Steviol Glycosides Modulate Glucose Transport in Different Cell Types

Author: maria rita moio
Date: 17/11/2014


Stevia rebaudiana Bertoni is a weak perennial shrub belonging to Asteraceae (Compositae) family, native to subtropical regions of Brazil and Paraguay. Its leaves have been used as a sweetener since ancient times and for many other medicinal purposes in Latin America and the Orient for centuries. The “sweet herb” has gained increasing interest from nutritional researchers and commercial area in the last years, due to the growing need to find new natural calorie-free sweeteners alternative to sugar. Indeed, in both industrialized and developing countries, the incidence of type 2 diabetes mellitus and obesity is sharply increasing as a result of dietary behaviours, reduced physical activities, and ageing. These metabolic disorders have become major public health problems worldwide.
Glycemic control is fundamental to the management of diabetes since it is associated with significantly decreased rates of retinopathy, nephropathy, neuropathy, and cardiovascular disease, the most common cause of death in diabetic patients. The effort to achieve near-normoglycemia through the key strategy of glycemic control includes recommendations for prevention and control of diabetes, for example, monitoring carbohydrate intake and limiting the consumption of sugar-sweetened beverages.
Stevia leaves and extracts are natural noncaloric sweeteners that can substitute sucrose. The main sweet components in leaves, approximately 200–400 times sweeter than sucrose as shown by organoleptic tests , are stevioside and rebaudioside A, steviol glycosides differing only by one glucose moiety. Stevioside is formed by 3 molecules of glucose and one molecule of the aglycone steviol, a diterpenic carboxylic alcohol; rebaudioside A holds one additional glucose molecule.
Steviol glycosides have been recently authorised as commercial sweeteners. US Food and Drug Administration (FDA) has allowed the use of Stevia extracts containing not less than 95% total steviol glycosides. Recently, the European Food Safety Authority (EFSA) approved the use of steviol glycosides as food additive. Considering the available toxicity data (in vitro and in vivo animal studies and some human tolerance studies), steviol glycosides are considered not carcinogenic, genotoxic, or associated with any reproductive/developmental toxicity. Joint Expert Committee on Food Additives (JECFA) established an accepted daily intake (ADI) for steviol glycosides (expressed as steviol equivalents) of 4 mg/kg bw/day.
Besides sweetness, steviol glycosides, in particular stevioside, have been shown to possess beneficial effects on human health. Pharmacological activities and therapeutical benefits include antitumour and anticancer, antiinflammatory, antihyperglycemic, antihypertensive, antidiarrheal, immunomodulatory, diuretic, and enzyme inhibitory actions. Stevia has also been used to help control weight in obese subjects; moreover, antioxidant properties have been described. Stevioside, rebaudioside A, and their metabolite steviol have been mostly investigated in in vivo animal studies and in humans. Results suggest that stevioside and related compounds affect plasma glucose modulating insulin secretion and sensitivity, which increase glucose removal from the plasma. In addition, it seems likely that stevioside inhibits gluconeogenesis in the liver of diabetic rats. These antihyperglycemic, insulinotropic, and glucagonostatic effects, especially for rebaudioside A, are largely plasma glucose level dependent, requiring high glucose levels. Despite the large number of studies on Stevia and steviol glycosides, very little is reported concerning the cellular and molecular mechanisms underpinning these effects.
It’s very intresting a study, in which they examined the role of steviol glycosides on cellular glucose transport in cultured cells. Glucose is a polar molecule and requires specific carrier proteins, located in the plasma membrane, to cross the lipid bilayer and enter the cell. Glucose is transported into the cells through two different types of membrane associated carrier proteins, the Na+-coupled glucose transporters (SGLT) and the facilitative glucose transporters (GLUT). The human GLUT family is integral membrane proteins widely distributed in probably all mammalian cells that regulate the movement of glucose between extracellular and intracellular compartments maintaining a constant supply of glucose available for metabolism. To date, GLUT family is constituted by 14 distinct isoforms, differently distributed in human tissues. GLUT1 is considered responsible for the basal uptake in many cell types, representing the most ubiquitously expressed isoform; GLUT4 is responsible for insulin-stimulated glucose uptake in peripheral tissues, but its expression has also been reported in the brain where glucose is an essential substrate for cerebral oxidative metabolism. It has recently been reported that in a human neuronal cell line, SH-SY5Y, GLUT1 translocation in response to insulin-like growth factor (IGF-I) occurs and, for the first time in a neuronal cell system, also GLUT4 is translocated to the plasma membrane in response to insulin. Also in many leukemia cell lines GLUT1 is recruited on the plasma membrane from intracellular compartments in response to different stimuli, greatly enhancing the rate of glucose uptake. Moreover, it is well known that impaired GLUT4 translocation is causally linked to insulin resistance and consequently to noninsulin-dependent diabetes mellitus.
In one study have been used cells of the neuroblastoma SH-SY5Y and the promyelocytic leukaemia line HL-60, both expressing insulin and insulin-like growth factor-1 (IGF-I) receptors to test some commercial Stevia extracts, in order to evaluate a possible effect of these compounds on glucose transport and to clarify the molecular mechanism of action. They used extracts from Stevia rebaudiana Bertoni supplied by Eridania Sadam SpA. The four extracts tested differ by the relative content of rebaudioside A and stevioside. In particular, according to the certificates of analysis of each sweetener, Reb A (R97) contains 97-98% rebaudioside A, Stevia RA60 (R60) contains about 60% rebaudioside A and about 20% stevioside; Steviol Glycosides SG95 (SG) contains 50% rebaudioside A and at least 25% stevioside; Truvia (TRU) contains a mixture of steviol glycosides not analytically quantified.
Glucose is the primary source of energy used by the brain and it is constantly delivered to individual cells (glial cells and neurons). In brain, the relationship among glucose metabolism, GLUT isoforms, modulation of glucose uptake, role of insulin, and distribution of insulin receptor (IR) is very complex, being dependent on specific regions of the brain and playing a key role also in cognitive functions. Recent studies report a close correlation between impaired glucose uptake/metabolism and neurodegenerative diseases such as Alzheimer’s disease.
It is also recognised that cancer cells frequently overexpress the GLUT family members, due to the uncontrolled proliferation requiring elevated energy, and they often express GLUT isoforms not present in normal conditions. Moreover, large hypoxic areas into the tumour cause an increase in glucose utilization by cancer cells through glycolysis. The requirement for energy is satisfied by an augmented sugar intake, realised by an increase in GLUT expression and an increment in the translocation of the transporters to the plasma membrane. For these reasons, cancer cells are a useful model system to study the glucose transport activity and its signalling transduction pathway, allowing to clarify the molecular mechanism underlying steviosides biological effects on glucose metabolism.
SH-SY5Y and HL-60 cells were treated with different concentrations (0.5–5 mg/mL, corresponding to 0.5–5 mM for R97, which can be assumed as a pure compound) of Stevia extracts for 24 h. 2 The extracts did not affect cell viability/proliferation, confirming that they are not cytotoxic within the concentration range tested. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay, which indicated that cell membrane integrity was not compromised and excluded cellular necrosis.
In order to evaluate a possible effect on apoptosis, the activity of caspase 3 was measured. Caspases play a central role in mediating various apoptotic responses and are activated in a sequential cascade of cleavages. Treatments of cells with Stevia extracts for 1 hour, 6 hours or for 24 hours did not influence caspase 3 activity, indicating that the compounds did not induce programmed cell death.

