Steviol glycosides affect trace element status in diabetic rats

doi:10.21203/rs.3.rs-4998370/v1

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Steviol glycosides affect trace element status in diabetic rats

https://doi.org/10.21203/rs.3.rs-4998370/v1

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Steviol glycosides (stevioside and rebaudioside A) have been reported to have lipid and glucose regulatory potential. The published literature presents conflicting results regarding the impact of hyperglycemia on Fe, Zn, and Cu levels, and almost no data exist on whether supplementary steviol glycosides can affect the status of trace elements in diabetes. This study aimed to evaluate the effect of hyperglycemia and dietary steviol glycosides supplementation on Fe, Zn, and Cu levels and the ratios of these elements in the liver and kidney of diabetic rats.

The experiment was conducted on 70 male Wistar rats, of which 60 were fed a high-fat diet for 8 weeks, followed by intraperitoneal streptozotocin injection to induce type 2 diabetes, while 10 healthy controls were fed the AIN-93M diet. Afterward, diabetic rats were allocated into the following 6 high-fat diet-fed experimental groups: untreated, supplemented with metformin, or supplemented with stevioside or rebaudioside A (0.5 or 2.5%) for 5 weeks. After the experiment, internal organs were harvested for mineral analyses. The content of Fe, Zn, and Cu in tissues was determined using the AAS method.

It was found that hyperglycemia significantly elevated the liver Zn/Cu ratio, simultaneously decreasing the kidney Fe level, as well as Fe/Zn and Zn/Cu ratios in diabetic rats. Supplementary steviol glycosides tended to normalize the kidney Zn/Cu ratio, while high doses of steviol glycosides tended to normalize the kidney Fe concentration in diabetic rats. The type of glycoside differentiated the kidney Zn level and the Fe/Zn ratio in diabetic rats.

stevia

steviol glycosides

trace elements

diabetes

rats

Type 2 diabetes mellitus (T2DM) is currently a serious global health issue, affecting ~ 462 million people in 2017 [1]. The prevalence of the disease is expected to increase due to many factors, such as a sedentary lifestyle and population aging [2]. As the largest proportion of patients is found in developing countries, the highest prevalence of T2DM is commonly observed in regions of the Middle East and North Africa [3]. While sub-Saharan regions of Africa still show relatively low prevalence, the rate is rapidly rising [4]. Finding new ways to improve the treatment of diabetic patients is currently a difficult challenge for professionals. Nevertheless, this effort is necessary for a few reasons. First, there is still no therapy that combines high efficacy, low risk of side effects, and affordability. Second, T2DM has become a significant economic burden on worldwide healthcare systems [5], expected to further increase in the future [6]. T2DM complications include retinopathy, neuropathy, nephropathy, impaired immunity to infections, and cardiovascular diseases [7]. The last-named complication is the leading cause of both morbidity and mortality among patients [8]. Overall lifestyle changes (improved diet and physical activity) and usually pharmacotherapy are commonly used in T2DM treatment [9, 10]. However, research is ongoing, and in-depth knowledge regarding the molecular basis of T2DM helps to develop more effective targeted treatment methods [11]. For example, new pharmacological agents, such as GLP-1 receptor agonists and/or SGLT2 inhibitors, are currently being introduced into the management strategies of the disease [12].

Stevia rebaudiana Bertoni is a plant that is a source of compounds that may be potentially useful in diabetes treatment. In particular, the potential lies in steviol glycosides (SG) that are extracted from the leaves of the plant, and nowadays commonly used as natural sweeteners in food industry [13]. Results of experimental studies confirmed the safety of SG and agencies such as the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) allowed to use these compounds as food additives (960) [14]. Apart from their high sweetness intensity, SG have been shown to have many health-promoting properties, especially antidiabetic properties, that can be used in managing T2DM, as they can regulate blood glucose concentrations, improve insulin sensitivity, and normalize lipid profile in the course of the disease [15–20].

It is commonly known that trace elements such as iron (Fe), zinc (Zn), and copper (Cu) are crucial for various metabolic processes. Abnormalities in their status can impact the development and management of T2DM and vice versa – the disease can affect their status [21, 22]. Fe is an essential element for oxygen transport. It is worth mentioning that both Fe deficiency and excess are risk factors for developing diabetes. This is most probably due to, among other factors, the role of Fe in oxidative stress and inflammation processes, which impair insulin secretion and increase insulin resistance [23]. Fe deficiency, however, can negatively affect glucose metabolism and amplify diabetic complications in patients [24]. Zn is vital for insulin-related processes, including the synthesis, storage, and secretion of insulin [25]. Experimental studies have shown that Zn deficiency is common in diabetic patients, and can amplify glycemic control difficulties and risk of diabetic complications. Zn supplementation has been shown to improve glycemia in type 1 & 2 diabetic patients [26]. Also, sufficient Zn body concentrations are associated with lowering the risk of developing T2DM, probably by improvement of insulin sensitivity [27]. Cu is involved in enzymatic processes, especially connected with oxidative stress. Abnormalities in Cu metabolism, including both deficiency and excess, are observed in T2DM. For instance, increased Cu body concentrations can increase oxidative stress and/or inflammation, which can contribute to insulin resistance and pancreas β-cell dysfunction [28]. However, decreased Cu body levels are associated with impairment of glucose metabolism and higher severity of diabetic complications [29]. Also, the balance between the aforementioned trace elements is crucial for glucose metabolism. High Fe intake can, for instance, impair Zn absorption, while sufficient Zn body concentrations protect against the toxicity of Cu. Thus, homeostasis in the status of these trace elements and their relevant ratios are important for the prevention, onset, and management of T2DM [30].

