Cadmium exposure decreases fasting blood glucose levels and exacerbates type-2 diabetes in a mouse model

Although the effects of cadmium (Cd) on the development of diabetes have been extensively investigated, the relationship between Cd exposure and the severity of established diabetes is unclear. Herein, we investigate the effects of long-term exposure to Cd in a streptozotocin-induced mouse model of type-2 diabetes mellitus (T2DM) and the underlying mechanism. C57BL/6 Mice were divided into the following four groups: (1) control group; (2) Cd-exposed group; (3) diabetic group; (4) Cd-exposed diabetic group. Cd exposure was established by the administration of 155 ppm CdCl2 in drinking water. After 25 weeks of treatment, serum fasting glucose and insulin were measured. Meanwhile, the liver and pancreas specimens were sectioned and stained with Hematoxylin and eosin. Gluconeogenesis, glycolysis, lactate concentration, and fibrosis in liver were evaluated. Clinical signs attributable to diabetes were more apparent in Cd-exposed diabetic mice, while no effects of Cd exposure were found on non-diabetic mice. Cd exposure significantly decreased fasting blood glucose (FBG) levels in diabetic group. We further demonstrated that the glycolysis related hepatic enzymes, pyruvate kinase M2 (PKM-2) and lactic dehydrogenase A (LDHA) were both increased, while the gluconeogenesis related hepatic enzymes, phosphoenolpyruvate-1 (PCK-1) and glucose-6-phosphatase (G6Pase) were both decreased in Cd exposed diabetic mice, indicating that Cd increased glycolysis and inhibited gluconeogenesis in diabetic model. Moreover, lactate accumulation was noted accompanied by the increased inflammation and fibrosis in the livers of diabetic mice following Cd exposure. Cd exposure disturbed glucose metabolism and exacerbated diabetes, providing a biological relevance that DM patients are at greater risk when exposed to Cd.


Introduction
Diabetes mellitus (DM) is a major contributor to morbidity, mortality, and disability, which represents a critical public health issue [1]. As other chronic diseases, DM is also genetic and environmentally-related [2]. Environmental pollutants, such as heavy metals, have been implicated as contributing to the pathogenesis of DM. Cadmium (Cd) is an environmental pollutant and has been associated with DM [3,4]. Prior research in animal models identified a dysregulation of glucose metabolism, specifically, a relationship between Cd exposure and increased blood glucose levels [5,6]. As reported, Cd exposure induced hyperglycemia, altered oxidative status and led to pancreatic β-cell dysfunction [7][8][9][10]. A recent study reported that Cd exposure changed the expression of multiple genes associated with diabetes susceptibility and glucose homeostasis [11]. Other studies found that Cd accumulation had to be reached threshold before detrimental effect on glycaemia and urinary Cd was associated with impaired fasting glucose in a dose-dependent manner after controlling for ethnicity, age, BMI, gender, and smoking [12]. Our research group also found that Cd decreased serum insulin concentrations and induced insulin resistance [13]. We further suggested that urinary Cd levels in humans were positively associated with DM [14]. In human, a cohort study suggested that individuals with high levels of urine Cd and plasma CRP (creactive protein) were at a greater risk of developing T2DM [15]. However, the underlying molecular and cellular mechanisms remain unclear to properly assess the actual relation between Cd exposure and DM.
Although the effects of Cd on the development of diabetes have been extensively investigated, the relationship between Cd exposure and the severity of established diabetes is unclear. Cd toxicity was identified as the cause of Itai-itai disease [16]. Currently, Cd continues to pose a significant threat to human health worldwide. Cd exists in the earth's crust and is widely distributed in the environment due to industrial and agricultural activities [17]. Apart from occupational exposure, drinking water, cigarette smoking, recharged nickel-Cd batteries and such foods as cereals, vegetables, potatoes, and meat products are the major sources of human Cd exposure [18,19]. Owing to the high level of Cd pollution and the increasing prevalence of DM in industrialized countries, the effects of Cd exposure on the severity of established DM need to be better understood.
In the present study, we analyzed the effects of Cd exposure on diabetes in a T2DM mouse model, and we included analyses to describe changes in key hepatic metabolic enzymes in these mice. Lactate, inflammation, and fibrosis were also analyzed in mouse livers after Cd exposure. These endpoints were used to better understand the possible mechanisms for an increased risk of diabetic severity caused by Cd exposure. Based on the widespread distribution of Cd in the environment and the increasing prevalence of DM in industrialized countries, our research provided biological relevance that DM patients are at greater risk for disease progression when exposed to Cd.

