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Magnesium-responsive genes are downregulated in diabetic patients after a three-month exercise program on a bicycle ergometer

Magnesium-responsive genes are downregulated in diabetic patients after a three-month exercise... Background: Exercise is an effective therapy for the management of diabetes because it helps regulate glucose and magnesium homeostasis. Nevertheless, the mechanisms by which exercise exerts effects on magnesium transport remain unclear. This study investigated the expression of genes encoding magnesium transporters (GMTs) after a three-month exercise program in diabetic patients. Methods: This study was conducted with a within-subject pre-post design. A total of 15 adult patients with type 2 diabetes mel- litus (T2DM) were recruited and underwent a three-month indoor bicycle exercise program. The expression of five GMTs (CNNM2, TRPM6, TRPM7, SLC41A1, and SLC41A3) was determined in blood samples. Relevant anthropometric values and biochemical parameters were also determined. Results: Although the body weight and body mass index decreased after three months exercise, there were no significant differences. Fasting blood glucose, glycated hemoglobin (HbA1c), waist circumference, and magnesium levels decreased after the exercise program (p < 0.05). The expression of SLC41A1 and SLC41A3 were downregulated after exercise, but only CNNM2, TRPM6, and TRPM7 showed significantly decreased expression levels compared with those before the exercise program (p < 0.05). Conclusion: The three-month exercise program ameliorated blood glucose levels and downregulated the expression of magne- sium-responsive genes in patients with T2DM. Keywords: Blood glucose; Exercise; Magnesium; Magnesium transporter; Type 2 diabetes mellitus healthy diet, and appropriate exercise. Appropriate exercise 1. INTRODUCTION improves blood pressure and glucose management, attenuates lipids, aids weight loss, enhances anti-inflammation, and plays a Type 2 diabetes mellitus (T2DM) is a widespread health prob- key role in the prevention and management of diabetes. Several lem with a dramatically increasing prevalence worldwide, and studies have revealed that regular exercise reduces glycated it is associated with increased cardiovascular risk. Three key hemoglobin (HbA1c), waist circumference, triglycerides (TG), aspects of diabetes management include suitable medications, 3,4 and systolic blood pressure in patients with T2DM. Over the past few decades, researchers have investigated the *Address correspondence: Dr. Sen-Wei Tsai, Department of Physical Medicine and Rehabilitation, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical ability of exercise to alter or regulate magnesium homeostasis, Foundation, 88, Section 1, Fengxing Road, Tanzi District, Taichung 427, Taiwan, including enzyme activation, nucleotide binding, and trans- ROC. E-mail address: tsaisenwei@gmail.com (S.-W. Tsai). membrane electrolyte flux. Nielsen and Lukaski suggested that Conflicts of interest: The authors declare that they have no conflicts of interest exercise may induce magnesium redistribution in the body to related to the subject matter or materials discussed in this article. compensate for metabolic demand. Additionally, exercise is Journal of Chinese Medical Association. (2019) 82: 495-499. suggested to impact magnesium transport, which may result Received February 9, 2019; accepted March 11, 2019. in depleted blood magnesium levels and increased demand for magnesium intake because of metabolic requirements. doi: 10.1097/JCMA.0000000000000112. Magnesium homeostasis plays an essential role in the regu- Copyright © 2019, the Chinese Medical Association. This is an open access lation of cellular physiology and biochemical functions. article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/). Altered blood magnesium levels may in turn shift the balance www.ejcma.org 495 <zdoi: 10.1097/JCMA.0000000000000112> Downloaded from http://journals.lww.com/jcma by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/03/2020 Chiang et al. J Chin Med Assoc 8–11 of other ions, resulting in metabolic or pathological changes. and the home bicycle exercise duration was 40 minutes, with a Magnesium homeostasis is maintained by regulation via hor- subsequent 5 minute cool-down period. During the study, diets mones and specific transporters, such as cyclin and CBS domain and medications were not changed. divalent metal cation transport mediator 2 (CNNM2), transient receptor potential melastatin 6 and 7 (TRPM6 and TRPM7), 2.2. Biochemical and anthropometric measurements solute carrier family 41 members 1–3 (SLC41A1, SLC41A2, Blood samples were collected for measurements of fasting blood 8,12 and SLC41A3), and by regulatory means such as hormones. glucose (FBG), HbA1c, lipoprotein profiles, and the expres- Previous studies have shown that exercise regulates magnesium sion of GMTs at fasting and resting statuses before and after and glucose transport by the interactions of cation channels (eg, the exercise program. Plasma magnesium levels were measured 2+ + + + + Ca , K , and Na ) and ion exchangers and/or pumps (eg, Na /K using a Spotchem EZ sp-4430 automatic analyzer (ARKRAY, + 2+ 8,13 and Na /Mg ). Inc., Kyoto, Japan). Blood glucose, TG, and cholesterol concen- Despite the evidence that magnesium is associated with blood trations were measured by using commercially available kits glucose regulation and is influenced by exercise, there is lim- (Beckman Coulter, Fullerton, USA). High-density lipoprotein ited knowledge regarding the molecular mechanisms by which (HDL) and low-density lipoprotein (LDL) levels were measured exercise modulates the molecular manipulation of magnesium by using commercially available kits (Roche Diagnostics GmbH, homeostasis. Mannheim, Germany). In addition, the anthropometric data, In our previous study, the downregulation of SLC41A1 including height, body weight, BMI, blood pressure, and waist + 2+ (encoding for the Na /Mg exchanger) following acute exercise circumference, were measured. was observed, and the level of SLC41A1 returned to basal status 24 hours after the acute exercise. However, the effects of exer- 2.3. GMT expression cise on the expression of genes encoding magnesium transporters The expression of five GMTs (CNNM2, TRPM6, TRPM7, (GMTs) and on the regulation of blood magnesium and glucose SLC41A1, and SLC41A3) was determined. Total RNA extrac- levels remain unclear. In the present study, we investigated the tions, RNA reverse transcription, quantitative real-time PCR status of magnesium (blood magnesium levels and the expression (qRT-PCR), and data analysis were performed as previously of GMTs) in T2DM patients. The exercise protocol consisted of described by Lin et al. Briefly, peripheral blood was collected a three-month indoor bicycle exercise program. Additionally, and centrifuged, total RNA was extracted from buffy coat layer anthropometric values and biochemical parameters of blood using RNAzol RT (Molecular Research Center, Inc., USA). An samples were determined. The aims of this study were to inves- RNA 6000 Nano LabChip analyzer (Agilent 2100 bioanalyzer; tigate changes in blood glucose and magnesium levels and the Agilent, Santa Clara, CA, USA) was used to assess the RNA expression of GMTs after a three-month exercise in T2DM integrity. A volume of 1 µg of total RNA was reverse transcribed patients. The results of this study may contribute to an improved to cDNA using SuperScript III First-Strand Synthesis SuperMix understanding of the role of magnesium in glucose regulation in for qRT-PCR (Invitrogen by Life Technologies, Carlsbad, CA, T2DM patients following a three-month exercise program. USA). The expression levels of the five GMTs were determined by SYBR Green-based qRT-PCR (Life Technologies, USA). The ∆∆Ct method was used for the relative quantification of GMT 2. METHODS expression. This prospective open-label study was approved by the ethical review board of the Taichung Veterans General Hospital (IRB 2.4. Statistical analysis TCVGH No: SG12147). Adult participants with T2DM were Categorical variables were analyzed according to the frequency recruited, and informed consent was obtained. In this study, the distribution and were expressed as numbers and percentages diagnosis of diabetes was based on the criteria of the American (n [%]). Continuous variables of a skewed distribution were Diabetes Association. Subjects were excluded if they had the expressed as the median and range (25th percentile to 75th following: (1) a hyperglycemic crisis or HbA1c > 12%, (2) acute percentile). In addition, a paired t-test was used to evaluate the or chronic renal diseases with serum creatinine levels > 200 difference between the means before and after the three-month mmol/L, (3) acute or chronic infectious diseases, (4) changes exercise protocol, and the data are presented as the mean values in medications for diabetes, hypertension, hyperlipidemia, or and SD. Pearson’s correlation (r) analysis was used to determine had antiplatelet or anti-inflammation therapy within the past the correlation between two continuous variables. A p-value < month, or (5) a limited ability to perform the regular exercise 0.05 was considered statistically significant. program, which was assessed according to the physician’s clini- cal judgment. 