Mimp/Mtch2, an Obesity Susceptibility Gene, Induces Alteration of Fatty Acid Metabolism in Transgenic Mice

Abstract

Objective: Metabolic dysfunctions, such as fatty liver, obesity and insulin resistance, are among the most common contemporary diseases worldwide, and their prevalence is continuously rising. Mimp/Mtch2 is a mitochondrial carrier protein homologue, which localizes to the mitochondria and induces mitochondrial depolarization. Mimp/Mtch2 single-nucleotide polymorphism is associated with obesity in humans and its loss in mice muscle protects from obesity. Our aim was to study the effects of Mimp/Mtch2 overexpression in vivo.

Methods: Transgenic mice overexpressing Mimp/Mtch2-GFP were characterized and monitored for lipid accumulation, weight and blood glucose levels. Transgenic mice liver and kidneys were used for gene expression analysis.

Results: Mimp/Mtch2-GFP transgenic mice express high levels of fatty acid synthase and of β-oxidation genes and develop fatty livers and kidneys. Moreover, high-fat diet-fed Mimp/Mtch2 mice exhibit high blood glucose levels. Our results also show that Mimp/Mtch2 is involved in lipid accumulation and uptake in cells and perhaps in human obesity.

Conclusions: Mimp/Mtch2 alters lipid metabolism and may play a role in the onset of obesity and development of insulin resistance.

Fig 1. Mimp/Mtch2-GFP expression in HEK-293 and in HEK-293T cells.

A. Western blot of control non-transfected HEK-293 cells, stable HEK-293 Mimp/Mtch2-GFP, non-transfected HEK-293T cells and from HEK-293T cells transiently transfected with Mimp/Mtch2-GFP. Western blot was performed using antibody specific for GFP, which recognizes Mimp/Mtch2-GFP expression (64kDa). Total cell actin served as internal control. B. Mitochondrial Membrane potential of HEK-293T cells transfected with Mimp/Mtch2-GFP and stained with JC-1. Images of each fluorescent emission are represented in (a) Mimp/Mtch2-GFP, (b) JC-1 green, (c) JC-1 red and (d) the overall overlay. (e) Overlay of only the JC-1 Green and Red staining. C. Alteration in the mitochondrial potential. The normalized R/G fluorescence ratio was calculated only for cells expressing Mimp/Mtch2-GFP (n = 50, P = 3.6 x 10⁻⁸, Student's t-test). “NT” represents non-transfected cells, served as control. Results are an average of 5 independent experiments and expressed as means ± SEM.

Fig 2. Characterization of Mimp/Mtch2-GFP Expression in Transgenic Mice.

A. Quantitative analysis of the fluorescent signal from CLSM images of exteriorized Mimp/Mtch2-GFP transgenic mice tissues (n = 6) and wild type (WT) mice kidney (n = 2), performed by MICA software. B. (a) Western blot analysis of Mimp/Mtch2-GFP levels in tissue lysates from transgenic mice organs. “+” represents HEK-293T cells transiently transfected with Mimp/Mtch2-GFP as a positive control; “Control” represents lysate obtained from control, non-transgenic mouse kidney; “N.T.” represents non-transfected cells. (b) Quantitative analysis of a representative western blot (presented in “a”), performed using Image J software. C. Mimp/Mtch2-GFP localization in mitochondria of exteriorized kidneys. (I) Red Mitotracker staining of the mitochondria. (II) GFP fluorescence. (III) Overlay showing co-localization of the two signals in the mitochondria. (a) Mimp/Mtch2-GFP transgenic mice. (b) WT mice. D. Mitochondrial membrane potential of transgenic mice kidney. (a) Images of excised kidneys stained in vivo with JC-1 and imaged 15 min after staining. (I) Mimp/Mtch2-GFP. (II) WT. (b) Quantification of the signals and the Red/Green ratios obtained from excised kidneys of WT (n = 7) and Mimp/Mtch2-GFP (n = 9) mice, compared by Student's t-test. All bar graphs results are expressed as means ± SEM. All Mice were 14 month old.

