyl acetate fractions of aqueous non-defatted extracts of seed and pulp, designated Sna1 and Pna1, acutely enhanced insulin-induced GLUT4 translocation. In accordance, both extracts increased insulin-stimulated cellular glucose uptake. Pna1, which displayed greater effects on GLUT4 and glucose uptake than Sna1, was further investigated and was demonstrated to increase GLUT4 translocation without changing the half-maximum dose (ED50) of insulin, nor changing GLUT4 translocation kinetics. At the molecular level, Pna1 was found to enhance insulin-induced PKB phosphorylation without changing phosphorylation of the insulin receptor. Pna1 appeared not to be toxic to cells and, like insulin, restored cell viability during serum starvation. By investigating the molecular composition of Pna1, nine compounds were identified that made up 87% of the mass of the extract, one of which is likely to be responsible for the insulin-enhancing effects of Pna1.

Conclusions

The C. colocynthis fruit possesses insulin-enhancing activity. This activity may explain in part its anti-diabetic effects in traditional medicine. It also identifies the C. colocynthis as a source of a potential novel insulin enhancer that may prove to be useful to reduce hyperglycemia in type 2 diabetes.

Introduction

Type 2 diabetes (T2D) prevalence is rapidly increasing around the world and associated with obesity, unhealthy diets, and a sedentary lifestyle (Lam and LeRoith, 2012). The onset of T2D is closely related to the desensitization of skeletal muscle, adipose tissue, and liver for insulin, also known as insulin resistance (Fujimoto, 2000; Thule, 2012). Insulin resistance is characterized by a reduced response of skeletal muscle and adipose tissue to regular insulin levels, in particular concerning the uptake of glucose from the blood during the postprandial phase. To compensate for this insulin resistance, the pancreas secretes high amounts of insulin, just until its β cells cannot further sustain the increased production of insulin, resulting in hyperglycemia. Glucose transporter GLUT4 is responsible for the insulin-regulated uptake of glucose from the blood during the postprandial phase (Fukumoto et al., 1989; James et al., 1988, 1989). In between postprandial phases, insulin levels are low and GLUT4 is retained within intracellular compartments in muscle and adipose tissue. Once insulin levels rise during the postprandial phase, intracellular signaling cascades are activated, leading to the translocation of GLUT4 from the intracellular compartments towards the cell surface, allowing these tissues to take up glucose (Bogan, 2012; Govers, 2014).

Despite the use of numerous drugs for the treatment of T2D, the search for novel anti-diabetic compounds is not over, mainly due to unsatisfactory results at long term and the serious side effects of certain treatments, for example those involving the insulin-sensitizing thiazolidinediones (TZDs) (Fonseca, 2013; Kumar et al., 2017). Plants have been scientifically studied for over half a century for their effects on diabetes and have appeared to be useful sources for the identification of novel bioactive compounds (e.g. metformin predecessors (Bailey and Day, 1989) and acarbose (Shu, 1998)). Moreover, plant extracts have been used as anti-diabetic remedy for even much longer, especially in traditional (herbal) medicine. Plants that have been intensely studied for their anti-diabetic properties include Allium sativum (garlic), Gymnema sylvestre (gurmar), Trigonella foenum graecum (fenugreek), Momordica charantia (bitter melon, ampalaya), Ficus benghalensis (banyan fig), Artemisia absinthium (wormwood) and Citrullus colocynthis (colocynth, bitter apple, bitter cucumber) (Bailey and Day, 1989; Li et al., 2015; Patel et al., 2012).