3. RESULTS 2.1. Indoor bicycle exercise program and exercise intensity 3.1. Baseline characteristics A cardiopulmonary exercise test (CPET) was performed, and A total of 15 adult patients (eight males and seven females) exercise intensity was determined according to our previous 16 with T2DM and a median age of 53 years were recruited from report. Briefly, the cardiopulmonary fitness of the patients Taichung Veteran’s General Hospital. The characteristics of the (maximum oxygen consumption, VO max) was obtained using 15 T2DM patients are shown in Table 1. a MasterScreen CPX system (CareFusion Respiratory Care, CA, USA) with a cycle ergometer. All recruited patients underwent 3.2. Clinical data, metabolic parameters, and plasma a three-month scheduled exercise program. In addition, the magnesium before and after exercise in-home training modality consisted of a commercially avail- able bicycle (Giant CS800, Taichung, Taiwan) with an indoor The body weight, BMI, metabolic parameters, and plasma mag- bicycle trainer (Giant Cyclotron Mag, Taichung, Taiwan) for at nesium levels that were collected before and after the three- least 3 days per week. For in-home exercise, an initial 5 minute month exercise program are shown in Table  2. Although the warm-up period at an intensity according to the Borg rating of body weight and body mass index (BMI) decreased after three- perceived exertion scale of 9 was suggested. The recommended month exercise, there was no significant difference. The FBG, training intensity was 60% VO max determined from the CPET, HbA1c, and waist circumference levels significantly decreased 496 www.ejcma.org Original Article. (2019) 82:6 J Chin Med Assoc 4. DISCUSSION Table 1 The characteristics of 15 patients with T2DM The results of this study provide evidence that a three-month in-home bike exercise program significantly downregulated the Median expression of CNNM2, TRPM6, and TRPM7, improved FBG (25th percentile– and HbA1c, and reduced the waist circumference. Exercise is an Variables n, % 75th percentile) essential factor in the prevention and management of diabetes. Age, y 53 (48–59) In this study, improved control of FBG and HbA1c levels and Sex a reduced waist circumference were observed. Several studies Male 8 (53.3) have reported that exercise ameliorates hyperglycemia in T2DM Female 7 (46.7) 19,20 patients. Boule et al observed decreases in HbA1c but no Duration of diabetes, y 5.0 (2.0–12.0) substantial changes in the BMI after an exercise program that Diabetes medication lasted 8 weeks or more in patients with T2DM. Jorge et al No medication 2 (13.3) reported that the postprandial plasma glucose, FBG, and HbA1c Oral hypoglycemic drug 8 (53.3) levels decreased by 25.0%, 13.5%, and 2.8%, respectively, after Insulin and oral hypoglycemic drug 5 (33.3) aerobic exercise (three times per week and 60 minutes per ses- Lipid-lowering medication sion) in T2DM patients. The results from the present study No medication 8 (53.3) agreed with these previous reports and confirmed that exercise Oral medication 7 (46.7) intensity and duration were properly performed in this study. In The categorical variables were analyzed according to the frequency distribution and expressed as this study, although the body weight and BMI decreased after numbers and percentages (n [%]). The continuous variables are presented as the median and range this three-month exercise intervention, there was no significant (25th percentile–75th percentile). difference. The duration of exercise, type of exercise, and diet T2DM = Type 2 diabetes mellitus. 21 control together play a role in weight reduction. Further study may be necessary to disclose the association between BMI reduc- tion and magnesium gene expression after exercise training. by 16%, 4.9%, and 2.0%, respectively, after the exercise pro- The downregulation of the expression of CNNM2, TRPM6, gram (p < 0.05). None of the patients had hypomagnesemia 2+ and TRPM7 was observed in this study. Studies have shown ([Mg ] < 0.75 mmol/L) before the exercise program. The that exercise not only affects glucose availability and the plasma magnesium levels decreased by 4.7%, and two patients action of insulin but also manipulates blood magnesium lev- had hypomagnesemia after the exercise program (p = 0.023). 