Therefore, the subject of this study was to verify the effect of SG supplementation on the levels of trace elements, such as Fe, Zn, and Cu, and their ratios in the liver and kidney, as the main storage organs, in rats with induced T2DM.

In-depth details regarding the materials and methods used in the experiment are described in the previous article [20], therefore will not be repeated at full length. This article presents information on the specifics related to the topic of trace elements.

EXPERIMENTAL AGENTS

The following dietary supplements were used in the experiment: metformin (Met; metformin hydrochloride from the antidiabetic drug Metformax; Teva Operations Poland Ltd., Kraków, Poland), stevioside (ST; Anhui Minmetals Development Co., Hefei, China) and rebaudioside A (RA; Anhui Minmetals Development Co., Hefei, China). Streptozotocin was used in the protocol of the laboratory model of T2DM induction (Sigma-Aldrich Ltd., Poznań, Poland).

STUDY PROTOCOL

The experimental stage of steviol glycoside supplementation lasted 5 weeks preceded by 8 weeks of feeding a high-fat (HF) diet in a group of diabetic rats to induce insulin resistance. Hyperglycemia was induced with intraperitoneal injections of streptozotocin. A detailed scheme of animal grouping is shown in Fig. 1.

Figure 1. Animal grouping scheme.

Note: C group: healthy rats, standard AIN-93M diet; Db Group: diabetic rats, HF diet; Db + Met Group: diabetic rats, HF diet + 0.15% metformin; Db + ST1 Group: diabetic rats, HF diet + 0.5% stevioside; Db + ST2 Group: diabetic rats, HF diet + 2.5% stevioside; Db + RA1 Group: diabetic rats, HF diet + 0.5% rebaudioside A; Db + RA2 Group: diabetic rats, HF diet + 2.5% rebaudioside A.

The experiment was conducted in the Association for Assessment and Accreditation of Laboratory Animal Care International approved Animal Care Facility in Poznań University of Life Sciences.

TRACE ELEMENT ANALYSIS

The organs were first pre-digested in 65% HNO₃ (Merck KGaA, Darmstadt, Germany) followed by mineralization in a Speedwave XPERT (Berghof Products, Instruments GmbH, Eningen, Germany) digestion system. The obtained samples were analyzed in terms of trace element concentrations using the atomic absorption spectrometry method with Carl-Zeiss Jena AAS-3 atomic absorption spectrometer (Jena, Germany). To validate the accuracy of the method, certified reference materials (INCT-SBF-4, Institute of Nuclear Chemistry and Technology, Warsaw, Poland; NCS ZC 73030, LGC Standards Ltd., Teddington, UK; BCR 679, Institute for Reference Materials and Measurements, Geel, Belgium; NIST1577C, NIST®, Gaithersburg, MD, USA) were used.

STATISTICAL ANALYSIS

All obtained values were first collected and organized in MS Excel 2019 (Microsoft Corporation, Redmond, USA). Data was then analyzed in Statistica 13.3 (TIBCO Software Inc., Palo Alto, USA). The following statistical methods were applied: Shapiro–Wilk test (p < 0.05), one–way analysis of variance (ANOVA) (p < 0.05), multivariate analysis of variance (MANOVA), and Fisher’s LSD post hoc test (p < 0.05). Results shown in the article refer to the stage of the experiment after T2DM induction.

Determining trace element status in both animals and humans is a difficult task due to the multitude of functions they serve and specific compartmentalization and distribution in the body. Trace element status is usually determined by measuring trace element concentrations in available fluids or tissues. In this experiment, the trace element status was evaluated by measuring Fe, Cu, and Zn concentrations in liver and kidney samples. Besides, since these particular trace elements play an important role in the maintenance of the antioxidant system, the Fe/Zn, Fe/Cu, and Zn/Cu ratios in these organs were calculated to verify their balance in diabetes.

LIVER TRACE ELEMENT STATUS

The effects of the tested diet on the liver trace element status are presented in Tables 1 & 2.

