Modulation of Zinc Transporter Expressions by Additional Zinc in C2C12 Cells Cultured in a High Glucose Environment and in the Presence of Insulin or Interleukin-6.

Zn status has been related to various chronic diseases presenting oxidative stress and inflammation, such as type 2 diabetes. Zn supplementation has been suggested to be a potential coadjuvant in the management of this condition. Zn transporters constitute a key component in the maintenance of Zn homeostasis. Our aim was to evaluate the modulatory effect of additional Zn (10 or 100 µM; as a ZnSO4*7H20) on the mRNA relative expression of selected Zn transporters (ZnT1, ZnT5, ZnT7, ZIP6, ZIP7, ZIP10, ZIP14), in myoblast (C2C12) cells cultured in normal (10 mM) and high glucose (30 mM), and in the absence or presence of insulin (1 nM), and interleukin-6 (IL-6; 5 nM) for 24 h. The main findings of our study were that in high glucose conditions in absence of insulin or IL-6, additional Zn increased ZnT1 and ZIP6, and decreased ZnT5 and ZIP7 expressions. However, this situation is modified by insulin, where incremental Zn induced increased expressions of ZnT1, ZnT5, and all the ZIP transporters studied. In high glucose conditions and in the presence of IL-6, additional Zn caused increased expressions of ZnT7, ZIP7, and ZIP14, compared with results in the absence of IL-6. This study provides preliminary evidence for the differential expression of selected Zn transporters in C2C12 cells subjected to high glucose and incremental Zn, suggesting that important changes in intracellular Zn distribution take place in response to inflammatory and high-insulin environments. Further study is necessary to understand the implications of these findings.

Vitamin D status and obesity in children from Chile.

BACKGROUND: Vitamin D [25(OH)D] is essential for normal bone development and maintenance. Furthermore, its deficiency has been associated with obesity, cardiovascular diseases, insulin resistance, autoimmune diseases, and certain cancers. OBJECTIVE: To determine the incidence of serum 25(OH)D deficiency (<20 ng/ml) among apparently healthy Chilean children (4-14 years old) from three Chilean geographic areas during May-September 2018. MATERIALS AND METHODS: Serum 25(OH)D levels were measured by a competitive protein-binding ELISA assay in 1134 children, and correlations between serum 25(OH)D levels, BMI, and geographic area were calculated. Individuals were grouped according to their serum 25-hydroxyvitamin D levels (ng/ml): severe deficiency: <5; moderate deficiency: 5-10.9; mild deficiency: 11-20.9; insufficiency: 21-29.9 and sufficiency: 30-100. RESULTS: We found 80.4% of children had serum 25(OH)D deficiency, with 1.7% severe, 24.6% moderate, and 54.1% mild. In the three cities, the percentage of serum 25(OH)D deficit was increased when comparing overweight or obesity with a healthy weight. Additionally, an interaction effect was observed between geographic area, nutritional status, and serum 25(OH)D levels using the factorial ANOVA test (p = 0.038). In Antofagasta, there were more overweight children and also a higher percentage of children with VitD deficiency (<30 ng/ml) compared to Santiago or Concepción. CONCLUSION: This study revealed a high prevalence of serum 25(OH)D deficiency in children between 4 and 14 years old in Chile (80.4%) during May-September 2018. Obese and overweight children had the highest prevalence of serum 25(OH)D deficiency.

Zinc Modulates the Response to Apoptosis in an In Vitro Model with High Glucose and Inflammatory Stimuli in C2C12 Cells.

Apoptosis is programmed cell death and its alteration is related to cancer, neurologic, autoimmune, and chronic diseases. A number of factors can affect this process. The aim of this paper is to study the effect of supplemental zinc on apoptosis-related genes in C2C12 myoblast cells after being challenged with a series of stimuli, such as high glucose, insulin, and an inflammatory agent. C2C12 myoblast cells were cultured for 24 h with zinc (Zn) (ZnSO4) 10 or 100 µM and/or glucose 10 or 30 mM. In addition to these stimuli, the cells were challenged with insulin 1 nM or interleukin-6 (IL-6) 5 nM. The mRNA expression of proapoptotic genes caspase 3 and Fas, the antiapoptotic genes, Xiap and Bcl-xL and the ratio of pro-/antiapoptotic genes Bax/Bcl-2, were determined by qRT-PCR. The expression of caspase-3 gene was significantly increased in the presence of the combination high Zn/high glucose with and without the presence of insulin and IL6 in the culture medium Fas expression instead, showed uneven responses. The expression of Bcl-xL and Xiap was increased in most conditions by having high Zn in the medium regardless of the presence of insulin or IL6. Bax/Bcl2 ratio was decreased in the presence of high Zn. Zn was able to stimulate the expression of antiapoptotic genes. This effect was specially noted in high-glucose conditions with and without the presence of insulin. This effect is partially overridden by the presence of an inflammatory agent such as IL-6.