22,23 els in T2DM patients. Magnesium transporters in different However, the total cholesterol, HDL, LDL, and TG levels in the parts of the body may play different functional roles. TRPM7, blood were not significantly different before and after the exer - cise program. SLC41A1, and SLC41A3 are ubiquitously expressed. TRPM6 and CNNM2 are significantly expressed in the kidney and intes- 8,24 3.3. The expression of magnesium transporter genes after tine. CNNM2 is predominantly expressed in the basolateral membrane of distal tubular segments; therefore, CNNM2 has exercise been speculated to regulate magnesium in the blood by manip- The expression of CNNM2, TRPM6, and TRPM7 was sig- ulating renal magnesium reabsorption. To the best of our nificantly downregulated after the exercise program (p < 0.05). knowledge, this is the first study to demonstrate the downregu- Although the expression levels of SLC41A1 and SLC41A3 lation of CNNM2 expression in the blood after a three-month decreased after the exercise program, there were no significant exercise program in T2DM patients. Exercise-induced hormones differences compared to the expression levels before the exercise and metabolite activities may cause urinary magnesium loss by program (Fig. 1.). reducing renal tubular magnesium reabsorption. Huang et al reported that high amounts of daily physical activity is related to low serum magnesium in diabetic patients. Indeed, our data Table 2 also revealed that blood magnesium levels slightly decreased Clinical data, metabolic parameters, plasma magnesium levels, while downregulating CNNM2 expression after the exercise and magnesium transporter gene expression before and after program. Therefore, it was hypothesized that long-term exercise the exercise training program may deplete magnesium in the blood and increase the demand Exercise training program for magnesium due to metabolic requirements and could there- Variables Before After p fore downregulate CNNM2 expression. Further research is nec- essary to clarify this issue. Body weight, kg 83.0 ± 11.6 81.6 ± 11.2 0.079 2 Both TRPM6 and TRPM7 transporters preferentially trans- BMI, kg/m 31.3 ± 4.6 30.8 ± 4.1 0.082 port magnesium and calcium into cells. The TRPM6 trans- Waist circumference, cm 103.9 ± 14.5 101.8 ± 14.8 0.033* porter has been shown to be specifically expressed in the colon, Metabolic parameters lung, and kidney and manipulates the reabsorption of magne- FBG, mg/dL 187.5 ± 15.2 159.0 ± 17.0 0.009* sium via an active and transcellular process in the distal convo- HbA1c, % 8.6 ± 0.4 8.3 ± 0.7 0.034* luted tubule. TRPM7 is ubiquitous and may play significant Total cholesterol, mg/dL 174.3 ± 6.8 178.7 ± 18.5 0.543 roles in the regulation of magnesium homeostasis in the body. HDL, mg/dL 53.5 ± 3.2 51.2 ± 5.5 0.532 In this study, TRPM6 and TRPM7 expression was significantly Triglyceride, mg/dL 120.5 ± 12.0 137.0 ± 19.1 0.123 downregulated after a three-month exercise program in T2DM LDL, mg/dL 106.1 ± 7.1 119.0 ± 14.0 0.199 patients. This implies that the three-month exercise likely trig- Plasma magnesium, mg/dL 2.07 ± 0.14 1.97 ± 0.18 0.023* gers the effective metabolic response. Downregulated TRPM6 *p < 0.05. and TRPM7 expression may establish blood calcium and mag- BMI = body mass index; FBG = fasting blood glucose; HbA1c = glycated hemoglobin; HDL-C = nesium levels and then maintain the effectiveness of glucose uti- high-density lipoprotein; LDL = low-density lipoprotein. a lization after exercise. A paired t-test was used to analyze the difference in the means before and after the exercise training Although the SLC41 family includes three members (A1, A2, program. The data are reported as means ± SD. Plasma magnesium levels were measured in 12 of the 15 patients. and A3), studies have primarily focused on the SLC41, A1, and A2 www.ejcma.org 497 Chiang et al. J Chin Med Assoc Fig. 1 The expression levels of magnesium transporters before and after a 3-month bike exercise program: A, CNNM2; B, TRPM6; C TRPM7; D, SLC41A1; and E, SLC41A3. 27 + 2+ isoforms. SLC41A1, a Na /Mg exchanger defined by Kolisek TRPM6, and TRPM7, which showed significant downregula- 27,28 et al, regulates magnesium efflux. In this study, although the tion after the exercise program. Future studies that are designed expression levels of SLC41A1 and SLC41A3 decreased, there to investigate the underlying mechanism of these downregulated were no statistically significant differences. Previous studies GMT genes and the effects of varied exercise programs may be have demonstrated that acute exercise increases glucose avail- beneficial for T2DM patients. ability and induces SLC41A1 downregulation. In our previous study, the expression of SLC41A1 was significantly down- REFERENCES regulated by 23.6% and led to temporarily increased plasma magnesium levels during acute exercise. 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Physiol 2012;302:C318–26. www.ejcma.org 499 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of the Chinese Medical Association Wolters Kluwer Health

