Diabetes-induced hypomagnesemia is not modulated by metformin treatment in mice

Approximately 30% of patients with type 2 diabetes mellitus (T2D) have hypomagnesemia (blood magnesium (Mg2+) concentration <0.7 mmol/L). In T2D patients, treatment with metformin is associated with reduced blood Mg2+ levels. To investigate how T2D and metformin affect Mg2+ homeostasis db/m and db/db mice were treated with metformin or placebo. Mice were housed in metabolic cages to measure food and water intake, and to collect urine and feces. Serum and urinary Mg2+ concentrations were determined and mRNA expression of magnesiotropic genes was determined in kidney and distal colon using RT-qPCR. Db/db mice had significantly lower serum Mg2+ levels than db/m mice. Mild hypermagnesuria was observed in the db/db mice at two weeks, but not at four weeks. Metformin-treatment had no effect on the serum Mg2+ concentration and on the urinary Mg2+ excretion. Both in kidney and distal colon of db/db mice, there was a compensatory upregulation in the mRNA expression of magnesiotropic genes, such as transient receptor potential melastatin 6 (Trpm6), whereas metformin treatment did not affect gene expression levels. In conclusion, we show that T2D causes hypomagnesemia and that metformin treatment has no effect on Mg2+ homeostasis in mice.

Approximately 30% of patients with type 2 diabetes mellitus (T2D) have hypomagnesemia (blood magnesium (Mg 2+ ) <0.7 mmol/L) 1,2 . Hypomagnesemia has serious clinical consequences as it increases the risk of complications such as retinopathy, nephropathy, micro and macrovascular disease and foot ulceration 3,4 . Moreover, Mg 2+ deficiency is correlated with insulin resistance, abrogated glucose metabolism and an increased risk of developing T2D [5][6][7] . However, the etiology and underlying mechanisms of hypomagnesemia in T2D patients remains largely unknown 8 .
As Mg 2+ is necessary for the activity of over 600 enzymes, it plays numerous vital physiological functions including macromolecule synthesis, energy balance and DNA transcription 9 . Moreover, Mg 2+ stabilizes ATP and is required for its phosphor transfer reactions 10 .
The intestine and kidney collaboratively regulate Mg 2+ balance and maintain its blood concentrations within a narrow range 11,12 . In the gut, the bulk of Mg 2+ absorption occurs in the small intestine via paracellular (passive) transport 11 . In the colon, the final absorption of Mg 2+ takes place by an active transcellular mechanism through transient receptor potential melastatin type 6/7 (TRPM6/TRPM7) cation channels 9 . In the kidney, 95-99% of filtered Mg 2+ is reabsorbed under physiological circumstances 9 . Approximately 85% of the filtered Mg 2+ is reabsorbed paracellularly by the proximal tubule and the thick ascending loop of Henle (TAL), where transport relies on tight junction permeability 13,14 . Active transport in the distal convoluted tubule (DCT) determines the final urinary Mg 2+ concentration, as this is the final segment where Mg 2+ is reabsorbed 15 . In physiological conditions, the DCT reclaims 5-10% of filtered Mg 2+ transcellularly via TRPM6/7 channels 14,16 . The expression and/or the activity of TRPM6 is affected by SNPs, dietary Mg 2+ intake, drugs and hormones, such as insulin and epidermal growth factor (EGF) 14,[17][18][19][20] . SNPs in TRPM6 that impair its response to insulin have been associated with an increased risk of developing T2D and gestational diabetes 7,19 .
Metformin, the first-line pharmacotherapy in T2D 21 , suppresses hepatic gluconeogenesis and improves insulin sensitivity 22 . Therefore, its major clinical benefit is reducing blood glucose levels with only a minimal risk of hypoglycemia 23,24 . The most common side effects of metformin treatment are lactic acidosis, nausea and diarrhea 25 . Recent cohort studies showed that metformin use in T2D patients is associated with reduced blood Mg 2+ levels 1,26 . However, the mechanism that underlies this correlation has not yet been elucidated. To investigate how T2D and metformin affect Mg 2+ homeostasis, control (db/m) and diabetic (db/db) mice were treated with placebo or metformin for four weeks. Serum and urinary electrolytes were measured and mRNA expression of magnesiotropic genes was evaluated in kidney and distal colon.