Since antioxidant properties of Stevia extracts have been described, the antioxidant activity of the commercial extracts was investigated in SH-SY5Y and HL-60 cells. Reactive oxygen species (ROS) levels were measured, and the resulti s that the compounds did not exhibit any antioxidant activity, since they were neither able to decrease basal ROS level at the highest concentration.
Since the increase in glucose uptake obtained with standards is consistent with that shown by the whole extracts, probably this effect is due to rebaudioside A and stevioside, the two major components of Stevia extracts. Furthermore, the influence of Stevia extracts on glucose transport activity was compared to the effect of 100 nM insulin. Results reveal that Stevia extracts and insulin behave similarly, being Stevia extracts as efficient as insulin in increasing glucose uptake. The cotreatment with insulin and Stevia extracts causes a rise of glucose transport significantly higher than the increase due to insulin alone.
It is well known that insulin induces the translocation of GLUT4 from cytosolic storage vesicles to the plasma membrane, enhancing glucose transport. This phenomenon was observed also for neural cells in vitro, in particular the human neuroblastoma SH-SY5Y cells. Translocation of GLUTs to the membrane has been reported as a consequence of various stimuli in many cellular types; in addition, changes in the expression of GLUTs have been described in response to several metabolic and oxidative stresses and in various physiological or pathological conditions.. The expression of GLUT1 and GLUT4 in neuroblastoma and leukaemia cells following treatments with Stevia extracts or insulin was assessed by Western blot analysis on cell lysates. The increase in GLUT1 and GLUT4 content obtained following exposure to Stevia extracts is similar to that obtained by insulin stimulation.
To clarify the molecular mechanism by which Stevia extracts enhance glucose transport, the phosphorylation status of PI3K and Akt was evaluated following Stevia extract treatment and insulin stimulation. Insulin activates the PI3K/Akt pathway, critical for neuronal survival and growth, synaptic plasticity and development, and learning. Indeed, stimulation of insulin receptor, localized in lipid rafts, produces the phosphorylation of tyrosine receptor kinases and the activation of a signal transduction pathway involving PI3K and Akt. The interaction of insulin with its receptor is a regulator of growth and differentiation of leukaemia cells. Highly specific insulin receptors have been identified on human promyelocytic leukaemia cells HL-60 [56]. Immunoblotting results (Figure 8) show an increase of phosphorylated forms of both PI3K and Akt following the treatment with insulin or Stevia extracts, indicating a possible similar mechanism of action or, at least, a common signalling pathway.
Thanks to this study, there is a demonstration that rebaudioside A and stevioside, the major glycosides in Stevia extracts, are able to enhance glucose uptake in both SH-SY5Y neuroblastoma and HL-60 myeloid leukaemia human cells, the raise being similar to that induced by insulin. Probably steviol glycosides act by modulating GLUT translocation through the PI3K/Akt pathway. Although further experiments are needed, these results support the hypothesis that steviol glycosides and insulin could share a similar mechanism in regulating glucose entry into cells. Stevia extracts, commercialised as zero-calorie natural sweeteners, are involved in insulin regulated glucose metabolism. These findings suggest that the use of Stevia extracts goes beyond their sweetening power and may also offer therapeutic benefits, supporting the use of botanicals dietary supplements to improve the quality of life.

Maria Rita Moio

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