Magnesium-responsive genes are downregulated in diabetic patients after a three-month exercise program on a bicycle ergometer

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Abstract

Background: Exercise is an effective therapy for the management of diabetes because it helps regulate glucose and magnesium homeostasis. Nevertheless, the mechanisms by which exercise exerts effects on magnesium transport remain unclear. This study investigated the expression of genes encoding magnesium transporters (GMTs) after a three-month exercise program in diabetic patients. Methods: This study was conducted with a within-subject pre-post design. A total of 15 adult patients with type 2 diabetes mel- litus (T2DM) were recruited and underwent a three-month indoor bicycle exercise program. The expression of five GMTs (CNNM2, TRPM6, TRPM7, SLC41A1, and SLC41A3) was determined in blood samples. Relevant anthropometric values and biochemical parameters were also determined. Results: Although the body weight and body mass index decreased after three months exercise, there were no significant differences. Fasting blood glucose, glycated hemoglobin (HbA1c), waist circumference, and magnesium levels decreased after the exercise program (p < 0.05). The expression of SLC41A1 and SLC41A3 were downregulated after exercise, but only CNNM2, TRPM6, and TRPM7 showed significantly decreased expression levels compared with those before the exercise program (p < 0.05). Conclusion: The three-month exercise program ameliorated blood glucose levels and downregulated the expression of magne- sium-responsive genes in patients with T2DM. Keywords: Blood glucose; Exercise; Magnesium; Magnesium transporter; Type 2 diabetes mellitus healthy diet, and appropriate exercise. Appropriate exercise 1. INTRODUCTION improves blood pressure and glucose management, attenuates lipids, aids weight loss, enhances anti-inflammation, and plays a Type 2 diabetes mellitus (T2DM) is a widespread health prob- key role in the prevention and management of diabetes. Several lem with a dramatically increasing prevalence worldwide, and studies have revealed that regular exercise reduces glycated it is associated with increased cardiovascular risk. Three key hemoglobin (HbA1c), waist circumference, triglycerides (TG), aspects of diabetes management include suitable medications, 3,4 and systolic blood pressure in patients with T2DM. Over the past few decades, researchers have investigated the *Address correspondence: Dr. Sen-Wei Tsai, Department of Physical Medicine and Rehabilitation, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical ability of exercise to alter or regulate magnesium homeostasis, Foundation, 88, Section 1, Fengxing Road, Tanzi District, Taichung 427, Taiwan, including enzyme activation, nucleotide binding, and trans- ROC. E-mail address: tsaisenwei@gmail.com (S.-W. Tsai). membrane electrolyte flux. Nielsen and Lukaski suggested that Conflicts of interest: The authors declare that they have no conflicts of interest exercise may induce magnesium redistribution in the body to related to the subject matter or materials discussed in this article. compensate for metabolic demand. Additionally, exercise is Journal of Chinese Medical Association. (2019) 82: 495-499. suggested to impact magnesium transport, which may result Received February 9, 2019; accepted March 11, 2019. in depleted blood magnesium levels and increased demand for magnesium intake because of metabolic requirements. doi: 10.1097/JCMA.0000000000000112. Magnesium homeostasis plays an essential role in the regu- Copyright © 2019, the Chinese Medical Association. This is an open access lation of cellular physiology and biochemical functions. article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/). Altered blood magnesium levels may in turn shift the balance www.ejcma.org 495 <zdoi: 10.1097/JCMA.0000000000000112> Downloaded from http://journals.lww.com/jcma by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/03/2020 Chiang et al. J Chin Med Assoc 8–11 of other ions, resulting in metabolic or pathological changes. and the home bicycle exercise duration was 40 minutes, with a Magnesium homeostasis is maintained by regulation via hor- subsequent 5 minute cool-down period. During the study, diets mones and specific transporters, such as cyclin and CBS domain and medications were not changed. divalent metal cation transport mediator 2 (CNNM2), transient receptor potential melastatin 6 and 7 (TRPM6 and TRPM7), 2.2. Biochemical and anthropometric measurements solute carrier family 41 members 1–3 (SLC41A1, SLC41A2, Blood samples were collected for