Effects of Zinc Deficiency in Male Mice on Glucose Metabolism of Male Offspring

Abstract

As an essential metal, zinc is central to insulin biosynthesis and energy metabolism. Zinc can not only maintain the activity of insulin, but also has insulin-like effect. When zinc is sufficient, the body needs less insulin. Zinc can correct abnormal glucose tolerance and even replace insulin to improve glucose metabolism disorder in diabetic rats. However, the effect of paternal zinc deficiency on glucose metabolism of offspring is still unclear. In the present study, sixteen 8-week-old male mice were randomly allocated into low-zinc group and control group (8 mice in each group), which were fed with low zinc and standard diet for 6 weeks, respectively. The mice were mated with female mice fed with standard diet to get the first generation of mice (F1) to explore the effect zinc deficiency on the glucose metabolism of offspring. Glucose tolerance, insulin sensitivity and insulin secretion in mice were determined by oral glucose tolerance test (OGTT), insulin tolerance test (ITT) and glucose stimulated insulin secretion test (GSIS), respectively. Compared with the control group, the fasting blood glucose levels of F1 generation male mice in the low zinc group increased at 15 and 30 min after glucose injection. The blood glucose of F1 generation male mice in the low zinc group of mice decreased at 60, 90, 120, 180, 240 min after insulin injection. Compared with the control group, serum insulin of F1 generation male mice in the low zinc group decreased at 15 min after glucose injection (all p values <0.05). However F1 female mice in the low zinc group did not show abnormal glucose metabolism. Marginal zinc deficiency in male mice can cause abnormal glucose homeostasis in male offspring, but not in female offspring.

Introduction

Zinc is an essential trace element of life, which regulates the expression and activation of various biomolecules, such as transcription factors, enzymes, aptamers, biological channels, growth factors and their receptors.^1,2) Zinc is a key structural or catalytic component of more than 300 enzymes involved in all aspects of cell signal transduction.³⁾ Zinc also regulates cell signal transduction, proliferation, differentiation and survival.^4,5) As a result, almost all important physiological processes in cells depend on proper function of zinc.

Although zinc is the second most abundant trace element in the human body, which is next only to iron.⁶⁾ Human body cannot store zinc, and needs continuously supply from diet.⁷⁾ Zinc deficiency occurs due to inadequate intake, reduced absorption or excretion loss, which is one of the major nutritional imbalance challenges for global population, ranking 11th among the world’s important health risk factors and 5th among developing countries.⁸⁾ Zinc deficiency is associated with a variety of diseases, and could cause systemic health problems, including growth and development, neurological dysfunction, reproductive and immune system damage etc.^9,10) Marginal zinc deficiency refers to zinc deficiency with zinc ion level reduced in blood circulation in the body, but no signs of skin injury, hair loss and other signs,¹¹⁾ which is the most common type of zinc deficiency in population, especially in children.

Recently, the relationship between zinc deficiency and diabetes mellitus has aroused people’s attention. Zinc maintains the activity of insulin, and has insulin-like effect itself.^12,13) Body needs less insulin when zinc is sufficient. Zinc can also correct abnormal glucose tolerance and even replace insulin to improve glucose metabolism disorder in diabetic rats.¹⁴⁾ Zinc deficiency can induce insulin resistance and even diabetes. Recent study founded that diabetic patients are generally in a state of zinc deficiency, and diabetic complication also is concerned with the decrease of cellular zinc or zinc-dependent antioxidant oxidase, such as diabetic retinopathy and diabetic peripheral neuropathy.¹⁵⁾ In addition, studies founded that zinc supplementation can significantly reduce some key blood glucose indicators.^16,17) Other studies have shown that zinc deficiency during pregnancy and lactation in Wistar female rats can lead to insulin secretion dysfunction in offspring.¹⁸⁾ However, the effect of paternal zinc deficiency on glucose metabolism of offspring is still unclear. In this study, C57BL/6J male mice were used to establish a mouse model of marginal zinc deficiency to explore its effect on glucose metabolism of offspring.