Table 1

Effects of SG on the liver and kidney trace element status in animals.
Parameter	Db	Db + Met		Db + ST1	Db + ST2	Db + RA1	Db + RA2
Liver
Fe (µg/g d.m.)	487.51 ± 68.79	543.82 ± 132.39	550.31 ± 139.79	559.57 ± 130.30	556.75 ± 85.21	514.94 ± 0.40	554.74 ± 70.53
Zn (µg/g d.m.)	108.89 ± 12.35	123.73 ± 17.53	110.36 ± 16.27	117.72 ± 19.35	111.48 ± 8.64	113.09 ± 12.40	113.98 ± 17.68
Cu (µg/g d.m.)	13.68 ± 1.78	13.01 ± 2.31	16.60 ± 5.53	14.43 ± 2.87	13.85 ± 1.47	13.60 ± 2.69	14.06 ± 1.36
Fe/Zn ratio	4.61 ± 0.93	4.41 ± 1.03	5.03 ± 1.19	4.74 ± 0.66	5.00 ± 0.65	4.57 ± 0.73	4.94 ± 0.87
Fe/Cu ratio	36.60 ± 10.11	42.96 ± 11.45	36.34 ± 15.06	38.95 ± 5.68	40.77 ± 8.04	39.73 ± 13.04	40.00 ± 7.56
Zn/Cu ratio	8.04 ± 1.15 ^ab	9.36 ± 1.07 ^c	7.14 ± 2.04 ^a	8.23 ± 0.73 ^abc	8.11 ± 0.85 ^ab	8.54 ± 1.58 ^bc	8.13 ± 1.10 ^abc
Kidney
Fe (µg/g d.m.)	314.24 ± 21.12 ^b	246.60 ± 37.18 ^a	269.50 ± 48.69 ^a	260.59 ± 30.75 ^a	282.17 ± 53.55 ^ab	262.35 ± 43.76 ^a	282.51 ± 35.30 ^ab
Zn (µg/g d.m.)	115.76 ± 8.86 ^a	122.20 ± 16.96 ^a	131.15 ± 8.74 ^ab	126.36 ± 19.99 ^a	145.08 ± 33.72 ^b	122.24 ± 13.50 ^a	114.00 ± 17.47 ^a
Cu (µg/g d.m.)	37.29 ± 7.20 ^a	50.56 ± 14.26 ^ab	129.19 ± 5.26 ^c	71.42 ± 38.66 ^b	49.13 ± 14.92 ^ab	74.88 ± 33.87 ^b	70.92 ± 34.22 ^b
Fe/Zn ratio	2.72 ± 0.23 ^c	2.03 ± 0.29 ^a	2.08 ± 0.46 ^a	2.11 ± 0.42 ^a	1.93 ± 0.51 ^a	2.15 ± 0.28 ^ab	2.52 ± 0.47 ^bc
Fe/Cu ratio	8.69 ± 1.65 ^c	5.57 ± 2.95 ^b	2.82 ± 2.52 ^a	4.61 ± 2.33 ^ab	6.38 ± 2.79 ^bc	4.68 ± 3.80 ^ab	4.89 ± 2.31 ^ab
Zn/Cu ratio	3.25 ± 0.58 ^c	2.65 ± 1.04 ^bc	1.37 ± 0.88 ^a	2.12 ± 0.80 ^ab	3.16 ± 1.05 ^c	2.14 ± 1.53 ^ab	1.93 ± 0.82 ^ab

Data are presented as mean ± standard deviation. Different letters (a–c) indicate statistically significant differences between values (Fisher’s LSD post-hoc test; a < b; p < 0.05).

Table 2

Main effects of SG (multivariate analysis).
Index	Main Effects
Index	Glycoside (ST vs RA)	p	Dose (0.5% vs 2.5%)	p
Liver
Fe (µg/g d.m.)	558.16 ± 106.81	NS	538.57 ± 112.21	NS
Fe (µg/g d.m.)	534.84 ± 80.98	NS	555.80 ± 76.21	NS
Zn (µg/g d.m.)	114.44 ± 14.63	NS	115.54 ± 16.13 112.59 ± 13.04	NS
Zn (µg/g d.m.)	113.54 ± 14.76	NS	115.54 ± 16.13 112.59 ± 13.04	NS
Cu (µg/g d.m.)	14.13 ± 2.20 13.83 ± 2.07	NS	14.04 ± 2.73 13.94 ± 1.39	NS
Fe/Zn ratio	4.87 ± 0.65 4.76 ± 0.80	NS	4.66 ± 0.68 4.97 ± 0.74	NS
Fe/Cu ratio	39.86 ± 6.82 39.86 ± 10.30	NS	39.32 ± 9.53 40.41 ± 7.58	NS
Zn/Cu ratio	8.17 ± 0.77	NS	8.38 ± 1.17	NS
Zn/Cu ratio	8.33 ± 1.33	NS	8.12 ± 0.94	NS
Kidney
Fe (µg/g d.m.)	271.38 ± 43.79	NS	261.36 ± 35.65	NS
Fe (µg/g d.m.)	273.10 ± 39.39	NS	282.33 ± 44.48	NS
Zn (µg/g d.m.)	136.21 ± 28.95	0.03905	124.56 ± 17.04	NS
Zn (µg/g d.m.)	117.85 ± 15.77^*	0.03905	131.27 ± 31.31	NS
Cu (µg/g d.m.)	59.69 ± 30.11 72.77 ± 32.89	NS	72.94 ± 35.49 58.81 ± 26.91	NS
Fe/Zn ratio	2.02 ± 0.46 2.35 ± 0.42^*	0.04612	2.13 ± 0.36 2.21 ± 0.56	NS
Fe/Cu ratio	5.49 ± 2.66 4.79 ± 2.98	NS	4.64 ± 2.94 5.68 ± 2.61	NS
Zn/Cu ratio	2.67 ± 1.06	NS	2.13 ± 1.13
Zn/Cu ratio	2.03 ± 1.16	NS	2.61 ± 1.12	NS
Data is presented as mean ± standard deviation (*p < 0.05)

The results indicate that induced diabetes did not affect the total content of any of the analyzed trace elements in the liver. However, the liver Zn/Cu ratio has been significantly elevated in the Db group, whereas supplement treatment tended to normalize this ratio in diabetic rats. The multivariate analysis did not reveal any additional effects of experimental factors on the liver and kidney trace element content and their relevant ratios in diabetic rats.

KIDNEY TRACE ELEMENT STATUS

The effects of the tested diets on the kidney trace element status are presented in Tables 1 & 2. The only trace element affected by T2DM in the kidney directly was Fe, because significantly lower Fe levels were observed in diabetic untreated rats (Db group). Furthermore, supplementary SG treatment was generally ineffective in restoring the liver Fe content, even though a slight (insignificant) elevation of this parameter is observed with higher doses of SG (Db + ST2, Db + RA2). Regarding the relevant kidney trace element ratios, some more statistically significant effects were observed, in particular, T2DM decreasing both Fe/Zn and Fe/Cu ratios in this organ. Generally, the administration of the diet enriched with SG was in most cases ineffective in mitigating these changes, however, a high dose of RA (Db + RA2) resulted in a normalization of the kidney Fe/Zn ratio in diabetic rats.