Short repeats in the heme oxygenase 1 gene promoter is associated with increased levels of inflammation, ferritin and higher risk of type-2 diabetes mellitus.

OBJECTIVE: We evaluated the relationship between the HO1 genotype, ferritin levels and the risk of type-2 diabetes and inflammation. RESEARCH METHODS: Eight hundred thirty-five individuals were evaluated and classified according to their nutritional status and the presence of type-2 diabetes: 153 overweight (OW); 62 obese (OB); 55 type-2 diabetes mellitus (DM); 202 OWDM; 239 OBDM and 124 controls (C). We studied biochemical (glycemia, insulin, lipid profile, liver enzyme, creatinine, hsCRP), hematological (hemoglobin, free erythrocyte protoporphyrin, transferrin receptor and serum Fe and ferritin) and oxidative stress (SOD, GHS and TBARS) parameters. We determined heme oxygenase activity and the (GT)n polymorphism in its gene promoter. RESULTS: Individuals with diabetes, independent of nutritional status, showed high levels of ferritin and HO activity compared to control subjects. Allelic frequency was not different between the groups (Chi(2), NS) however, genotypes were different (Chi(2), P<0.001). The SS (short-short) genotype was higher in all DM individuals compared to controls and MM was higher in controls. SM (short-medium) genotype was an independent risk factor for DM in logistic regression analysis. We observed high risk for type-2 diabetes mellitus in the presence of SM genotype and high levels of ferritin (OR adjusted: 2.7; 1.9-3.6; p<0.001; compared to control group). It was also significantly related to inflammation. CONCLUSION: The SM genotype in HO1 gene promoter and ferritin levels were associated with higher risk for type-2 diabetes and for having a higher marker of inflammation, which is the main risk factor for the development of chronic diseases.

Glycemic control and oxidative stress markers and their relationship with the Thioredoxin Interacting Protein (TXNIP) gene in type 2 diabetic patients / Control glicémico y marcadores de estrés oxidativo y su relación con el gen de la proteína interactuante con la tioredoxina (TXNIP) en suejtos diabéticos tipo 2

Objective: To investigate the relationship between oxidative stress and biochemical parameters and the expression of TXNIP, IL-6, IL-1β and TNF-α in peripheral mononuclear cells (PMCs) from type-2 diabetic patients. Methods: We studied 60 males: 20 normal-weight type- 2 diabetic patients (NW), 20 obese diabetic patients (OB) and 20 controls (C). Biochemical and oxidative stress parameters were evaluated. PMCs were isolated and total RNA was extracted in order to determine the expression of TXNIP, IL-6, IL-1β and TNF-α by qRT-PCR. Results: OB had higher weight, BMI and abdominal circumference (One way ANOVA, p<0.0001). NW had higher fasting glycemia (One way ANOVA, p=0.0034) however OB had higher HbA1c (One way ANOVA, p<0.0001). OB also had higher hsCRP (One way ANOVA, p=0.0158). TBARS and AGES were elevated in both NW and OB (One way ANOVA, p<0.0001 and p=0.0008, respectively). Compared to OB and C participants, the expression of TXNIP was significantly higher in NW (Kruskal Wallis, p=0.0074); IL-1β, IL-6 and TNF-α transcripts were higher in NW and OB (Kruskal Wallis, p<0.0001, for all). In NW patients, the expression of TXNIP was positively correlated with fasting glycemia and AGES and negatively correlated with HOMA-β (r=0.72; r=0.59; r=-0.44, respectively, for all p<0.05), in OB there was correlation only with 8-Isoprostanes (r=0.42, p=0.046). Conclusions: Our results suggest that fasting glycemic control, independent of adiposity and nutritional status, represents a risk factor for β-cell dysfunction, increases oxidative stress markers and it is related with an elevation of TXNIP expression (AU)