measurements of fasting blood 8,12 and SLC41A3), and by regulatory means such as hormones. glucose (FBG), HbA1c, lipoprotein profiles, and the expres- Previous studies have shown that exercise regulates magnesium sion of GMTs at fasting and resting statuses before and after and glucose transport by the interactions of cation channels (eg, the exercise program. Plasma magnesium levels were measured 2+ + + + + Ca , K , and Na ) and ion exchangers and/or pumps (eg, Na /K using a Spotchem EZ sp-4430 automatic analyzer (ARKRAY, + 2+ 8,13 and Na /Mg ). Inc., Kyoto, Japan). Blood glucose, TG, and cholesterol concen- Despite the evidence that magnesium is associated with blood trations were measured by using commercially available kits glucose regulation and is influenced by exercise, there is lim- (Beckman Coulter, Fullerton, USA). High-density lipoprotein ited knowledge regarding the molecular mechanisms by which (HDL) and low-density lipoprotein (LDL) levels were measured exercise modulates the molecular manipulation of magnesium by using commercially available kits (Roche Diagnostics GmbH, homeostasis. Mannheim, Germany). In addition, the anthropometric data, In our previous study, the downregulation of SLC41A1 including height, body weight, BMI, blood pressure, and waist + 2+ (encoding for the Na /Mg exchanger) following acute exercise circumference, were measured. was observed, and the level of SLC41A1 returned to basal status 24 hours after the acute exercise. However, the effects of exer- 2.3. GMT expression cise on the expression of genes encoding magnesium transporters The expression of five GMTs (CNNM2, TRPM6, TRPM7, (GMTs) and on the regulation of blood magnesium and glucose SLC41A1, and SLC41A3) was determined. Total RNA extrac- levels remain unclear. In the present study, we investigated the tions, RNA reverse transcription, quantitative real-time PCR status of magnesium (blood magnesium levels and the expression (qRT-PCR), and data analysis were performed as previously of GMTs) in T2DM patients. The exercise protocol consisted of described by Lin et al. Briefly, peripheral blood was collected a three-month indoor bicycle exercise program. Additionally, and centrifuged, total RNA was extracted from buffy coat layer anthropometric values and biochemical parameters of blood using RNAzol RT (Molecular Research Center, Inc., USA). An samples were determined. The aims of this study were to inves- RNA 6000 Nano LabChip analyzer (Agilent 2100 bioanalyzer; tigate changes in blood glucose and magnesium levels and the Agilent, Santa Clara, CA, USA) was used to assess the RNA expression of GMTs after a three-month exercise in T2DM integrity. A volume of 1 µg of total RNA was reverse transcribed patients. The results of this study may contribute to an improved to cDNA using SuperScript III First-Strand Synthesis SuperMix understanding of the role of magnesium in glucose regulation in for qRT-PCR (Invitrogen by Life Technologies, Carlsbad, CA, T2DM patients following a three-month exercise program. USA). The expression levels of the five GMTs were determined by SYBR Green-based qRT-PCR (Life Technologies, USA). The ∆∆Ct method was used for the relative quantification of GMT 2. METHODS expression. This prospective open-label study was approved by the ethical review board of the Taichung Veterans General Hospital (IRB 2.4. Statistical analysis TCVGH No: SG12147). Adult participants with T2DM were Categorical variables were analyzed according to the frequency recruited, and informed consent was obtained. In this study, the distribution and were expressed as numbers and percentages diagnosis of diabetes was based on the criteria of the American (n [%]). Continuous variables of a skewed distribution were Diabetes Association. Subjects were excluded if they had the expressed as the median and range (25th percentile to 75th following: (1) a hyperglycemic crisis or HbA1c > 12%, (2) acute percentile). In addition, a paired t-test was used to evaluate the or chronic renal diseases with serum creatinine levels > 200 difference between the means before and after the three-month mmol/L, (3) acute or chronic infectious diseases, (4) changes exercise protocol, and the data are presented as the mean values in medications for diabetes, hypertension, hyperlipidemia, or and SD. Pearson’s correlation (r) analysis was used to determine had antiplatelet or anti-inflammation therapy within the past the correlation between two continuous variables. A p-value < month, or (5) a limited ability to perform the regular exercise 0.05 was considered statistically significant. program, which was assessed according to the physician’s clini- cal judgment. 