The multivariate analysis revealed that the type of SG differentiated content of Zn in the kidney and the kidney Fe/Zn ratio in diabetic rats. Interestingly enough, diabetic rats treated with RA, regardless of the dose, had significantly lower kidney Zn levels, while the Fe/Zn ratio was higher, as compared to ST.

Diabetes (T2DM) can significantly alter the body status of trace elements in organs including the liver and kidneys. The main purpose of this study was the evaluation of tissular essential trace elements, namely Fe, Zn, and Cu, in diabetic rats subjected to SG supplementation, since it is known that these metals are involved in processes of lipid peroxidation and play an important role in the pathogenesis and exacerbation of diabetic complications [31]. Usually, the assessment of trace element status, particularly in human subjects, is based on a limited number of biomarkers in blood, mainly by measuring their total or fractional concentration in blood or serum, depending on the element. For example, Fe status can be evaluated conveniently using an array of hematologic indices, or the serum ferritin level, and comparing them with the reference values. On the other hand, for Zn and Cu, the available biomarkers in blood or serum have certain limitations, as they do not always correspond to their metabolic or storage pools. In the available literature, there is an abundance of conflicting results regarding trace element levels in the blood/serum in diabetic human subjects that will not be repeated in detail in this study. The variability of data may result from the differences in sample sizes, the age of patients, the surrounding environment, duration of the disease, ethnicity, nutritional habits and status, and glycemic control of the patients enrolled in the study.

Oxidative stress has been associated with T2DM since the generation of reactive oxygen species (ROS) is a common phenomenon in this disease [32]. Oxidative damage to DNA, proteins, and lipids has been observed and correlated to diabetic complications [33, 34]. Several studies have shown that hyperglycemia plays a role in inducing oxidative stress in diabetes [35]. However, high levels of glucose are not the only aspect responsible for the generation of ROS. Some studies imply an important role of transition metals as catalysts of oxidative stress. The biology of transition metals, such as Cu, Zn, Mn, Mo, Cr, V, and Fe, has been evaluated in the context of T2DM [36, 37]. The mechanisms of generation of ROS induced by transition elements have been reported in a variety of publications, and therefore will not be discussed in detail in this paper [38–44]. In brief, increased ROS production brought about by chronic hyperglycemia (glucotoxicity) leads to oxidative stress that has been implicated as a contributor to both the onset and progression of diabetes and its associated complications [45]. Some of the consequences of an oxidative environment are the development of insulin resistance, β-cell dysfunction, impaired glucose tolerance, and mitochondrial dysfunction, which can ultimately lead to a diabetic disease state. Experimental and clinical data suggest an inverse association between insulin sensitivity and ROS levels.

Essential trace elements (Fe, Zn, Cu) in the organism usually take the form of metalloproteins distributed between a few metabolic pools that remain in a dynamic balance by homeostatic mechanisms. It has been reported that aberrant alterations in their homeostasis can lead to the release of free species that in turn undergo redox cycling reactions (e.g., Fenton reaction) to produce ROS [46]. Trace elements (Fe, Zn, Cu) in the forms of metalloproteins are among the protective agents preventing oxidative stress and the development of diabetes and diabetic complications [47].

The role of Fe in diabetes and its complications has been widely discussed in the literature [48]. The central importance of Fe in the pathophysiology of disease is attributed to the ease with which Fe is reversibly oxidized and reduced. This property, while essential for its metabolic functions, makes Fe potentially hazardous because of its ability to participate in the generation of powerful oxidant species such as hydroxyl radical [48]. In the liver specifically, T2DM may disturb Fe homeostasis. Some patients exhibit elevated hepatic Fe concentrations and thus have an increased risk of liver damage and excessive oxidative stress [49]. Experimental studies suggest that Fe liver overload is associated with insulin resistance development and can amplify both liver inflammation and fibrosis [50]. T2DM can significantly change the trace element status also in the kidneys. Increased Fe deposition can contribute to nephropathy and further kidney damage through oxidative stress-related mechanisms [48].

As an antioxidant trace element, Zn not only potentiates the action of insulin but is also crucial for insulin production [51]. Zn deficiency reported in T2DM is believed to result from increased urinary losses of this trace element and reduced Zn status is negatively correlated with hyperglycemia and poor glycemic control [52]. In the course of T2DM, body Zn concentrations are frequently lowered, which can result in impaired insulin signaling and glucose metabolism [53]. Decreased Zn concentrations in the liver may be associated with oxidative damage and abnormal liver regeneration processes [54]. Kidney Zn levels are often observed to be lowered in the course of T2DM. This can result in impaired antioxidant defense mechanisms in the organ and further development of diabetic nephropathy [55]. Therefore, adequate kidney Zn levels are crucial for renal health and the prevention of oxidative damage in T2DM [56].