Objetivo: Investigar la relación existente entre parámetros bioquímicos y de estrés oxidativo y la expresión de TXNIP, IL-6, IL-1β y TNF-α en células mononucleares periféricas (CMPs) de sujetos diabéticos. Material y métodos: Se estudió 60 sujetos hombres: 20 con peso normal y diabetes tipo 2 (NW), 20 sujetos obesos con diabetes (OB) y 20 sujetos controles (C). Se evaluaron parámetros bioquímicos y de estrés oxidativo. Además se aislaron CMPs para la extracción de RNA total y se determinó la expresión de los genes TXNIP, IL-6, IL- 1β y TNF-α mediante PCR de tiempo real cuantitativo. Resultados: Los OB presentaron mayor IMC y circunferencia abdominal que los NW y los C (ANOVA de una vía, p<0.0001). Los NW tuvieron mayor glicemia en ayuna (ANOVA de una vía, p=0.0034) sin embargo, los OB presentaron mayor HbA1c (ANOVA de una vía, p<0.0001). Los OB además presentaron mayores nivel de PCRus (ANOVA de una vía, p=0.0158). Los TBARS y los AGES se observaron elevadas tanto en OB como en NW (ANOVA de una vía, p<0.0001 y p=0.0008, respectivamente). Comparado con los OB y C la expresión de TXNIP fue significativamente más alta en los NW (Kruskal Wallis, p=0.0074); la expresión de IL-1β, IL-6 y TNF-α se observó más elevada en los NW y OB (Kruskal Wallis, p<0.0001). En los NW la expresión de TXNIP se correlacionó positivamente con la glicemia en ayunas y con los AGES y negativamente con HOMA-β (r=0.72; r=0.59; r=-0.44, respectivamente, p<0.05), en los OB hubo correlación solamente con los 8-isoprostanos (r=0.42, p=0.046). Conclusiones: Nuestros resultados sugieren que la alteración de la glicemia en ayunas, independiente de la adiposidad y del estado nutricional, representa un factor de riesgo para la disfunción de la célula beta, aumenta los marcadores de estrés oxidativo y se relaciona con el aumento de la expresión de TXNIP (AU)

Association between ferritin and hepcidin levels and inflammatory status in patients with type 2 diabetes mellitus and obesity.

OBJECTIVE: The aim of this study was to determine the association between iron parameters and inflammation in obese individuals with and without type 2 diabetes mellitus (T2DM). METHODS: We studied 132 obese individuals (OB), 60 individuals with T2DM, 106 obese individuals with T2DM (T2DOB), and 146 controls (C). All of were men aged >30 y. Biochemical, iron nutrition, and oxidative stress parameters were determined. Peripheral mononuclear cells were isolated and total RNA was extracted to quantify tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, interleukin (IL)-6, toll-like receptor (TLR)-2/4 and hepcidin by quantitative reverse transcription polymerase chain reaction. RESULTS: OB, T2DM, and T2DOB individuals had higher ferritin, retinol-binding protein 4, and thiobarbituric acid reactive substance (TBAR) levels than controls. T2DOB and T2DM individuals showed high high-sensitivity C-reactive protein (hsCRP) levels and OB with and without T2DM had elevated levels of serum hepcidin. Heme oxygenase activity was high in OB and T2DM and there were no differences observed in superoxide dismutase and glutathione parameters. A correlation between TBARS and ferritin in T2DOB was observed (r = 0.31; P < 0.006). Multiple linear regression analysis showed an association between diabetes and obesity with ferritin, TBARS, and hsCRP levels. The upper quartiles of ferritin, TBARS and hepcidin showed an adjusted odd ratio for T2DM of 1.782, 2.250, and 4.370, respectively. TNF-α, IL-6, hepcidin, NF-κB, TLR-2/4 mRNA abundances were increased in T2DM and T2DOB. CONCLUSION: Elevated hsCRP and hepcidin levels, and increased gene expression of TNF-α, IL-6, NF-κB, and TLR-2/4 in patients with diabetes, obesity, or both exacerbate and perpetuate the insulin resistance and inflammatory state.

Association between ferritin, high sensitivity C-reactive protein (hsCRP) and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects.

Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM); 30 obese subjects without diabetes (OB) and 30 healthy subjects (Cn). We isolated peripheral mononuclear cells (PMCs) and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-, IL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL- 6), and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state.