Methods
Animal study. The animal study was approved by the animal ethics board of the Radboud University Nijmegen (RU DEC 2015-0073) and by the Dutch Central Commission for Animal Experiments (CCD, AVD103002015239). Experimental procedures were conducted in accordance with the institutional guidelines and in compliance with Dutch and European laws and policies. Twenty diabetic (db/db) and twenty control (db/m) male mice (Charles River, Germany), aged 8-10 weeks, were acclimatized for two weeks in a temperatureand light-controlled room two mice per cage (Eurostandard Type IIL), with ad libitum access to tap water and standard pellet chow. At day 0, diets were changed to a diet containing 0.05% (w/w) MgO (#S9074-E1107, Ssniff Spezialdiäten, GmbH, Germany) and drinking water to demineralized water. At days-2, 12 and 26 mice were housed individually in metabolic cages for 48 hours (24 hours adaptation, 24 hours collection) to measure food and water intake and to collect urine and feces. Mice were weighed twice weekly and blood was collected via the submandibular vein at days -2 and 15. Mice were randomly divided into four experimental groups of ten mice per group, of which half received metformin hydrochloride (0.5 mg/ml, Sigma Aldrich, MI, USA), dispersed in the drinking water. Researchers and animal caretakers were blinded for the metformin treatment. After 28 days of treatment, mice were anaesthetized by 4% (v/v) isoflurane and exsanguinated by orbital sinus bleeding, and death was confirmed by cervical dislocation. Colon and kidney tissues were cleaned with ice-cold PBS and snap-frozen in liquid nitrogen. Analytical measurements. Serum and urinary Mg 2+ concentrations were determined using a spectrophotometric assay (Roche/Hitachi, Tokyo, Japan), according to manufacturer's protocol. Ca 2+ concentrations were determined by the o-cresophthalein complexone method. Absorbance for the Mg 2+ and Ca 2+ assays was measured at 600 nm and 570 nm, respectively, on a Bio-Rad Benchmark plus microplate spectrophotometer (Bio-Rad Laboratories, CA, USA). Serum and urinary Na + and K + concentrations were measured at the clinical chemistry department applying standardized methods 1 . Serum and urinary glucose concentrations were determined by a spectrophotometric assay according to the manufacturer's protocol (Instruchemie, Delfzijl, the Netherlands).

RT-qPCR.
statistical analyses. Interaction between the two main variables (genotype and treatment) was investigated using a two-way ANOVA test. If there was a significant interaction effect, an unpaired multiple t test, with the Holm-Sidak method for multiple comparisons, was used. In the absence of a significant interaction effect, a two-way ANOVA approach with a Tukey's multiple comparisons test was used. Statistical significance was assessed using Graphpad Prism v7 (La Jolla, CA, USA, RRID: SCR_002798. A p-value of ≤0.05 was considered statistically significant. Results are presented as mean ± SEM.

Metformin reduces food intake of db/db mice without affecting body weight. To investigate how
T2D and its first-line treatment metformin affect Mg 2+ homeostasis, control (db/m) and diabetic (db/db) mice were treated with metformin or placebo for four weeks. Db/db mice were significantly heavier than db/m mice (Fig. 1a,b; 27.0 ± 0.3 vs. 45.6 ± 0.6 gr. for db/m and db/db mice at four weeks, respectively, p ≤ 0.05). Metformin treatment had no effect on body weight in both db/m and db/db mice (Fig. 1a,b). Metformin treatment reduced the food intake only in the db/db mice (Fig. 1c). The lower food intake was accompanied by a decreased feces weight, water intake and urinary volume in the metformin-treated db/db mice (Fig. 1d-f). Metformin did not influence non-fasting serum glucose levels in both genotypes (Fig. 1g). However, the glycosuria of the db/db mice was attenuated by metformin treatment (Fig. 1h).
Db/db mice have an enhanced colonic expression of Trpm6. When serum Mg 2+ levels decrease, intestinal uptake of Mg 2+ is enhanced 15 . Colonic mRNA expression of Trpm6, the major channel for regulated Mg 2+ absorption, was elevated in db/db compared to db/m mice (Fig. 3a). There was no difference in mRNA expression of the ubiquitous Mg 2+ channel Trpm7 and of the Mg 2+ transport regulator Cyclin m4 (Cnnm4) (Fig. 3b,c). The colonic gene expression of the basolateral Mg 2+ transporter solute carrier family 41 (Slc41a1) was lower in both db/db groups (Fig. 3d).