Cu is a controversial element, due to its bidirectional functions in the body. On the one hand, Cu, together with Zn, is an integral component of superoxide dismutase (SOD), one of the key endogenous enzymes protecting against ROS. On the other hand, it is a strong pro-oxidant, thus elevated levels of Cu are not only implicated in increased oxidative stress in T2DM but also contribute to insulin resistance and hyperglycemia [57, 58]. Despite these observations, data regarding serum levels of Cu in T2DM are inconsistent with claims that Cu concentrations are not altered in T2DM [59]. As regards the liver Cu levels in T2DM, research shows that disease complications may lead to either an accumulation or deficiency of this element in the organ, increasing the risk of disrupted liver function and increased oxidative stress [60]. Cu imbalance is common in the kidneys of T2DM patients. This state can significantly impair renal function and contribute to diabetic complications progression mostly by amplifying oxidative stress [61].

In this experiment, the liver and kidney Fe, Zn, and Cu levels were used to evaluate trace element status, as they better reflect the storage pools. Furthermore, the liver and kidney Fe/Zn, Fe/Cu, and Zn/Cu ratios were taken into account, based on the working assumption that any significant deviations in the above ratios (as compared with the reference, here the control group of healthy rats) can suggest a metabolic distress, particularly associated with the increased oxidative stress in the tissues. The presented study showed that the liver and kidney Fe, Zn, and Cu levels, and the ratios of some of these elements, were altered in the diabetic rats compared to the healthy control group. There is a scarcity of literature data discussing tissular trace element ratios as presented in this article, which poses a challenge in further interpretation of the obtained results. From the available reports, some experimental studies performed on diabetic rats (similar models to the present study) were selected and the liver and kidneys Fe/Zn, Fe, Cu, Zn/Cu ratios were calculated revealing diverse patterns, i.e. both increasing and decreasing trends in the alteration of trace element ratios in these organs of diabetic rats vs. the healthy control rats. For example, in the liver of diabetic rats, increasing trends for Fe/Zn ratio [62–64], Fe/Cu ratio [62–65], Zn/Cu ratio [62–64], with decreasing trends for Zn/Cu [66] ratio vs. the healthy control rats were reported. Furthermore, in the kidneys of diabetic rats, increasing trends for Fe/Zn ratios [65, 66], and Fe/Cu ratios [62, 63], were reported whereas decreased trends were observed for Fe/Cu and Zn/Cu ratios [64–66] vs. the healthy control rats.

In light of scientific reports evaluating the metabolic consequences of trace element ratios in tissues (here: Fe/Zn, Fe/Cu, and Zn/Cu), an attempt has been made to cautiously interpret the experimental data obtained, based on the available knowledge about the role of these elements in maintaining oxidant-antioxidant balance in the biological systems. Therefore, it can be hypothesized that altered (increased or decreased) tissular Fe/Zn, Fe/Cu, and Zn/Cu ratios may imply some metabolic abnormalities associated with oxidative stress

The results of this experiment showed that the type of SG significantly differentiated only the kidney Zn level, as well as the kidney Fe/Zn ratios. Particularly RA, irrespective of its dose, was found to give markedly lower values compared to ST. The reason for this differentiation is unclear, but taking into account previous findings by the authors [20], showing that RA appeared to be less effective in correction of histological damage compared to ST, it can be hypothesized that the efficacy of mitigation of metabolic distress by SG correlates with the changes in the Fe/Zn ratio.

The main limitation of this study is the lack of information on trace element levels in other tissues (e.g. blood, serum, muscles) and their respective biomarkers (e.g. ferritin, Zn, and Cu-dependent enzymes, SOD), however, this weakness was taken into account in the discussion section and the final conclusions. The strength of this study was, however, the use of the storage tissues (liver, kidneys) of Fe, Zn, and Cu, which remain in homeostatic dynamics with other functional pools of these elements (e.g. blood, muscles).

Chronic hyperglycemia itself significantly elevated the liver Zn/Cu ratio, while decreasing the kidney Fe level, as well as Fe/Zn and Zn/Cu ratios in diabetic rats. Supplementary SG (ST, RA) tended to normalize the kidney Zn/Cu ratio, while high doses of SG tended to normalize the kidney Fe concentration in diabetic rats. Interestingly enough, the type of SG (ST, RA), irrespective of its dose, appears to differentiate the kidney Zn level and the Fe/Zn ratio in diabetic rats.

The results of this study contribute to the body of knowledge about the functional properties of SG and their potential to mitigate metabolic alteration caused by hyperglycemia in diabetes. Nevertheless, more studies are warranted to focus on the mechanisms and the significance of these effects in both animal and human subjects.

Ethics Approval:
The experimental protocol was approved by the Local Ethical Commission in Poznań (approval #31/2019).

Conflicts of interest
The authors have no conflicts of interest to declare that are relevant to the content of this article.

FUNDING SOURCE

The presented work is an integral part of the research project (National Science Centre, Poland, NCN 2017/27/B/NZ9/00677).

Author Contribution

Study conception was designed by Zbigniew Krejpcio. Material preparation, data collection and analysis were performed by Jakub Michał Kurek, Ewelina Król and Halina Staniek. The draft of the manuscript was written by Jakub Michał Kurek and Zbigniew Krejpcio. All authors read and approved the final manuscript.