3. RESULTS 2.1. Indoor bicycle exercise program and exercise intensity 3.1. Baseline characteristics A cardiopulmonary exercise test (CPET) was performed, and A total of 15 adult patients (eight males and seven females) exercise intensity was determined according to our previous 16 with T2DM and a median age of 53 years were recruited from report. Briefly, the cardiopulmonary fitness of the patients Taichung Veteran’s General Hospital. The characteristics of the (maximum oxygen consumption, VO max) was obtained using 15 T2DM patients are shown in Table 1. a MasterScreen CPX system (CareFusion Respiratory Care, CA, USA) with a cycle ergometer. All recruited patients underwent 3.2. Clinical data, metabolic parameters, and plasma a three-month scheduled exercise program. In addition, the magnesium before and after exercise in-home training modality consisted of a commercially avail- able bicycle (Giant CS800, Taichung, Taiwan) with an indoor The body weight, BMI, metabolic parameters, and plasma mag- bicycle trainer (Giant Cyclotron Mag, Taichung, Taiwan) for at nesium levels that were collected before and after the three- least 3 days per week. For in-home exercise, an initial 5 minute month exercise program are shown in Table  2. Although the warm-up period at an intensity according to the Borg rating of body weight and body mass index (BMI) decreased after three- perceived exertion scale of 9 was suggested. The recommended month exercise, there was no significant difference. The FBG, training intensity was 60% VO max determined from the CPET, HbA1c, and waist circumference levels significantly decreased 496 www.ejcma.org Original Article. (2019) 82:6 J Chin Med Assoc 4. DISCUSSION Table 1 The characteristics of 15 patients with T2DM The results of this study provide evidence that a three-month in-home bike exercise program significantly downregulated the Median expression of CNNM2, TRPM6, and TRPM7, improved FBG (25th percentile– and HbA1c, and reduced the waist circumference. Exercise is an Variables n, % 75th percentile) essential factor in the prevention and management of diabetes. Age, y 53 (48–59) In this study, improved control of FBG and HbA1c levels and Sex a reduced waist circumference were observed. Several studies Male 8 (53.3) have reported that exercise ameliorates hyperglycemia in T2DM Female 7 (46.7) 19,20 patients. Boule et al observed decreases in HbA1c but no Duration of diabetes, y 5.0 (2.0–12.0) substantial changes in the BMI after an exercise program that Diabetes medication lasted 8 weeks or more in patients with T2DM. Jorge et al No medication 2 (13.3) reported that the postprandial plasma glucose, FBG, and HbA1c Oral hypoglycemic drug 8 (53.3) levels decreased by 25.0%, 13.5%, and 2.8%, respectively, after Insulin and oral hypoglycemic drug 5 (33.3) aerobic exercise (three times per week and 60 minutes per ses- Lipid-lowering medication sion) in T2DM patients. The results from the present study No medication 8 (53.3) agreed with these previous reports and confirmed that exercise Oral medication 7 (46.7) intensity and duration were properly performed in this study. In The categorical variables were analyzed according to the frequency distribution and expressed as this study, although the body weight and BMI decreased after numbers and percentages (n [%]). The continuous variables are presented as the median and range this three-month exercise intervention, there was no significant (25th percentile–75th percentile). difference. The duration of exercise, type of exercise, and diet T2DM = Type 2 diabetes mellitus. 21 control together play a role in weight reduction. Further study may be necessary to disclose the association between BMI reduc- tion and magnesium gene expression after exercise training. by 16%, 4.9%, and 2.0%, respectively, after the exercise pro- The downregulation of the expression of CNNM2, TRPM6, gram (p < 0.05). None of the patients had hypomagnesemia 2+ and TRPM7 was observed in this study. Studies have shown ([Mg ] < 0.75 mmol/L) before the exercise program. The that exercise not only affects glucose availability and the plasma magnesium levels decreased by 4.7%, and two patients action of insulin but also manipulates blood magnesium lev- had hypomagnesemia after the exercise program (p = 0.023). 