Experimental

Animals and Reagents

C57BL/6J mice (6 weeks of age, both males and females) were purchased from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. D-Glucose and bovine insulin were purchased from Sigma-Aldrich (MO, U.S.A.). Ultra Sensitive Mouse Insulin Assay (MO, U.S.A.) (ELISA) kit was purchased from Crystal Chem Inc., IL, U.S.A. General tissue fixative liquid was purchased from Wuhan Servicebio Biotechnology Co., Ltd., China. Hematoxylin eosin (H&E) staining kit was purchased from Beijing Solaibao Biotechnology Co., Ltd., Beijing, China. Blood glucose meter and blood glucose test paper were purchased from ROCHE, Switzerland. Serum interleukin-6 (IL-6) and Superoxide Dismutase (SOD) activity detection kits were purchased from NanJing JianCheng Bioengineering Research Institute Co., Ltd., NanJing, China. The diets used in this experiment were standard diet (D08080401, zinc content: 29 mg/kg) and zinc deficient diet (D08080403, zinc content: 0 mg/kg) customized by Research Diets Inc., NJ, U.S.A. The protocol for the use of animals was approved by the Animal Ethics Board of Zhengzhou University.

Preparation of Low Zinc and Control Diets

Firstly, the diet was stired into powder in a blender, and the zinc deficiency diet (827.6 g) and standard diet (172.4 g) were fully mixed, and an appropriate amount of ultra-pure water was added to mix together, and then the low zinc diet (5 mg/kg) was made and dried at room tempreture.⁷⁾ In order to maintain consistency, 1000 g standard diet was processed in the same way, which was used as control diet. All diet was stored at −20 °C.

Establishment and Experimental Grouping of Male Mice with Marginal Zinc Deficiency

Sixteen male mice were randomly divided into two groups, as low zinc group and the control group (8 for each group), which were the Parental mice (F0 generation male mice), fed with low-zinc and standard diet for 6 weeks respectively. Then they were mated with female mice (fed with standard diet) in the cage from 8 p.m. to 6 a.m. in the next morning, the mice had no diet but water during the cage. The female mice were checked every morning and be separated with male when vaginal plug was found, to obtain the first generation of mice (F1 generation mice), the weight and survival rate of F1 offspring mice were recorded. Female and F1 generation mice were given control diet throughout the research.

Measurement of Zinc Levels in Serum and Other Tissues

Zinc ion level was detected by inductively coupled plasma mass spectrometry (ICP-MS) in Guangzhou Jinyu Medical Laboratory Group Co., LTD.

Detection of IL-6 in Serum

Serum samples were taken, and serum IL-6 level was detected according to the instructions of ELISA kit.

Determination of SOD Activity

SOD activity in serum was measured using the xanthine oxidase method. The procedures were conducted in accordance with kit instructions.

Histological Observation of Pancreas

The pancreas tissues were taken and placed in 4% paraformaldehyde fixation solution for more than 24 h. The sections were routinely embedded in paraffin and stained with hematoxylin eosin. The pathological changes of the tissue structure were observed under light microscope.

Oral Glucose Tolerance Test (OGTT)

After fasting for 16 h, 20% D-glucose solution was prepared and gavaged to the mice at a dose of 2 g/kg. Blood was collected from tail vein, and fasting blood glucose was recorded at 0, 15, 30, 60, 90 and 120 min after gavage.

Insulin Tolerance Test (ITT)

After fasting for 2 h, mice were injected with 50 U/L insulin saline at a dose of 0.5 U/kg, and blood was collected from tail vein. Blood glucose values were recorded at 0 min (before injection), 15, 30, 60, 120 min, 180, and 240 min after injection.

Glucose Stimulated Insulin Secretion Test (GSIS)

After fasting for 16 h, inner canthal blood was extracted from mice, and about 50 µL (2–3 drops) was placed in EP tube at room temperature. Twenty percent D-glucose solution was prepared and the mice were given intragastric administration at a dose of 2 g/kg. Blood was taken in the same way after intragastric administration 15 and 30 min, respectively. The movement should be gentle during the whole process to avoid stress reaction of the mice.

Statistical Analysis

SPSS 25.0 was used for statistical analysis, and data were obtained from three independent experiments and presented as means ± standard deviation (S.D.) Comparisons were performed using one-way ANOVA with Tukey’s post hoc test following appropriate transformation of raw data to achieve a normal distribution and equalized variance when necessary, and the Student’s t-test was used to compare the difference between two groups. p < 0.05 was considered statistical significant.

Results

Low Zinc Diet Induced Zinc Decreased in Whole Body Tissues of F0 Generation Male Mice

As shown in Fig. 1A, the zinc content in control and low zinc diet were 28.36 ± 1.13 and 5.01 ± 0.34 mg/kg, respectively. Serum zinc in control group was 1.93 ± 0.15 ng/µL, serum zinc in low zinc group was decreased 32.6% compared with the control group (Fig. 1B), and the zinc content in tissues of low zinc group mice decreased to varying degrees (p < 0.05) (Fig. 1C).

