Introduction

Neuropathy is one of most commonly occurring diabetic complications with an overall prevalence of 50–60 % in diabetic patients [1]. Some of the functional consequences of diabetic neuropathy could be alleviated by insulin treatment in insulin-dependent diabetes mellitus [2]. Oxidative stress plays an important role in the pathogenesis of diabetes through reactive oxygen species (ROS) that may initiate inflammatory response because they stimulate a number of genes regulating the inflammatory signaling cascades [3]. These genes may be upregulated by ROS-mediated activation of nuclear factor-kappa B (NF-κB), one of the primary transcription factors initiating inflammatory response and contributing to inflammatory damage in chronic diseases including diabetes [4]. It mediates numerous inflammatory pathways in multiple cells and organ systems. Inflammation is now recognized to exacerbate many neurodegenerative conditions including diabetic neuropathy [5]. Inhibition of NF-κB activity in spinal glia alleviates pain behaviors in rats with chronic nerve constriction injuries [6]. Nuclear factor erythroid 2-related factor 2 (Nrf2), involved in combating against oxidative stress and neuroinflammation, is a basic leucine zipper transcription factor known to regulate the expression of a number of detoxifying and antioxidant genes. This has also been claimed to regulate various inflammatory processes [7, 8]. Several studies ascertained pivotal role of Nrf2 in modulation of inflammation in insulin resistance and diabetes [9, 10].

Chromium (Cr) was proposed to be an essential element about 50 years when its role was believed to be associated with glucose metabolism and insulin action. This role has recently been questioned as Cr has been proposed to act as a pharmacological agent [11, 12]. To achieve pharmacological effects, it is clear that supranutritional dose is necessary or subjects that carry risks for impaired glucose and/or lipid metabolism. Indeed, there is an interrelationship between chronic diseases and various micronutrients such as Cr, selenium (Se), and zinc (Zn) [13–16]. Significant alterations of these elements in diabetic individuals and animals have been attributed to insulin deficiency [13]. Patients with diabetes have lower serum Cr, Zn, and Se levels than healthy subjects [13]. Moreover, absorption and excretion of Cr are higher in diabetic subjects than in nondiabetic subjects [17, 18].

Various Cr chelates are available. The Cr histidinate (CrHis) complex is a form developed to enhance stability and absorption of Cr, which is shown in a human study [19]. The present study tested the hypothesis that CrHis is more efficacious than CrPic for improving glucose metabolism in diabetic subjects, with respect to the IκB/NF-κB pathway and Nrf2 responses in the brain. Inhibition of the IκB/NF-κB pathway or Nrf2 response may be involved in the amelioration of insulin resistance during chromium supplementation in the brain of diabetic rats [20]. Therefore, a combination of a high-fat diet (HFD) and low-dose streptozotocin (STZ) injection were used to create a type 2 diabetic animal model [16] to investigate the effect of CrHis/CrPic supplementation on changes in IκB/NF-κB pathway and Nrf2 expression in the brain under diabetic conditions.

Materials and Methods

Animals, Diets, and Experimental Design

Male Wistar rats (n = 90, 8 weeks old) weighing 200–250 g were purchased from Firat University Laboratory Animal Research Centre (Elazig, Turkey) and reared at the temperature of 22 ±2 °C, humidity of 55 ± 5 %, and with a 12/12-h light/dark cycle. The experiment was conducted under the protocol approved by the Ethical Committee of Firat University. All procedures involving rats were conducted in strict compliance with relevant laws, the Animal Welfare Act, Public Health Services Policy, and guidelines established by the Institutional Animal Care and Use Committee of the university. Rats consumed a standard diet and tap water ad libitum.

Ingredients and chemical composition of the basal (control) diet are shown in Table 1. The diets were stored at 4 °C cold chamber. Animals were fed with a diet consisting of either 8 % fat (control) or 40 % fat (HFD). CrPic and CrHis (Nutrition 21, Inc., Purchase, NY, USA) chelates were dissolved in water to assure daily consumption of 8 μg elemental Cr via drinking water for 12 weeks. This amount was calculated based on 560 μg Cr that is needed for a 70-kg adult human after adjusting doses based on metabolic body size (700.70 = 19.60 kg, needing 560 μg Cr; ~0.2500.70 = 0.38 kg needing 10.8 μg Cr).

Table 1 Composition of experimental diets
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A rat model of type 2 diabetes created by feeding with a HFD and STZ treatment developed by Reed et al. [21] provides a novel animal model for type 2 diabetes that is applicable in testing antidiabetic compounds. In this model, established hyperglycaemia status (glucose level >140 mg/dl) after STZ injection (40 mg/kg i.p., for 2 weeks) in high-fat fed rats was not due to a greater decline in β-cell function. Before STZ injection, glucose concentrations of rats were measured and compared to controls.