Animal model and treatment
Eight-week-old C57BL/6 male mice were purchased from the Experimental Animal Centre of Soochow University (Suzhou, China). All mice were housed five per cage in poly-carbonate cages and maintained under a natural light/ dark cycle at 18-28°C and 40-60% humidity. High fat diet combined with administration of streptozotocin (STZ) is frequently used to produce experimental T2DM [20]. After acclimatization to laboratory conditions for 1 week, the mice were fed by high fat diet (26.2% protein, 26.3% carbohydrate and 34.9% fat; Biopike company, D12492).
Eight weeks later, mice were administered STZ (100 mg/ kg) once by intraperitoneal injection. One week later, mice with fasting blood glucose (FBG) levels were measured by an automatic glucometer (Roche, Switzerland, Accu-Chek) and ≥16.7 mmol/L were defined as T2DM mice. Mice were then subdivided into the following four groups: (1) non-diabetic mice (CON, n = 10);(2) non-diabetic mice administered 155 ppm CdCl 2 in the drinking water (Cd, n = 10); (3) diabetic mice (DM, n = 15); (4) diabetic mice administered 155 ppm CdCl 2 in the drinking water (DM + Cd, n = 15). After 25 weeks of treatment, all mice were sacrificed by cervical dislocation. Blood samples were collected from the retroorbital plexus and allowed to clot at room temperature for 10 min and then centrifuged at 3000 × g at 4°C for 10 min to obtain serum. Tissues were removed and fixed in 10% buffered formalin or frozen at −80°C. The study protocol was approved by the Soochow University Institutional Animal Care and Use Committee (SCKK2017-0006). All procedures were conducted in accordance with the guidelines of the care and use of laboratory animals [21].

Assessment of clinical signs
Animal had been observed at least once a day by the staff who had at least 2 years of experience in mice studies, and body weight was measured once a week. The food intake and water consumption of each cage were monitored regularly. Mice showed signs of distress had been monitored frequently and carefully observed. Any mouse appeared to be in grave distress or moribund condition were euthanized.

H&E staining
Liver and pancreas specimens from all experimental groups that were fixed in 10% neutral buffered formalin were dehydrated and embedded in paraffin. The tissues were sectioned (5 μm thickness) and stained with hematoxylin and eosin (H&E, Beyotime, China). Histological features were observed using light microscopy (CKX41, OLYM-PUS, Tokyo, Japan).

Masson trichrome collagen staining
The liver sections were stained with Weigert's iron hematoxylin solution for 5 min, acid ethanol for 10 s for differentiation, and then washed with tap water. Sections were counterstained with Masson trichrome stain (Sbjbio, China) for 4 min, then washed with tap water for 1 min. Sections were then stained with Ponceau stain for 5 min and washed with phosphomolybdic acid for 2 min. Next, a weak acid working solution (pH 5.8) was used to wash the sections for 1 min. Sections were then stained with aniline blue for 2 min and washed with the weak acid working solution for 1 min. Finally, sections were washed with 95% and 100% ethyl alcohol three times for 10 s and xylene for 1 min, respectively. Sections were evaluated microscopically as described above.

Enzyme-linked immunosorbent (ELISA) assay
The level of insulin in serum was measured by ELISA (Elabscience Biotechnology, Wuhan, China) according to the manufacturer's instructions. Optical density (OD) was measured at 450 nm. Insulin concentrations were expressed as pg/ml.

Lactate assay
Tissue lactate concentrations were determined using the Lactate Assay Kit (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China). The tissues were homogenized with a grinding miller, and the supernatants were extracted and centrifuged (10,000◊g, 10 min), and then collected for further evaluation. The solutions from the assay kit were mixed and incubated for 10 min at 37°C, and the reaction was stopped by stop solution. The OD was measured at 530 nm. The concentrations of lactate in tissue were normalized and expressed as mmol/g protein, serum lactate levels were expressed as mmol/L.

Western blot
Tissues were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitors. Protein concentrations were determined using a BCA protein assay kit (Beyotime). Equal amounts of sample were separated on SDS-PAGE and transferred to PVDF membranes. The samples were blocked with 5% skimmed milk in PBS-T, and then incubated with antibodies for PKM-2, LDHA, and GAPDH as a loading control (all rabbit monoclonal antibodies, diluted 1:1000). Blots were incubated with goat antirabbit IgG secondary antibody (diluted 1:3000). Reactive signals were detected by a chemiluminescence imager (GeneGnomeXRQ).