Data Availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Khan MAB, Hashim MJ, King JK et al (2020) Epidemiology of Type 2 Diabetes – Global Burden of Disease and Forecasted Trends. J Epidemiol Glob Health 10:107–111. https://doi.org/10.2991/jegh.k.191028.001
NCD Risk Factor Collaboration (2016) Worldwide trends in diabetes since 1980: a pooled analysis of 751 population-based studies with 4.4 million participants. Lancet Lond Engl 387:1513–1530. https://doi.org/10.1016/S0140-6736(16)00618-8
Namazi N, Moghaddam SS, Esmaeili S et al (2024) Burden of type 2 diabetes mellitus and its risk factors in North Africa and the Middle East, 1990–2019: findings from the Global Burden of Disease study 2019. BMC Public Health 24:98. https://doi.org/10.1186/s12889-023-16540-8
Pastakia SD, Pekny CR, Manyara SM, Fischer L (2017) Diabetes in sub-Saharan Africa – from policy to practice to progress: targeting the existing gaps for future care for diabetes. Diabetes Metab Syndr Obes Targets Ther 10:247–263. https://doi.org/10.2147/DMSO.S126314
Butt MD, Ong SC, Rafiq A et al (2024) A systematic review of the economic burden of diabetes mellitus: contrasting perspectives from high and low middle-income countries. J Pharm Policy Pract 17:2322107. https://doi.org/10.1080/20523211.2024.2322107
Bommer C, Heesemann E, Sagalova V et al (2017) The global economic burden of diabetes in adults aged 20–79 years: a cost-of-illness study. Lancet Diabetes Endocrinol 5:423–430. https://doi.org/10.1016/S2213-8587(17)30097-9
Yu MG, Gordin D, Fu J et al (2023) Protective Factors and the Pathogenesis of Complications in Diabetes. Endocr Rev 45:227–252. https://doi.org/10.1210/endrev/bnad030
Rawshani A, Rawshani A, Franzén S et al (2017) Mortality and Cardiovascular Disease in Type 1 and Type 2 Diabetes. N Engl J Med 376:1407–1418. https://doi.org/10.1056/NEJMoa1608664
Davies MJ, D’Alessio DA, Fradkin J et al (2018) Management of Hyperglycemia in Type 2 Diabetes, 2018. A Consensus Report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care 41:2669–2701. https://doi.org/10.2337/dci18-0033
Einarson TR, Acs A, Ludwig C, Panton UH (2018) Prevalence of cardiovascular disease in type 2 diabetes: a systematic literature review of scientific evidence from across the world in 2007–2017. Cardiovasc Diabetol 17:83. https://doi.org/10.1186/s12933-018-0728-6
Zinman B, Wanner C, Lachin JM et al (2015) Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N Engl J Med 373:2117–2128. https://doi.org/10.1056/NEJMoa1504720
Marso SP, Daniels GH, Brown-Frandsen K et al (2016) Liraglutide and Cardiovascular Outcomes in Type 2 Diabetes. N Engl J Med 375:311–322. https://doi.org/10.1056/NEJMoa1603827
Huang C, Wang Y, Zhou C et al (2024) Properties, extraction and purification technologies of Stevia rebaudiana steviol glycosides: A review. Food Chem 453:139622. https://doi.org/10.1016/j.foodchem.2024.139622
EFSA Panel on Food Additives and Flavourings (FAF), Younes M, Aquilina G et al (2020) Safety of a proposed amendment of the specifications for steviol glycosides (E 960) as a food additive: to expand the list of steviol glycosides to all those identified in the leaves of Stevia Rebaudiana Bertoni. EFSA J 18. https://doi.org/10.2903/j.efsa.2020.6106
Anton SD, Martin CK, Han H et al (2010) Effects of stevia, aspartame, and sucrose on food intake, satiety, and postprandial glucose and insulin levels. Appetite 55:37–43. https://doi.org/10.1016/j.appet.2010.03.009
Chatsudthipong V, Muanprasat C (2009) Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol Ther 121:41–54. https://doi.org/10.1016/j.pharmthera.2008.09.007
Das S, Das AK, Murphy RA et al (1992) Evaluation of the cariogenic potential of the intense natural sweeteners stevioside and rebaudioside A. Caries Res 26:363–366. https://doi.org/10.1159/000261469
Goyal SK, Samsher, Goyal RK (2010) Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int J Food Sci Nutr 61:1–10. https://doi.org/10.3109/09637480903193049
Jeppesen PB, Gregersen S, Poulsen CR, Hermansen K (2000) Stevioside acts directly on pancreatic beta cells to secrete insulin: actions independent of cyclic adenosine monophosphate and adenosine triphosphate-sensitive K+-channel activity. Metabolism 49:208–214. https://doi.org/10.1016/s0026-0495(00)91325-8
Kurek JM, Król E, Krejpcio Z (2020) Steviol Glycosides Supplementation Affects Lipid Metabolism in High-Fat Fed STZ-Induced Diabetic Rats. Nutrients 13:112. https://doi.org/10.3390/nu13010112
Kurek JM, Król E, Staniek H, Krejpcio Z (2024) Steviol glycosides, L-arginine and chromium(III) affect trace element status in diabetic rats. Acta Sci Pol Technol. https://doi.org/10.17306/J.AFS.001258
Prasad AS (2008) Zinc in Human Health: Effect of Zinc on Immune Cells. Mol Med 14:353–357. https://doi.org/10.2119/2008-00033.Prasad
Jiang R, Manson JE, Meigs JB et al (2004) Body iron stores in relation to risk of type 2 diabetes in apparently healthy women. JAMA 291:711–717. https://doi.org/10.1001/jama.291.6.711
Thomas MC, MacIsaac RJ, Tsalamandris C, Jerums G (2004) Elevated iron indices in patients with diabetes. Diabet Med J Br Diabet Assoc 21:798–802. https://doi.org/10.1111/j.1464-5491.2004.01196.x
Li YV (2014) Zinc and insulin in pancreatic beta-cells. Endocrine 45:178–189. https://doi.org/10.1007/s12020-013-0032-x
Ranasinghe P, Pigera S, Galappatthy P et al (2015) Zinc and diabetes mellitus: understanding molecular mechanisms and clinical implications. DARU J Pharm Sci 23:44. https://doi.org/10.1186/s40199-015-0127-4
Chausmer AB (1998) Zinc, insulin and diabetes. J Am Coll Nutr 17:109–115. https://doi.org/10.1080/07315724.1998.10718735
Eljazzar S, Abu-Hijleh H, Alkhatib D et al (2023) The Role of Copper Intake in the Development and Management of Type 2 Diabetes: A Systematic Review. Nutrients 15:1655. https://doi.org/10.3390/nu15071655
DiNicolantonio JJ, Mangan D, O’Keefe JH (2018) Copper deficiency may be a leading cause of ischaemic heart disease. Open Heart 5:e000784. https://doi.org/10.1136/openhrt-2018-000784
Sanjeevi N, Freeland-Graves J, Beretvas SN, Sachdev PK (2018) Trace element status in type 2 diabetes: A meta-analysis. J Clin Diagn Res JCDR 12:OE01–OE08. https://doi.org/10.7860/JCDR/2018/35026.11541
Özenç S, Saldir M, Sarı E et al (2015) Selenium, zinc, and copper levels and their relation with HbA1c status in children with type 1 diabetes mellitus. Int J Diabetes Dev Ctries 35:514–518. https://doi.org/10.1007/s13410-015-0327-y