The 2 × 3 factorially arranged experimental groups were: group I (rats fed with the control diet only), group II (rats fed with the control diet and supplemented with CrPic), group III (rats fed with the control diet and supplemented with CrHis), group IV (diabetic rats fed with the HFD diet), group V (diabetic rats fed with the HFD diet and supplemented with CrPic), and group VI (diabetic rats fed with the HFD diet and supplemented with CrHis).

Western Blot Analyses

In all groups, brains were removed from sacrificed rats by cervical dislocation. Small pieces of samples in each group of animals were pooled together for Western blot analysis. Protein extraction was performed as follows: The sample was homogenized in an ice-cold 1 ml of hypotonic buffer A [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mM ethylene diamine tetraacetic acid (EDTA), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)]. A solution of 80 μl of 10 % Nonidet P-40 (NP-40) was added to the homogenates, and the mixture was centrifuged for 2 min at 14,000×g. The supernatant was collected as a cytosolic fraction for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα) and 4-hydroxynonenal (4-HNE) assays. The precipitated nuclei were washed once with 500 μl of buffer A plus 40 μl of 10 % NP-40, centrifuged, resuspended in 200 μl of buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA), 1 mM DTT, 0.1 mM PMSF, and 20 % glycerol], and centrifuged for 5 min at 14,800×g. The supernatant containing nuclear proteins was collected for Nrf2 and NF-κB p65 [22].

Concentration of the protein was determined according to the procedure described by Lowry using a commercial protein assay kit (Sigma, St. Louis, MO, USA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer containing 2 % β-mercaptoethanol was added to the supernatant. Equal amounts of protein (50 μg) were electrophoresed and subsequently transferred to nitrocellulose membranes (Schleicher and Schuell Inc., Keene, NH, USA). Nitrocellulose blots were washed twice for 5 min each in phosphate-buffered saline (PBS) and blocked with 1 % bovine serum albumin in PBS at room temperature for 1 h prior to application of the primary antibody. The antibody against Nrf-2 and 4-HNE adducts were the purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and 4 Alpha Diagnostics (San Antonio, TX), respectively. Antibody against IκBα and NF-κB p65 was purchased from Abcam (Cambridge, UK). Primary antibody was diluted (1:1,000) in the same buffer containing 0.05 % Tween-20. The nitrocellulose membrane was incubated overnight at 4 ºC with protein antibody. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK). Specific binding was detected using diaminobenzidine and H2O2 as substrates. Protein loading was controlled using a monoclonal mouse antibody against β-actin antibody (A5316; Sigma). Blots were performed at least three times to confirm data reproducibility. Bands were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, USA).

Statistical Analysis

The data were analyzed using the general linear model procedure of SAS software [23]. The least square means of the groups were compared at a significant probability of less than 0.05 using ANOVA. Treatments were also compared using Student's unpaired t test for comparison of individual treatment. Between-group differences in latencies were analyzed by the analysis of variance for repeated measurements followed by Fisher's post hoc test for all groups.

Results

The brain NF-κB p65 subunit expression increased significantly in diabetic rats (Fig. 1a). Although Cr chelates did not alter brain NF-κB level in nondiabetic rats, both chelates significantly decreased brain NF-κB level in nondiabetic rats, at a greater extent in diabetic rats supplemented with CrHis than those supplemented with CrPic (P < 0.05).

Fig. 1
figure 1

Western blot analysis of cerebral nuclear factor-kappa B (NF-κB p65) (a), nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor (IκBα) (b), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (c), and 4-hydroxynonenal (HNE) protein adducts (d) in nondiabetic (fed control diet, c) and diabetic (injected with streptozotocin, STZ, and fed with high-fat diet, HFD) rats receiving 8 μg elemental Cr via drinking water in the form of chromium picolinate (CrPic) or chromium histidinate (CrHis). A representative blot is shown (n = 3). Actin was included to ensure equal protein loading. Densitometric analyses of these bands are represented as percent of control. Values are means ± standard error of the mean. Different letters indicate group mean differences

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The diabetes decreased cerebral IκBα level (Fig. 1b). Despite no alteration in nondiabetic rats, Cr chelates increased cerebral IκBα level in diabetic rats. This increase was more in rats supplemented with CrHis than rats supplemented with CrPic, but did not reach level of nondiabetic rats.

The protein concentration of Nrf2 in the brain tissues for diabetic rats was lower than that for nondiabetic rats (Fig. 1c). Neither CrPic nor CrHis affected cerebral Nrf2 protein level in nondiabetic rats. The extent of protein level increase in diabetic rats supplemented with CrHis was greater than those supplemented with CrPic.