RNA extraction from tissue and rt-PCR
Total RNA was extracted from frozen tissues using the Total RNA Extraction Kit (Invitrogen, USA). Extracted RNA was used as a template for reverse transcription using the RT Reagent Kit. Real-time quantitative PCR was performed using the ABI 7300 Real-time PCR System and SYBR Premix Ex Taq TM II. The reaction was performed in a final volume of 20 μL. The cycle began with an initial denaturing step for 1 min at 95°C followed by 40 PCR cycles: 15 s at 95°C and 25 s at 63°C. Relative gene expression was calculated using the 2−ΔΔCt method. The sequences of the forward (F) and reverse (R) primers are presented in Table 1.

Statistical analysis
All quantitative data are expressed as the mean ± SD of three or more independent experiments. Comparisons between two groups were analyzed using Student's t test, and comparisons between more than two groups were made using one-way ANOVA to identify differences among means. A value of P < 0.05 was considered statistically significant. Statistical analyses were performed, and graphs were created, using a GraphPad cameyo statistical package.

Cd exacerbated clinical signs in diabetic mice
Cd mice showed no sign of Cd toxicity, whose body weight were similar as control mice. Both DM mice and DM + Cd mice showed diabetic signs including polyuria, polydipsia, and polyphagia, whose body weights dropped gradually after STZ injection at 8th week. The body weight of the DM + Cd mice was significantly less than the DM mice ( Fig. 1, Table 2). Moreover, significantly decreased locomotor activity were found in DM and Cd + DM group mice, especially in Cd + DM group mice. Six mice were euthanized since these mice appeared to be in grave distress in DM + Cd mice. There was no death in other three groups ( Table 2).  Figure 1C). The fasting serum insulin levels in the DM + Cd mice were also decreased compared with DM mice (67.12 ± 9.29 vs 81.60 ± 10.50, P < 0.01. Figure 1D), and this finding was supported histopathologically by the abnormal shapes of islets in the diabetic mice after Cd treatment (Fig. 1E).

Cd promotes liver lactate accumulation in diabetic mice
Lactate is an end-product of glycolysis. As shown in Fig. 3A, there were significant increase of lactate concentration in DM mice and DM + Cd mice compared with CON mice (0. 13