22,23 els in T2DM patients. Magnesium transporters in different However, the total cholesterol, HDL, LDL, and TG levels in the parts of the body may play different functional roles. TRPM7, blood were not significantly different before and after the exer - cise program. SLC41A1, and SLC41A3 are ubiquitously expressed. TRPM6 and CNNM2 are significantly expressed in the kidney and intes- 8,24 3.3. The expression of magnesium transporter genes after tine. CNNM2 is predominantly expressed in the basolateral membrane of distal tubular segments; therefore, CNNM2 has exercise been speculated to regulate magnesium in the blood by manip- The expression of CNNM2, TRPM6, and TRPM7 was sig- ulating renal magnesium reabsorption. To the best of our nificantly downregulated after the exercise program (p < 0.05). knowledge, this is the first study to demonstrate the downregu- Although the expression levels of SLC41A1 and SLC41A3 lation of CNNM2 expression in the blood after a three-month decreased after the exercise program, there were no significant exercise program in T2DM patients. Exercise-induced hormones differences compared to the expression levels before the exercise and metabolite activities may cause urinary magnesium loss by program (Fig. 1.). reducing renal tubular magnesium reabsorption. Huang et al reported that high amounts of daily physical activity is related to low serum magnesium in diabetic patients. Indeed, our data Table 2 also revealed that blood magnesium levels slightly decreased Clinical data, metabolic parameters, plasma magnesium levels, while downregulating CNNM2 expression after the exercise and magnesium transporter gene expression before and after program. Therefore, it was hypothesized that long-term exercise the exercise training program may deplete magnesium in the blood and increase the demand Exercise training program for magnesium due to metabolic requirements and could there- Variables Before After p fore downregulate CNNM2 expression. Further research is nec- essary to clarify this issue. Body weight, kg 83.0 ± 11.6 81.6 ± 11.2 0.079 2 Both TRPM6 and TRPM7 transporters preferentially trans- BMI, kg/m 31.3 ± 4.6 30.8 ± 4.1 0.082 port magnesium and calcium into cells. The TRPM6 trans- Waist circumference, cm 103.9 ± 14.5 101.8 ± 14.8 0.033* porter has been shown to be specifically expressed in the colon, Metabolic parameters lung, and kidney and manipulates the reabsorption of magne- FBG, mg/dL 187.5 ± 15.2 159.0 ± 17.0 0.009* sium via an active and transcellular process in the distal convo- HbA1c, % 8.6 ± 0.4 8.3 ± 0.7 0.034* luted tubule. TRPM7 is ubiquitous and may play significant Total cholesterol, mg/dL 174.3 ± 6.8 178.7 ± 18.5 0.543 roles in the regulation of magnesium homeostasis in the body. HDL, mg/dL 53.5 ± 3.2 51.2 ± 5.5 0.532 In this study, TRPM6 and TRPM7 expression was significantly Triglyceride, mg/dL 120.5 ± 12.0 137.0 ± 19.1 0.123 downregulated after a three-month exercise program in T2DM LDL, mg/dL 106.1 ± 7.1 119.0 ± 14.0 0.199 patients. This implies that the three-month exercise likely trig- Plasma magnesium, mg/dL 2.07 ± 0.14 1.97 ± 0.18 0.023* gers the effective metabolic response. Downregulated TRPM6 *p < 0.05. and TRPM7 expression may establish blood calcium and mag- BMI = body mass index; FBG = fasting blood glucose; HbA1c = glycated hemoglobin; HDL-C = nesium levels and then maintain the effectiveness of glucose uti- high-density lipoprotein; LDL = low-density lipoprotein. a lization after exercise. A paired t-test was used to analyze the difference in the means before and after the exercise training Although the SLC41 family includes three members (A1, A2, program. The data are reported as means ± SD. Plasma magnesium levels were measured in 12 of the 15 patients. and A3), studies have primarily focused on the SLC41, A1, and A2 www.ejcma.org 497 Chiang et al. J Chin Med Assoc Fig. 1 The expression levels of magnesium transporters before and after a 3-month bike exercise program: A, CNNM2; B, TRPM6; C TRPM7; D, SLC41A1; and E, SLC41A3. 27 + 2+ isoforms. SLC41A1, a Na /Mg exchanger defined by Kolisek TRPM6, and TRPM7, which showed significant downregula- 27,28 et al, regulates magnesium efflux. In this study, although the tion after the exercise program. Future studies that are designed expression levels of SLC41A1 and SLC41A3 decreased, there to investigate the underlying mechanism of these downregulated were no statistically significant differences. Previous studies GMT genes and the effects of varied exercise programs may be have demonstrated that acute exercise increases glucose avail- beneficial for T2DM patients. ability and induces SLC41A1 downregulation. In our previous study, the expression of SLC41A1 was significantly down- REFERENCES regulated by 23.6% and led to temporarily increased plasma magnesium levels during acute exercise. 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Journal

Journal of the Chinese Medical Association – Wolters Kluwer Health

Published: Jun 1, 2019

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