Db/db mice have an elevated renal expression of genes involved in Mg 2+
handling. Db/db mice had an enhanced gene expression of the DCT-specific apical Mg 2+ channel Trpm6, and the basolateral Mg 2+ extruder Slc41a3 (Fig. 4a,b). While both db/db groups showed a higher expression of Slc12a3, encoding for NCC, metformin further enhanced the expression of this gene in db/db mice (Fig. 4c). The driving force for paracellular Mg 2+ uptake in the TAL is generated by NKCC2, encoded by Slc12a1, which is expressed higher in db/db mice (Fig. 4d). A significant genotype effect indicated a decreased expression of Claudin 10b (Cldn10b) in db/db mice (Fig. 4e, 1.00 ± 0.05 vs. 0.83 ± 0.02 relative gene expression in db/m vs. db/db mice, two-way ANOVA p ≤ 0.05). In contrast, the mRNA expression of Cldn14, Cldn16 and Cldn19 was enhanced in db/db mice ( Fig. 4f-h). The gene expression of the ubiquitous Mg 2+ channel Trpm7 was elevated in the placebo-treated db/db mice and the expression of Fxyd2, encoding for the gamma subunit of the Na + -K + -ATPase, was enhanced in both db/db groups (Fig. 4i,j).

Discussion
Hypomagnesemia is a common clinical feature in T2D patients 1,3 . Metformin use is associated with a lower blood Mg 2+ concentration in these patients 1,26 . In this study, db/db mice developed hypomagnesemia with compensatory upregulation of key renal and colonic magnesiotropic genes. Metformin treatment had no effect on Mg 2+ homeostasis in either control or diabetic mice. Our data demonstrate that hypomagnesemia is a consequence of T2D and is not modulated by metformin treatment in mice. INSERT ENTER Metformin is the first-line therapy for T2D 27 . In large-scale observational cohort studies metformin-use is associated with lower serum Mg 2+ levels and reduced renal Mg 2+ wasting in T2D patients 1,26,[28][29][30] . In a small intervention study in T2D patients, metformin treatment resulted in a minor reduction in the serum Mg 2+ concentration (from 0.72 to 0.70 mmol/L), despite major improvements in the blood glucose concentration 29 . In our study, metformin treatment did not affect the serum Mg 2+ concentration and urinary Mg 2+ excretion in db/db and db/m mice. In addition, metformin did not alter gene expression of colonic and renal Mg 2+ transporters. This is in line with a study that observed no effect of a two-week metformin treatment on serum Mg 2+ levels in streptozotocin-induced diabetic rats 31 . Possibly, a two-to four-week treatment duration is too short to detect effects on Mg 2+ homeostasis. The association between metformin and lower serum Mg 2+ levels in T2D patients could also be caused by other factors that were not included in the analyses. For instance, a well-known side effect of metformin-treatment is chronic diarrhea, leading to intestinal malabsorption and hypomagnesemia 28 . Hypomagnesemia is prevalent in over 30% of T2D patients [32][33][34][35] . A remaining question is whether hypomagnesemia is the cause or the consequence of T2D 8   indicating that hypomagnesemia is a consequence of T2D. At the fourth week of the experiment, db/db mice developed massive glycosuria but no renal Mg 2+ wasting. This finding is against the leading hypothesis that renal Mg 2+ wasting in T2D patients is a result of glycosuria 2,3,36 . Indeed, metformin treatment noticeably decreased glycosuria in db/db mice but did not modify the urinary Mg 2+ excretion. This is in line with recent observations that glycosuria-causing SGLT2 inhibitors, lead to a mild increase in serum Mg 2+ levels 37,38 . Therefore, it is unlikely that glycosuria underlies hypermagnesuria-induced hypomagnesemia in T2D. As