Fig 3. Detection of fatty livers and kidneys and blood glucose levels in Mimp/Mtch2-GFP transgenic mice.

A. (a) Ultrasound analysis of control (I) and Mimp/Mtch2-GFP transgenic mouse (II), 14 month old. (b) Calculation of % of mice showing fatty changes in H&E stained fixed sections from Mimp/Mtch2-GFP (n = 18) and WT (n = 7) mice. P = 0.0003 for both liver and kidney (Chi test). All mice were maintained on HFD. B. (a) H&E staining of fixed sections. (I) Control mouse liver. (II) Liver section of Mimp/Mtch2-GFP transgenic mouse. Fat vesicles are indicated by arrow. (III) Control mouse showing a normal kidney. (IV) Kidney section obtained from a Mimp/Mtch2-GFP transgenic mouse shows fatty vesicles that accumulate mainly in the proximal convoluted tubules of the kidney (indicated by arrow). Bars represent 0.10 mm. (b) Oil-Red-O staining of Mimp/Mtch2-GFP transgenic mouse showing fatty liver (II) and fatty kidney (IV), and of control mouse showing normal liver (I) and kidney (III). All mice were between the ages of 12 and 14 month and maintained on HFD from birth. C. Glucose levels and weight in Mimp/Mtch2-GFP mice. Mimp/Mtch2-GFP transgenic mice (n = 16) and age matched control mice (n = 12) consuming high or low fat diets were monitored weekly in the mornings for their (a) blood glucose levels and (b) their weight. Measurements were performed between the ages of 9 to 16 month and then averaged for all time points and all mice. All bar graphs results are expressed as means ± SEM. Groups were compared using ANOVA with Tukey’s post hoc.

Fig 4. Cluster analysis of cDNA microarray and gene expression in Mimp/Mtch2-GFP mice.

A. K-Means clustering over 57 probes that change significantly (p<0.05) in fatty (n = 2) compared to non-fatty (n = 2) kidneys of Mimp/Mtch2-GFP transgenic mice consuming HFD. Rows, genes; Columns, samples. Down-regulation is colored in green and up-regulation is colored in red. B. Quantitative real time PCR (qRT-PCR) for gene expression of (a) Mimp/Mtch2, (b) MCAD and Thiolase and (c) FASN, in livers (I) and kidneys (II) of control (WT) and Mimp/Mtch2-GFP transgenic mice, in the ages of 21–23 month, consuming low fat diet. The transgenic mice were divided into 2 groups of low and high Mimp/Mtch2 according to Mimp/Mtch2 expression levels. Each group consists of at least 3 mice. Results are represented as averaged relative quantity (RQ) of the mice in each group. All bar graphs results are expressed as means ± SEM. Groups were compared using ANOVA with Tukey’s post hoc.

Fig 5. Mimp/Mtch2 increases expression of lipids, lipid uptake and lipid metabolism genes in HEK-293T cells.

A. Oil-red-O staining of HEK-293T cells transiently expressing Mimp/Mtch2-GFP (b) and control non-transfected cells (a). B. HEK-293T cells were transfected with Mimp/Mtch2-GFP or an empty GFP vector. RNA was extracted and measured by qRT-PCR for the expression of MCAD, Thiolase and FASN. Expression between the groups was compared using Student’s t-test. C. HEK-293T cells were transfected with Mimp/Mtch2-GFP or an empty GFP vector. Cells medium was supplemented with 100 or 200 μM oleic acid for 24 hours and cells were stained for triglycerides using oil-red-O. Cells were imaged using Leica and oil-red-O signal intensity was quantified using Image J software. Each bar represents an average of at least 6 fields and experiment was repeated 3 times. Groups were compared using ANOVA with Tukey’s post hoc. All bar graphs results are expressed as means ± SEM.

Fig 6. Gene expression in obesity.

The expression levels of: A. Mimp/Mtch2 and B. MCAD and Thiolase, in pre-published cDNA microarray from human skeletal muscle of non-obese, obese and morbidly obese patients [19]. All bar graphs results are expressed as means ± SEM. The non-obese and morbidly obese groups were compared using Student’s t-test.