Discussion
In this study, we observed that Cd exacerbated the clinical signs and progression of diabetes in the T2DM mice. STZinduced mice are characterized by a significant hyperglycemia, loss of body weight, polyphagia and polydipsia, which is classic diabetic symptoms. Cd exposure decreased body weight, serum insulin level, and locomotor activity in diabetic mice. The phenomenon is consistent with the clinical picture of advanced diabetes in humans, which is characterized by decreased serum insulin content, physical weakness, and high mortality [22]. The results provide a biological relevance that DM patients are at greater risk when exposed to Cd. Surprisingly, Cd decreased the FBG levels in diabetic mice despite the serum insulin concentrations being decreased. This is in contrast with some published studies where Cd was reported to increase FBG levels in normal mice or diabetic mice [23,24]. However, our data were consistent with other studies showing that Cd decreased FBG levels [25,26]. The reasons for this discrepancy, and the anomaly of FBG and insulin both decreasing, are unclear. A full resolution to this apparent discrepancy will be the focus of future research.
Liver plays a key role in blood glucose regulation by glycolysis and gluconeogenesis. To determine whether Cd exposure existed effects on glycolysis and gluconeogenesis, contents of several enzymes in liver were investigated. The PKM enzyme is a rate-controlling key glycolytic enzyme in glycolysis, which catalyzes the conversion of phosphoenolpyruvate to pyruvate [27]. LDHA is one of the endpoint of glycolysis pathway, catalyzing the formation of lactate from pyruvate [28]. We found that Cd stimulated the expression of PKM-2 and LDHA in the liver of diabetic mice, indicating Cd increased liver glycolysis in diabetic mice. Studies reported that Cd stimulated liver glycolysis in aquatic organism [29]. However, there is no report about the effect of Cd exposure on liver glycolysis in mammals. Published studies suggested that Cd promoted glycolysis in lung and neuronal cells in rats [30,31]. In the present study, increased lactate production in liver of Cd-exposed mice supported the idea that Cd increased glycolysis.
Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme of gluconeogenesis in the liver [32,33]. We found no significant difference in expression of PCK-1 between diabetic mice and control mice. However, Cd exposure significantly decreased the expression of PCK-1 in diabetic mice liver. PEPCK is a rate-limiting step in gluconeogenesis, which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate. So, activity of PEPCK determines the amount of glucose produced. Thus, the decreased expression of PCK-1 indicates Cd decreased gluconeogenesis in diabetic mice. Besides, G6Pase is an another essential enzyme to gluconeogenesis, which induce the hydrolysis from glucose-6-phosphate to glucose [34]. In this study, the mRNA levels of both PCK-1 and G6Pase were inhibited by Cd exposure, which indicated that Cd inhibited hepatic gluconeogenesis in diabetic mice.
Based on these results, it can be concluded that the marked decrease in blood glucose level in Cd exposed mice, as observed in the present study, may result from promoted glucose metabolism by glycolysis and inhibiting hepatic endogenous glucose production by gluconeogenesis. Fig. 4 Cd promoted liver fibrosis in diabetic mice. A Relative mRNA levels of Collagen I and Collagen III in liver. B Representative images of Masson trichrome staining in liver. Areas of fibrosis indicated with black arrows. Data are represented as mean ± SD. Statistical significance is considered at ***P < 0.001, **P < 0.01, *P < 0.05, compared with CON; ### P < 0.001, ## P < 0.01, # P < 0.05, compared with Cd; &&& P < 0.001, && P < 0.01, & P < 0.05, compared with DM It is paradoxical that improved glucoregulation and exacerbated diabetic symptoms. Next, lactate concentration was studied. As expected, significant increased lactate production was found in the liver of diabetic mice after Cd exposure, which may result from decreased gluconeogenesis and increased glycolysis. Lactate, the end product of glycolysis, is acknowledged as an energy source and intermediary metabolic product [35]. Excessive lactate production results in lactic acidosis, which is a rare but lifethreatening complication of DM [36]. However, it is not clear the side effects of elevated lactate concentration which is below the lactic acidosis level. Researches focus on the metabolic flexibility of lactate in disease. However, little is known about the underlying mechanism of the nonmetabolic functions and accumulation of lactate in pathological progression. Healthy liver exhibits a higher lactate clearance than any other organ [37]. A report found that blood lactate might be an independent risk factor for the development of type-2 diabetes [38]. A prospective study reported that blood lactate predicted incident diabetes independent of many other risk factors and was strongly related to insulin resistance. In human, lactate accumulation was found in chronic liver disease [39]. Published studies reported that increased liver lactate levels stimulated hepatic stellate cells activation and contributed to the severity of fibrosis. It was reported that lactate augmented LPS-stimulated inflammatory gene expression. Nonalcoholic fatty liver disease is a common complication of DM with incidence rate 50% [40]. Therefore, the mice liver fibrosis was investigate in the present study. We found that Cd exposure indeed worsen liver fibrosis in diabetic mice. Consistent with the effects of Cd on fibrosis, we also found that Cd increased the expression of IL-6, TGF-β, Collagen I and Collagen III in diabetic mice liver. These cytokines may involve in the process of worsen fibrosis induced by Cd. Besides, published studies had shown that hepatic lactate was related to inflammatory stress [41], which is consistent with this study. In general, Cd increased the lactate concentrations and promoted liver inflammation in diabetic mice liver, and increased inflammatory stress is the possible reason resulted in liver fibrosis. Together, our data support several possible mechanisms for the exacerbated clinical signs associated with Cd exposure in T2DM mice.
The results of our study have potentially broad biological relevance. The numbers of people who live in Cd contaminated areas or who are exposed to Cd in the workplace and those who are diagnosed with DM has increased rapidly worldwide [42][43][44][45][46]. Greater insight into the interaction of Cd and DM is essential.
However, there were some limitations of this study. Firstly, the sample of mice in this study was not enough to fully define the creative idea reported in this study. Secondly, blood pH and concentrations of HCO3-were not detected since insufficient blood from mice, so it is hard to verify lactic acidosis in the experiment mice. Hence, this idea needs to be further investigated with a larger sample size and multiple species of animals. Specifically, we did not further explore those mechanisms related to the changes reported for blood glucose and insulin. All of these would be the focus of future research.

Conclusion
In a T2DM mouse model, Cd exposure disturbed glucose metabolism and exacerbated diabetes. Increased hepatic lactate accumulation, inflammation and fibrosis may contribute to the effects of Cd. The data support the hypothesis that Cd exposure is a risk factor for the exacerbation of diabetes.