db/db mice develop severe hyperinsulinemia, the observed hypomagnesemia could be a consequence of a Mg 2+ -shift towards the intracellular compartment, induced by insulin [39][40][41] . Future studies should focus on measuring intracellular Mg 2+ concentrations in diabetic mice. The kidneys are essential in maintaining the serum Mg 2+ concentration within the physiological range 15 . The DCT is the final segment where Mg 2+ can be reabsorbed 9 . In the DCT, regulated Mg 2+ reabsorption takes place transcellularly via TRPM6 18 . Mg 2+ uptake by TRPM6 is dependent on NCC, although the underlying mechanism remains largely unknown 42,43 . Gene expression levels of Trpm6 and Slc12a3, encoding for NCC, were enhanced in db/db mice, indicative of compensation in the DCT. As only a minor hypermagnesuria is observed at two-weeks, and no hypermagnesuria at four-weeks, there appears to be proper renal compensation in the db/db mice. The TAL is responsible for the bulk of renal Mg 2+ reabsorption 9 . In the TAL, paracellular Mg 2+ and Ca 2+ reabsorption is regulated by the Cldn14/16/19 complex 44,45 . Cldn14 mRNA expression is strongly regulated by dietary Ca 2+ intake 46,47 . The high food intake, and therefore high Ca 2+ intake, of db/db mice is likely the underlying cause of the extensive upregulation of Cldn14 expression. The high expression of Cldn14 will have a negative effect on Mg 2+ reabsorption in the TAL, leading to a compensatory increase in Cldn16/19 expression 48 . In contrast, gene expression of Cldn10b was decreased. Cldn10b enhances the Na + -permeability of the TAL, and thereby indirectly increases uptake of Mg 2+ and Ca 2+ in the TAL. Therefore, Cldn10b-deficient mice develop hypermagnesemia and hypomagnesuria. Likely, the observed reduction in Cldn10b expression in the db/db mice is a response to the high osmolality of the pro-urine. INSERT ENTER The strength of this study is that using oral metformin treatment in diabetic mice closely resembles the human situation. Db/db mice developed hypomagnesemia making them an excellent model to study the mechanisms of hypomagnesemia in T2D. Moreover, this study extensively investigated differences in expression of all known genes involved in Mg 2+ transport, in both kidney and colon. Some limitations have to be considered. The fact that metformin treatment did not affect Mg 2+ homeostasis raises the question whether the dose and duration of metformin treatment were sufficient. However, the metformin treatment reduced the food intake of db/db mice, a known positive effect of metformin. Moreover, the dosage of metformin that the db/db received (0.5 mg/ml, equivalent to a daily intake of approximately 165 mg/kg bodyweight) is similar to previous studies investigating the metabolic effects of metformin in mice [49][50][51][52] . A second limitation is that the expression of genes such as Cldn10b/14, Slc12a1 and Slc12a3 is regulated by both dietary intake and serum levels of K + , Na + and Ca 2+ 43,44,53 . As db/db mice have hyperphagia, their dietary intake of ions is also increased. Despite the higher food intake, db/db mice still develop hypomagnesemia. However, for other differences between db/m and db/db mice it is difficult to differentiate whether they are caused by T2D-related factors or by a higher food intake.
In conclusion, hypomagnesemia is a consequence of T2D, which is not affected by metformin treatment. The reason that metformin-users have lower serum Mg 2+ concentrations is likely mediated by other factors, and not by a direct effect of metformin on Mg 2+ (re)absorption.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.