The Citrullus colocynthis (L.) Schrad is a member of the Cucurbitaceae family. Plants of this family are tolerant to drought, but intolerant to wet and poorly drained soil and frost (Hussain et al., 2014). The C. colocynthis, of which the appearance resembles that of watermelon, is a perennial herbaceous vine that possesses yellow flowers, producing 15–30 round fruits that are 7–10 cm in size (Shi et al., 2014). It is widely distributed in desert areas around the world, especially in the Sahara desert and the South-West part of Asia, including the Arabian desert, Pakistan, Iran, and India, but also in China (Hussain et al., 2014). In all these places, parts of this plant have been used in traditional medicine, especially for the treatment of constipation. More generally, its seeds, pulp, rind, leaves, and roots have been reported to be used to treat diabetes, intestinal disorders (constipation, indigestion, ulcer, dysentery, gastroenteritis, colic pain), bacterial infections, cancer, hypertension, rheumatism, common cold, cough, toothache, wounds, jaundice, asthma, and bronchitis (Dhakad et al., 2017; Hussain et al., 2014). At present, the plant (especially its fruit) is the topic of pharmacological investigations, mainly concerning its anti-diabetic and (cucurbitacin-related) anti-cancer effects (Hussain et al., 2014). Other current areas of investigation include those concerning its anti-hyperlipidemic (cholesterol- and triglyceride-lowering), anti-inflammatory, analgesic, insecticidal, and anti-microbial activities (Dhakad et al., 2017; Gurudeeban et al., 2010; Hussain et al., 2014). In addition to its medicinal value, the Citrullus colocynthis is also well-appreciated for its marked nutritional value, due to its high protein amount and the elevated concentration of essential amino acids (Gurudeeban et al., 2010; Hussain et al., 2014).

In clinical studies, the powder of dried colocynth fruits appeared to reduce fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) levels in T2D patients (Barghamdi et al., 2016; Huseini et al., 2009). In accordance, in animal studies, seed, pulp and rind from the colocynth fruit but even the plant roots and leaves have been demonstrated to possess anti-diabetic activity (Abdel-Hassan et al., 2000; Agarwal et al., 2012; Amin et al., 2017; Benariba et al., 2012; Dallak et al., 2009; Ebrahimi et al., 2016; Huseini et al., 2009; Ostovar et al., 2020; Shi et al., 2014). Long-term treatments with colocynth extracts have been demonstrated to improve hyperglycemia through preservation and restoration of β cell mass (Amin et al., 2017; Sebbagh et al., 2009) and stimulation of synthesis and secretion of insulin (Dallak et al., 2009). Interestingly, four animal studies have suggested that extracts of this plant also have acute blood glucose lowering effects. First, in normoglycemic rats, the administration of an aqueous root extract 30 min before an oral glucose tolerance test (OGTT) reduced glycemia already within the first 30 min of the OGTT (Agarwal et al., 2012). Second, seed and rind extracts reduced glycemia in overnight food-starved normal and streptozotocin (STZ)/alloxan-induced diabetic rats and rabbits within 60–90 min of administration (Abdel-Hassan et al., 2000; Azzi et al., 2015; Lahfa et al., 2017). For the moment, these acute effects remain unexplored. Hypothetically, these extracts might acutely affect glycemia via insulinotropic, insulin-mimetic, or insulin-enhancing/sensitizing actions. In favor of an insulinotropic action is the finding that colocynth extracts acutely induce the release of insulin from isolated pancreatic islets (Benariba et al., 2013; Ebrahimi et al., 2016; Nmila et al., 2000). However, their acute effects in STZ/alloxan-treated animals rather provides evidence for an insulin-mimetic action (Abdel-Hassan et al., 2000; Azzi et al., 2015; Lahfa et al., 2017). At present, there are no indications that support the presence of insulin-enhancing or insulin-sensitizing activity in colocynth extracts.

In the current study, the acute anti-diabetic effects of the colocynth were investigated at the cell biological level. Various colocynth extracts were prepared and tested for their acute effects on insulin-induced GLUT4 translocation in adipocytes. The results of the present study highlight two promising non-toxic ethyl acetate extracts from the colocynth fruit, Sna1 from seed and Pna1 from pulp, which enhance the effect of insulin towards both GLUT4 translocation and cellular glucose uptake. The molecular composition of these extracts was determined in order to provide insight into the nature of the compound responsible for this insulin-enhancing activity.