La obesidad y la diabetes tipo 2 comparten un perfil pro-inflamatorio crónico leve. Recientemente ha sido reconocido que el hierro es un factor de riesgo para el desarrollo de diabetes tipo 2. El objetivo de este estudio fue evaluar la relación entre el estado nutricional de hierro y la inflamación con el riesgo de desarrollar diabetes mellitus tipo 2 en sujetos obesos. Estudiamos 30 hombres con obesidad y diabetes mellitus tipo 2 (OBDM); 30 sujetos obesos sin diabetes (OB) y 30 sujetos sanos (Cn). Aislamos células mononucleares periféricas (PMCs) y las desafiamos con altas concentraciones de hierro. Se aisló el RNA y se determinó la abundancia relativa de los RNAm de TNF-, IL-6 y de hepcidina mediante RT-PCR cuantitativo. También se determino el marcadores de la nutrición de hierro, parámetros bioquímicos, inflamatorios y de estrés oxidativo. Los sujetos OBDM y OB mostraron elevados niveles de PCRus comparado con el grupo Cn. OBDM además eran portadores de elevados niveles de ferritina comparado con el grupo Cn. La expresión de TNF-e IL-6 estuvieron aumentadas en las PMCs de sujetos OBDM que fueron tratadas con altas concentraciones de hierro. Se encontró que el mayor cuartil de ferritina se asoció con un aumentado riesgo de desarrollo de diabetes cuando fue ajustado por BMI y HOMA-IR; esta asociación fue independiente del estado pro-inflamatorio. Los mas altos niveles de expresión de hepcidina tambien mostraron una tendencia a aumentar el riesgo de desarrollo de diabetes, sin embargo éste no fue significativo. Niveles de PCRus por sobre los 2 mg/L mostraron que también tienden a aumentar el riesgo de desarrollo de diabetes mellitus tipo 2. Como conclusión, el hierro puede estimular la expresión de genes pro-inflamatorios (TNF-, IL-6), y tanto la ferritina como el aumento de expresión de hepcidina podrían ser factores de riesgo para el desarrollo de diabetes mellitus tipo 2. Sujetos con un alto riesgo cardiovascular medido por PCRus también muestran un riesgo aumentado de desarrollar diabetes mellitus tipo 2.

Association between ferritin, high sensitivity c-reactive protein (hsCRP) and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects / Relación entre ferritina, pcrus y expresion de hepcidina con el riesgo de desarrollo de diabetes mellitus tipo 2 en sujetos obesos

Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM); 30 obese subjects without diabetes (OB) and 30 healthy subjects (Cn). We isolated peripheral mononuclear cells (PMCs) and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-α, IL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL- 6), and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state (AU)

La obesidad y la diabetes tipo 2 comparten un perfil pro-inflamatorio crónico leve. Recientemente ha sido reconocido que el hierro es un factor de riesgo para el desarrollo de diabetes tipo 2. El objetivo de este estudio fue evaluar la relación entre el estado nutricional de hierro y la inflamación con el riesgo de desarrollar diabetes mellitus tipo 2 en sujetos obesos. Estudiamos 30 hombres con obesidad y diabetes mellitus tipo 2 (OBDM); 30 sujetos obesos sin diabetes (OB) y 30 sujetos sanos (Cn). Aislamos células mononucleares periféricas (PMCs) y las desafiamos con altas concentraciones de hierro. Se aisló el RNA y se determinó la abundancia relativa de los RNAm de TNF-α, IL-6 y de hepcidina mediante RT-PCR cuantitativo. También se determinó el marcador de la nutrición de hierro, parámetros bioquímicos, inflamatorios y de estrés oxidativo. Los sujetos OBDM y OB mostraron elevados niveles de PCRus comparado con el grupo Cn. OBDM además eran portadores de elevados niveles de ferritina comparado con el grupo Cn. La expresión de TNF-α e IL-6 estuvieron aumentadas en las PMCs de sujetos OBDM que fueron tratadas con altas concentraciones de hierro. Se encontró que el mayor cuartil de ferritina se asoció con un aumentado riesgo de desarrollo de diabetes cuando fue ajustado por BMI y HOMA-IR; esta asociación fue independiente del estado pro-inflamatorio. Los más altos niveles de expresión de hepcidina también mostraron una tendencia a aumentar el riesgo de desarrollo de diabetes, sin embargo éste no fue significativo. Niveles de PCRus por sobre los 2 mg/L mostraron que también tienden a aumentar el riesgo de desarrollo de diabetes mellitus tipo 2. Como conclusión, el hierro puede estimular la expresión de genes pro-inflamatorios (TNF-α, IL-6), y tanto la ferritina como el aumento de expresión de hepcidina podrían ser factores de riesgo para el desarrollo de diabetes mellitus tipo 2. Sujetos con un alto riesgo cardiovascular medido por PCRus también muestran un riesgo aumentado de desarrollar diabetes mellitus tipo 2 (AU)

Glycemic control and oxidative stress markers and their relationship with the thioredoxin interacting protein (TXNIP) gene in type 2 diabetic patients.