Fig. 1. Low Zinc Diet Induced Low Zinc State in Tissues of F0 Generation Male Mice

(A) Zinc content in diet, (B) Serum Zinc, (C) Zinc content in tissues of F0 generation male mice (Control group: n = 5; Low zinc group n = 6). Bars represent the Means  ±  S.D. *represents p < 0.05, ** represents p < 0.01, compared with the control group.

Low Zinc Diet Caused Spleen Enlarged and IL-6 Increased in F0 Generation Male Mice

As shown in Fig. 2A, In F0 generation, there was no statistical difference in liver/body weight coefficient between the low zinc group and control group (p > 0.05). The spleen/body weight ratio increased, and the difference was statistically statistical compared with the control group (p < 0.05) (Fig. 2B). Serum IL-6 level in low zinc group was increased 36.5%, and the difference was statistically differences (p < 0.05) (Fig. 2C), while SOD activity was no statistical difference from that in control group (p > 0.05) (Fig. 2D). After 6 weeks of low zinc diet, there was no statistical change in OGTT in the low zinc group compared with the control group (p > 0.05) (Fig. 2E). These results suggest that marginal zinc deficiency had a slight effect on F0 generation mice.

Fig. 2. Low Zinc Diet Caused Spleen Enlarged and IL-6 Increased in F0 Generation Male Mice

Organ coefficient (A and B), inflammatory factors (C), oxidative stress levels (D) and OGTT (E) of F0 generation male mice. Bars represent the Means  ±  S.D. * represents p < 0.05 compared with the control group.

Low Zinc Diet Reduced Survival Rate of F1 Offspring Mice

The survival rate of F1 offspring mice in the zinc deficient group was 75.1%, which was significantly lower than that in the control group (p < 0.05) (Fig. 3A). The weight of surviving F1 generation mice was recorded, and the results showed that compared with the control group, the weight of female and male F1 generation mice did not change significantly (p > 0.05), as shown in Figs. 3B and 3C.

Fig. 3. Low Zinc Diet Reduced Survival Rate and Had No Effect on Body Weight of F1 Offspring

The Survival rate (A) and body weight of F1 female offspring mice (B) and F1 female offspring mice (C) (n = 15 mice/group). Bars represent the Means  ±  S.D. *represents p < 0.05, ** represents p < 0.01, compared with the control group.

Low Zinc Diet Attenuates Glucose Metabolism in F1 Male Offspring

As shown in Fig. 4A, compared with the control group, the fasting blood glucose of F1 generation male mice in the low zinc group increased 15 and 30 min after glucose injection, with statistical significance (p < 0.05). Compared with the control group, the glucose levels of F1 generation male mice in the low zinc group decreased at 60, 90, 120, 180 and 240 min after insulin injection, with statistically statistical differences (p < 0.05), which as shown in Fig. 4B. Insulin secretion of F1 generation male mice in the low zinc group decreased 23.2% after glucose injection 15 min, compared with the control group (p < 0.05), as shown in Fig. 4C. There was no difference in pancreatic tissue morphology of F1 generation male mice in the low zinc group compared with the control group (Figs. 4D, 4E).

Fig. 4. Low Zinc Diet Attenuates Glucose Metabolism in F1 Male Offspring

OGTT in mice following with 2 g/kg glucose challenge (A), ITT in mice post 0.5 IU/kg insulin injection(B), insulin secretion function (C), Pancreatic histomorphology of Control group (D) and Low zinc group (E) of F1 male offspring (100×). (Control group: n = 5; Low zinc group n = 6). Bars represent the Means  ±  S.D. *represents p < 0.05, ** represents p < 0.01, compared with the control group.

Low Zinc Diet Had No Effect on Glucose Metabolism in F1 Female Offspring

As shown in Fig. 5A, there was no statistical difference in OGTT of F1 generation female mice in low zinc group compared with the control group (p > 0.05). ITT of F1 generation female mice in low zinc group was not significantly different from that in control group (p > 0.05) (Fig. 5B). There was no statistical difference in GSIS of F1 generation female mice in low zinc group compared with the control group (p > 0.05) (Fig. 5C). There was no difference in pancreatic tissue morphology of F1 generation male mice in the low zinc group compared with the control group (Figs. 5D, 5E).

Fig. 5. Low Zinc Diet Had No Effect on Glucose Metabolism in F1 Female Offspring

OGTT in mice following with 2 g/kg glucose challenge (A), ITT in mice post 0.5 IU/kg insulin injection (B), insulin secretion function (C), Pancreatic histomorphology of Control group (D) and Low zinc group (E) of F1 female offspring (100×), (Control group: n = 5; Low zinc group n = 6). Bars represent the Means  ±  S.D.