The brain 4-HNE protein adducts increased significantly in diabetic rats (Fig. 1d). Supplemental Cr chelates did not affect cerebral level of 4-HNE protein adducts. However, both Cr forms decreased the level of 4-HNE protein adducts in brains of diabetic rats at a similar extent.

Discussion

The purpose of this study was to determine the effects of CrHis or CrPic on inflammatory markers in the brain of diabetic rats. Similar to the present study, other reports have shown that diabetes causes alterations in synthesis or concentrations of inflammatory cytokines [24–26]. In this experiment, CrHis or CrPic decreased NF-κB activation in rats with HFD/STZ-induced brain injury, suggesting that CrPic and CrHis decrease lipid peroxidation via the Nrf2/ARE-mediated pathway, as reflected by 4-HNE protein adducts. Jain et al. [25] reported that chromium niacinate supplementation lowered the blood levels of tumor necrosis-α (TNF-α), interleukin-6 (IL-6), C-reactive protein, and cholesterol and CrPic supplementation caused a decrease in TNF-α, IL-6, and lipid peroxidation in rats.

Various extracellular signals can initiate NF-κB pathways by activating IκB kinase complex (IKK). The activation of IκB kinase complex leads to the phosphorylation, ubiquitination, and degradation of IκB, which allows NF-κB to enter the nucleus where it regulates the expression of specific genes [4, 27]. Zhang et al. [28] hypothesized that IKKβ/NF-κB pathway is linked to dysfunction of hypothalamic signaling induced by overnutrtion. They [28] studied the connection between IKKβ/NF-κB and central dysregulation of energy balance (insulin/leptin pathway) in the hypothalamus and found that chronic high-fat feeding up-activated NF-κB in hypothalamus. In a previous study, we reported that NF-κB p65 increased in rats fed with HFD compared to rats fed with standard diet, but reduced by the CrHis administration [29].

Nrf2 is considered as the axis of defense against oxidative stress, and there is a clear correlation between pathogenesis of diabetic neuropathy and Nrf2 pathway [8, 10]. Nrf2 pathway has been implicated to play a significant role in contributing to the antioxidant defense of the body. Excess production of ROS is considered to cause abnormal axon morphology and altered neuronal membrane permeability along with causing functional modification of various cellular proteins [26, 30]. Nrf2 and HO-1 have been shown to possess protective effect against STZ-induced diabetes and diabetic neuropathy [8]. In the present work, Nrf2 level in the HFD/STZ-induced diabetes group was lower than those of controls in brain, whereas Cr treatment induced activation of Nrf2 and enhanced nuclear translocation and subsequent ARE binding, suggesting that Cr may be involved in stabilization and activation of Nrf2. Yet, this needs further studies to be substantiated. In a previous study, it was shown that hepatic Nrf2 and HO-1 levels increased by supplementation of CrHis in rats fed with HFD [29].

Oxidative stress plays a major role in diabetes as well as in diabetic neuropathy [31, 32]. The reaction of free radicals with membrane lipids causes the formation of lipid peroxidation products including several aldehydic compounds, one of which is highly toxic and called 4-HNE. This is frequently measured as an indicator of lipid peroxidation and oxidative stress in vivo and considered as an index of oxidative stress. Moreover, it is one of the most effective activators of Nrf2 [33]. 4-HNE forms adducts with key neuronal proteins [34] and these adducts have been shown to be increased in the peripheral nerves of STZ diabetic rats [34, 35]. In the present study, 4-HNE protein adducts, indicator of lipid peroxidation, in the brain of diabetic rats decreased when dietary CrHis or CrPic was supplemented. CrHis supplementation did not alter these parameters in nondiabetic rats. The current study appears to be the first to examine the specific association between dietary Cr intake and 4-HNE protein adducts in diabetic rats. Significantly lower levels of 4-HNE adducts observed in diabetic rats may indicate an association between Cr intake and 4-HNE adducts for diabetic rats. Indeed, previous findings have shown that the production of 4-HNE is altered in diabetes, resulting in increased susceptibility of the tissues to injury [36]. Cr is postulated to function to augment antioxidant defense system, as confirmed by decreases in lipid peroxidation, TNF-α, and IL-6 [16, 25]. Preuss et al. [37] also reported a decrease in hepatic TBARS formation by supplementation of CrPic and Cr nicotinate in rats.

In conclusion, diabetes affected the IκB/NF-κB pathway and Nrf2 responses in brain tissue, as reflected by increased cerebral NF-κB and 4-HNE protein adducts levels and decreased cerebral Nrf2and IκBα. Cr chelates (CrPic and CrHis) exerted protective role in diabetic rats. Histidinate form of Cr was superior to picolinate form of Cr in reversing brain injury in diabetes, as reflected by a greater reduction in level of NF-κB and greater increases in levels of IκBα and Nrf2 in the brain.