Magnesium deficiency prevents high-fat-diet-induced obesity in mice

Aims/hypothesis Hypomagnesaemia (blood Mg2+ <0.7 mmol/l) is a common phenomenon in individuals with type 2 diabetes. However, it remains unknown how a low blood Mg2+ concentration affects lipid and energy metabolism. Therefore, the importance of Mg2+ in obesity and type 2 diabetes has been largely neglected to date. This study aims to determine the effects of hypomagnesaemia on energy homeostasis and lipid metabolism. Methods Mice (n = 12/group) were fed either a low-fat diet (LFD) or a high-fat diet (HFD) (10% or 60% of total energy) in combination with a normal- or low-Mg2+ content (0.21% or 0.03% wt/wt) for 17 weeks. Metabolic cages were used to investigate food intake, energy expenditure and respiration. Blood and tissues were taken to study metabolic parameters and mRNA expression profiles, respectively. Results We show that low dietary Mg2+ intake ameliorates HFD-induced obesity in mice (47.00 ± 1.53 g vs 38.62 ± 1.51 g in mice given a normal Mg2+-HFD and low Mg2+-HFD, respectively, p < 0.05). Consequently, fasting serum glucose levels decreased and insulin sensitivity improved in low Mg2+-HFD-fed mice. Moreover, HFD-induced liver steatosis was absent in the low Mg2+ group. In hypomagnesaemic HFD-fed mice, mRNA expression of key lipolysis genes was increased in epididymal white adipose tissue (eWAT), corresponding to reduced lipid storage and high blood lipid levels. Low Mg2+-HFD-fed mice had increased brown adipose tissue (BAT) Ucp1 mRNA expression and a higher body temperature. No difference was observed in energy expenditure between the two HFD groups. Conclusions/interpretation Mg2+-deficiency abrogates HFD-induced obesity in mice through enhanced eWAT lipolysis and BAT activity. Electronic supplementary material The online version of this article (10.1007/s00125-018-4680-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

were injected with D-glucose (Invitrogen, The Netherlands) between 15 and 30 minutes post insulin-injection. For the subsequent time points, these mice were given the same glucose values as the lowest values within that group. After 15 weeks on the diets, three mice per group per day underwent an intraperitoneal glucose tolerance test (IPGTT), over a period of four days (n=12 mice/group). After an overnight fast (from 18:00 to 09:00), mice were injected with 2 g/kg body weight D-glucose (Invitrogen, The Netherlands) dissolved in PBS, with an injection volume of 50ul/10g body weight. Blood glucose levels were measured at 0, 15, 30, 60 and 120 minutes after the glucose injection.

RNA-Sequencing
Total RNA was extracted from epididymal white adipose tissue using TRIzol reagent (Invitrogen, UK) according to manufacturer's protocol. RNA integrity was validated by investigating the 18S/28S bands on a 2 w/v% agarose gel. Five randomly selected samples of each group were RNA-sequenced (RNA-Seq). Quality control and RNA-Seq were performed by Beijing Genomics Institute (BGI), Hong Kong). Quality control was performed by using Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System to qualify and quantify the sample library. One sample from the NormalMg-LFD group failed the quality control, and was excluded from subsequent sequencing and analyses. One sample from the LowMg-HFD group showed a small contamination with pancreatic tissue and was excluded from subsequent analyses. 13 million reads were sequenced using the Hiseq 4000 platform (Illumina, USA) using a 50 bp single-end module. Clean reads were mapped to Mus Musculus transcriptome (GRCm38/mm10) using HISAT/Bowtie2 tool [24,25]. RSEM software v1.2.31 was used to quantify gene expression levels (FPKM values)[26]. FPKM values were log 2 transformed and further analysed in R (www.r-project.org, RRID:SCR_001905). In order to filter non-expressed transcripts from the data, only transcripts that showed an average expression level of 8 within a group and for which the transcript levels were above 8 in at least four replicates from an experimental group were retained, yielding a total of 8808 transcripts. To calculate the differences between expression levels for genes belonging to the same Gene Ontology group, the fold change between the LowMg and NormalMg condition for both the HFD and LFD groups were collected for each gene in the group. Subsequently a ttest was used to test for the hypothesis of equal means. The procedure was repeated for all GO terms and the p-values for the tests were corrected for multiple testing using the Benjamini-Hochberg method as implemented in the p.adjust method in R. Heatmaps for individual GO terms were created using the ggplot2 library (RRID:SCR_014601) [27].

9-Week replication mouse study -MRC Harwell Institute
All experimental procedures were conducted in compliance with the UK Animals Scientific Procedures Act (1986) and University of Oxford ethical guidelines. 39 male C57BL6/J mice (MRC Harwell, UK) were randomly allocated into 4 groups of n=10 mice (n=9 in the LowMg-LFD group) housed with five per cage (1284L and 1285L IVC, Tecniplast, Italy). Mice had ad libitum access to demineralized chlorinated tap water and standard pellet chow. At 8 weeks old, mice were put on experimental diets identical to the first animal experiment at the Radboudumc, for a period of 9 weeks. At day 14, mice were housed individually in metabolic cages (Tecniplast, Italy) for 24 hours for the collection of urine and faeces and determining food and water intake. Mice were weighed twice weekly and blood was collected via tail bleed at day -1 and 14. Respiration metabolic cages (TSE Phenomaster Cages, Germany) were used at day 28 and 56 of the experiment and body temperatures were measured by rectal probe (ATP-instrumentation, UK). Data were averaged per hour and plotted from 6:30 PM to 9:30 AM. After 9 weeks on the diets, mice were anaesthetized by 4 v/v% isoflurane and exsanguinated via orbital sinus bleeding. Death was confirmed by cervical dislocation.
Tissues were stored in 10 v/v% formalin or snap frozen in liquid nitrogen.   Symbols: NormalMg-LFD (white circle), LowMg-LFD (light grey circle), NormalMg-HFD (white square), LowMg-HFD (light grey square). Data are mean ± SEM. Depending on the absence or presence of a significant interaction effect between dietary fat and Mg 2+ content, either a two-way ANOVA (Tukey's multiple comparison test) or a multiple t-test (Holm-Sidak multiple comparison test) approach, respectively, was used to determine statistical significance. * Indicates p<0.05.
ESM Table 2. List of Differentially Regulated GO-Terms from the RNA-SEQ on White Adipose Tissue Between NormalMg-HFD and LowMg-HFD. A negative log 2 fold change indicates a higher expression in the NormalMg-HFD group compared to the LowMg-HFD.