Mohamed AK, Bierhaus A, Schiekofer S et al (1999) The role of oxidative stress and NF-kappaB activation in late diabetic complications. BioFactors Oxf Engl 10:157–167. https://doi.org/10.1002/biof.5520100211
Dinçer Y, Akçay T, Alademir Z, Ilkova H (2002) Assessment of DNA base oxidation and glutathione level in patients with type 2 diabetes. Mutat Res 505:75–81. https://doi.org/10.1016/s0027-5107(02)00143-4
Ziegler D, Sohr CGH, Nourooz-Zadeh J (2004) Oxidative stress and antioxidant defense in relation to the severity of diabetic polyneuropathy and cardiovascular autonomic neuropathy. Diabetes Care 27:2178–2183. https://doi.org/10.2337/diacare.27.9.2178
Forbes JM, Coughlan MT, Cooper ME (2008) Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 57:1446–1454. https://doi.org/10.2337/db08-0057
Viktorínová A, Tošerová E, Križko M, Ďuračková Z (2009) Altered metabolism of copper, zinc, and magnesium is associated with increased levels of glycated hemoglobin in patients with diabetes mellitus. Metabolism 58:1477–1482. https://doi.org/10.1016/j.metabol.2009.04.035
Zheng Y, Li X-K, Wang Y, Cai L (2008) The role of zinc, copper and iron in the pathogenesis of diabetes and diabetic complications: therapeutic effects by chelators. Hemoglobin 32:135–145. https://doi.org/10.1080/03630260701727077
Fenton HJH (1894) LXXIII.—Oxidation of tartaric acid in presence of iron. J Chem Soc Trans 65:899–910. https://doi.org/10.1039/CT8946500899
Haber F, Weiss J, Pope WJ (1997) The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Lond Ser - Math Phys Sci 147:332–351. https://doi.org/10.1098/rspa.1934.0221
Halliwell B, Gutteridge JM (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1–85. https://doi.org/10.1016/0076-6879(90)86093-b
Henle ES, Linn S (1997) Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. J Biol Chem 272:19095–19098. https://doi.org/10.1074/jbc.272.31.19095
Koppenol WH (1993) The centennial of the Fenton reaction. Free Radic Biol Med 15:645–651. https://doi.org/10.1016/0891-5849(93)90168-t
Minotti G, Aust SD (1987) The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide. J Biol Chem 262:1098–1104. https://doi.org/10.1016/S0021-9258(19)75755-X
Walling C (1975) Fenton’s reagent revisited. Acc Chem Res 8:125–131. https://doi.org/10.1021/ar50088a003
Rains JL, Jain SK (2011) Oxidative stress, insulin signaling, and diabetes. Free Radic Biol Med 50:567–575. https://doi.org/10.1016/j.freeradbiomed.2010.12.006
Valko M, Jomova K, Rhodes CJ et al (2016) Redox- and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol 90:1–37. https://doi.org/10.1007/s00204-015-1579-5
Wei W, Liu Q, Tan Y et al (2009) Oxidative stress, diabetes, and diabetic complications. Hemoglobin 33:370–377. https://doi.org/10.3109/03630260903212175
Swaminathan S, Fonseca VA, Alam MG, Shah SV (2007) The role of iron in diabetes and its complications. Diabetes Care 30:1926–1933. https://doi.org/10.2337/dc06-2625
Fernández-Real JM, Manco M (2014) Effects of iron overload on chronic metabolic diseases. Lancet Diabetes Endocrinol 2:513–526. https://doi.org/10.1016/S2213-8587(13)70174-8
Simcox JA, McClain DA (2013) Iron and diabetes risk. Cell Metab 17:329–341. https://doi.org/10.1016/j.cmet.2013.02.007
Chistiakov DA, Voronova NV (2009) Zn(2+)-transporter-8: a dual role in diabetes. BioFactors Oxf Engl 35:356–363. https://doi.org/10.1002/biof.49
Bandeira VDS, Pires LV, Hashimoto LL et al (2017) Association of reduced zinc status with poor glycemic control in individuals with type 2 diabetes mellitus. J Trace Elem Med Biol 44:132–136. https://doi.org/10.1016/j.jtemb.2017.07.004
Puri M, Gujral U, Nayyar SB, COMPARATIVE STUDY OF SERUM ZINC, MAGNESIUM AND COPPER LEVELS AMONG PATIENTS OF TYPE 2 DIABETES MELLITUS WITH AND WITHOUT MICROANGIOPATHIC COMPLICATIONS (2013). Innov J Med Health Sci
Chasapis CT, Loutsidou AC, Spiliopoulou CA, Stefanidou ME (2012) Zinc and human health: an update. Arch Toxicol 86:521–534. https://doi.org/10.1007/s00204-011-0775-1
Gembillo G, Visconti L, Giuffrida AE et al (2022) Role of Zinc in Diabetic Kidney Disease. Nutrients 14:1353. https://doi.org/10.3390/nu14071353
Takeda A, Tamano H (2009) Insight into zinc signaling from dietary zinc deficiency. Brain Res Rev 62:33–44. https://doi.org/10.1016/j.brainresrev.2009.09.003
Bo S, Durazzo M, Gambino R et al (2008) Associations of dietary and serum copper with inflammation, oxidative stress, and metabolic variables in adults. J Nutr 138:305–310. https://doi.org/10.1093/jn/138.2.305
Park K, Gross M, Lee D-H et al (2009) Oxidative stress and insulin resistance: the coronary artery risk development in young adults study. Diabetes Care 32:1302–1307. https://doi.org/10.2337/dc09-0259
Car N, Car A, Granić M et al (1992) Zinc and copper in the serum of diabetic patients. Biol Trace Elem Res 32:325–329. https://doi.org/10.1007/BF02784618
Gembillo G, Labbozzetta V, Giuffrida AE et al (2022) Potential Role of Copper in Diabetes and Diabetic Kidney Disease. Metabolites 13:17. https://doi.org/10.3390/metabo13010017
Arredondo M, Núñez MT (2005) Iron and copper metabolism. Mol Aspects Med 26:313–327. https://doi.org/10.1016/j.mam.2005.07.010
Król E, Krejpcio Z, Michalak S et al (2012) Effects of combined dietary chromium(III) propionate complex and thiamine supplementation on insulin sensitivity, blood biochemical indices, and mineral levels in high-fructose-fed rats. Biol Trace Elem Res 150:350–359. https://doi.org/10.1007/s12011-012-9515-5
Król E, Jeszka-Skowron M, Krejpcio Z et al (2016) The Effects of Supplementary Mulberry Leaf (Morus alba) Extracts on the Trace Element Status (Fe, Zn and Cu) in Relation to Diabetes Management and Antioxidant Indices in Diabetic Rats. Biol Trace Elem Res 174:158–165. https://doi.org/10.1007/s12011-016-0696-1
Salvador ÂC, Król E, Lemos VC et al (2016) Effect of Elderberry (Sambucus nigra L.) Extract Supplementation in STZ-Induced Diabetic Rats Fed with a High-Fat Diet. Int J Mol Sci 18:13. https://doi.org/10.3390/ijms18010013
Król E, Krejpcio Z, Okulicz M, Śmigielska H (2020) Chromium(III) Glycinate Complex Supplementation Improves the Blood Glucose Level and Attenuates the Tissular Copper to Zinc Ratio in Rats with Mild Hyperglycaemia. Biol Trace Elem Res 193:185–194. https://doi.org/10.1007/s12011-019-01686-7
Król E, Krejpcio Z, Iwanik K (2014) Supplementary Chromium(III) Propionate Complex Does Not Protect Against Insulin Resistance in High-Fat-Fed Rats. Biol Trace Elem Res 157:147–155. https://doi.org/10.1007/s12011-013-9877-3