Discussion

The relationship between nutrients and nutrient sensing pathway and diabetes has also attracted great attention, and the role of nutrients in diabetes is most concerned with macronutrients, such as carbohydrates and fat. While micronutrients, such as iron and zinc, which are easy to be ignored, are also closely related to diabetes.^19–21) Studies have reported that zinc deficiency can induce insulin resistance and even diabetes in the body.^22,23)

Zinc deficiency not only affects individual health, but also adversely affects the health of their offspring. We used zinc deficient diet and control diet (5 and 30 mg/kg, respectively) to intervene F0 generation mice,²⁴⁾ and the serum zinc in low zinc group decreased by about 32.6%, and the zinc content in tissues of low zinc group mice decreased to varying degrees. However, there were no signs of severe zinc deficiency such as skin ulceration and hair loss, indicating that the mouse model of marginal zinc deficiency was successfully constructed. The serum inflammatory factor IL-6 in male zinc-deficient mice of F0 generation increased, and the organ coefficient of spleen increased compared with the control group, indicating splenomegaly, which is consistent with previous reports that zinc deficiency has been confirmed to cause damage to human immune function.²⁵⁾ The activity of SOD enzyme in serum of female Zinc-deficient mice of F0 generation decreased, which confirmed that zinc deficiency would affect SOD enzyme activity.^26,27) Previous literatures have reported that zinc ion can improve the catalytic efficiency and stability of SOD enzyme.^28,29) F0 generation male mice did not show glucose tolerance impairment after the low zinc diet intervention, which may be due to the marginal zinc deficiency of mice, which is relatively mild and does not cause the disorder of glucose metabolism.

The survival rate of F1 generation mice in the low zinc group reduced, indicating that zinc deficiency in male mice affected the growth and development of their offspring. However, most of the died mice died soon after birth, which were too young to accurately identify their sex, which is a limitation of our study. The growth curve of the surviving F1 generation mice showed no abnormality, which maybe because the normal diet was restored in the later stage, which alleviated the influence of zinc deficiency in early life on offspring to some extent. Consistent with our study, some studies have shown that zinc deficiency will affect the growth and development of offspring, but zinc supplementation can recover to a certain extent.^18,30)

F1 generation of male mice impaired glucose tolerance and insulin secretion in damage, but the body of insulin sensitivity enhancement, speculation may be when the mice blood glucose, insulin secretion from the body reflective. Whereas, as a result of mice impaired insulin secretion, insulin levels, body compensatory to adjust the insulin sensitivity. Compensatory regulation could not completely compensate for the functional impairment, and finally resulted in dyshomeostasis of glucose in mice. There was no tissue damage in the pancreas of F1 generation male mice, and this phenotype and sex difference may be involved in epigenetic mechanism. Zinc ion is an essential metal element in the epigenome and is necessary for various epigenetic enzymes to exert their activities. Sperm has specific epigenetic characteristics, including DNA methylation, DNA-related proteins, post-translational histone modification and so on.^31–34) Dysregulation of zinc homeostasis can lead to changes in epigenetic characteristics.²⁴⁾ Population studies have shown that men with normal zinc intake have higher sperm quality and less DNA damage than men with low zinc intake.³⁵⁾

Female mice of F1 generation did not show abnormal glucose metabolism. Previous literature also pointed out that female mice are less sensitive to metabolic effects caused by zinc deficiency compared with males, while males are more sensitive to malnutrition.^36,37) Gender differences in the effects of zinc deficiency on offspring have been reported,^38,39) and epidemiological studies have shown that the risk of diabetes in males is higher than that in females.^40,41)

This study is demonstrated that the effects of paternal zinc deficiency on offspring. The results presented interesting results that low zinc diet increased serum IL-6 level, paternal marginal zinc deficiency lowered offspring survival rate, and attenuated glucose metabolism through insulin decrease in F1 male offspring but not in F1 female. This study provides a theoretical basis for gender differences in diabetes patients, and provides the most direct evidence that parental zinc deficiency causes abnormal glucose metabolism in male offspring.

In conclusion, marginal zinc deficiency in male C57/BL6J mice can lead to abnormal glucose metabolism in male offspring, but has no effect on female offspring, and the exact mechanism needs to be further explored.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant Nos. 31750110463, 21577119 and 81801547).

Conflict of Interest

The authors declare no conflict of interest.

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