OBJECTIVE: To investigate the relationship between oxidative stress and biochemical parameters and the expression of TXNIP, IL-6, IL-1ß and TNF-α in peripheral mononuclear cells (PMCs) from type-2 diabetic patients. METHODS: We studied 60 males: 20 normal-weight type- 2 diabetic patients (NW), 20 obese diabetic patients (OB) and 20 controls (C). Biochemical and oxidative stress parameters were evaluated. PMCs were isolated and total RNA was extracted in order to determine the expression of TXNIP, IL-6, IL-1ß and TNF-α by qRT-PCR. RESULTS: OB had higher weight, BMI and abdominal circumference (One way ANOVA, p<0.0001). NW had higher fasting glycemia (One way ANOVA, p=0.0034) however OB had higher HbA1c (One way ANOVA, p<0.0001). OB also had higher hsCRP (One way ANOVA, p=0.0158). TBARS and AGES were elevated in both NW and OB (One way ANOVA, p<0.0001 and p=0.0008, respectively). Compared to OB and C participants, the expression of TXNIP was significantly higher in NW (Kruskal Wallis, p=0.0074); IL-1ß, IL-6 and TNF-α transcripts were higher in NW and OB (Kruskal Wallis, p<0.0001, for all). In NW patients, the expression of TXNIP was positively correlated with fasting glycemia and AGES and negatively correlated with HOMA-ß (r=0.72; r=0.59; r=-0.44, respectively, for all p<0.05), in OB there was correlation only with 8-Isoprostanes (r=0.42, p=0.046). CONCLUSIONS: Our results suggest that fasting glycemic control, independent of adiposity and nutritional status, represents a risk factor for ß-cell dysfunction, increases oxidative stress markers and it is related with an elevation of TXNIP expression.

Objetivo: Investigar la relación existente entre parámetros bioquímicos y de estrés oxidativo y la expresión de TXNIP, IL-6, IL-1y TNF-en células mononucleares periféricas (CMPs) de sujetos diabéticos. Material y métodos: Se estudió 60 sujetos hombres: 20 con peso normal y diabetes tipo 2 (NW), 20 sujetos obesos con diabetes (OB) y 20 sujetos controles (C). Se evaluaron parámetros bioquímicos y de estrés oxidativo. Además se aislaron CMPs para la extracción de RNA total y se determinó la expresión de los genes TXNIP, IL-6, IL- 1y TNF-mediante PCR de tiempo real cuantitativo. Resultados: Los OB presentaron mayor IMC y circunferencia abdominal que los NW y los C (ANOVA de una vía, p.

Effects of high iron and glucose concentrations over the relative expression of Bcl2, Bax, and Mfn2 in MIN6 cells.

Type 2 diabetes is characterized by hyperglycemia and oxidative stress. Hyperglycemia is linked to mitochondrial dysfunction and reduced ß-cell mass due to the reduced expression of genes such as Mfn2 as well as the participation of the Bcl2 gene family, responsible for increased apoptosis. The purpose of this study was to describe the effect of different iron and/or glucose concentrations over Mfn2, Bax, and Bcl2 expressions in a ß-pancreatic cell line (MIN6 cells). MIN6 cells were pre-incubated with different iron and/or glucose concentrations, and the relative mRNA abundance of the Bcl2/Bax ratio and of Mfn2 genes was measured by qRT-PCR. Heme oxygenase (HO) activity, iron uptake, superoxide dismutase activity, and glutathione content were also determined. The Bcl2/Bax ratio increased and Mfn2 expression decreased in MIN6 cells after glucose stimulation. These effects were higher when glucose and iron were incubated together. Additionally, treatment with glucose/iron showed a higher HO activity. Our study revealed that high glucose/Fe concentrations in MIN6 cells induced an increase of the Bcl2/Bax ratio, an indicator of increased cell apoptosis.