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Steviol glycosides affect trace element status in diabetic rats

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Version 1

Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

EXPERIMENTAL AGENTS

STUDY PROTOCOL

TRACE ELEMENT ANALYSIS

STATISTICAL ANALYSIS

RESULTS

LIVER TRACE ELEMENT STATUS

KIDNEY TRACE ELEMENT STATUS

DISCUSSION

CONCLUSIONS

Declarations

Ethics Approval:
The experimental protocol was approved by the Local Ethical Commission in Poznań (approval #31/2019).

Conflicts of interest
The authors have no conflicts of interest to declare that are relevant to the content of this article.

FUNDING SOURCE

Author Contribution

Data Availability

References

Additional Declarations

Status:

Version 1

Steviol glycosides affect trace element status in diabetic rats

Status:

Version 1

Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

EXPERIMENTAL AGENTS

STUDY PROTOCOL

TRACE ELEMENT ANALYSIS

STATISTICAL ANALYSIS

RESULTS

LIVER TRACE ELEMENT STATUS

KIDNEY TRACE ELEMENT STATUS

DISCUSSION

CONCLUSIONS

Declarations

Ethics Approval: The experimental protocol was approved by the Local Ethical Commission in Poznań (approval #31/2019).

Conflicts of interest The authors have no conflicts of interest to declare that are relevant to the content of this article.

FUNDING SOURCE

Author Contribution

Data Availability

References

Additional Declarations

Status:

Version 1

Ethics Approval:
The experimental protocol was approved by the Local Ethical Commission in Poznań (approval #31/2019).

Conflicts of interest
The authors have no conflicts of interest to declare that are relevant to the content of this article.