Multidrug resistance-associated protein 4 (Mrp4) is a novel genetic factor in the pathogenesis of obesity and diabetes

Abstract

Multidrug resistance protein 4 (Mrp4) is an efflux transporter known to transport several xenobiotics and endogenous molecules. We recently identified that the lack of Mrp4 increases adipose tissue and body weights in mice. However, the role of Mrp4 in adipose tissue physiology are unknown. The current study aimed at characterizing these specific roles of Mrp4 using wild-type (WT) and knockout (Mrp4^-/- ) mice. Our studies determined that Mrp4 is expressed in mouse adipose tissue and that the lack of Mrp4 expression is associated with adipocyte hypertrophy. Furthermore, the lack of Mrp4 increased blood glucose and leptin levels, and impaired glucose tolerance. Additionally, in 3T3-L1 cells and human pre-adipocytes, pharmacological inhibition of Mrp4 increased adipogenesis and altered expression of adipogenic genes. Lack of Mrp4 activity in both of our in vivo and in vitro models leads to increased activation of adipose tissue cAMP response element-binding protein (Creb) and decreased plasma prostaglandin E (PGE) metabolite levels. These changes in Creb activation, coupled with decreased PGE levels, together promoted the observed metabolic phenotype in Mrp4^-/- mice. In conclusion, our results indicate that Mrp4 as a novel genetic factor involved in the pathogenesis of metabolic diseases, such as obesity and diabetes.

Keywords: Adipogenesis; Drug transporters; Mrp4.

Figure 1-. Mrp4-deficiency in mice promotes the development of the obese phenotype.

Phenotype characterization of WT and Mrp4^−/− mice (n=5 mice per group). Bodyweight (A) and adipose (Epididymal/gonadal fat pad) tissue weights, and adipose to bodyweight ratios (B) of both male and female mice. C) Representative photographs of male WT and Mrp4^−/− mice. D) Western blot analysis of Mrp4 in different tissues. E) Representative images (200X) of H&E stained white adipose tissue sections of WT and Mrp4^−/− mice. Data are presented as mean ± SEM and p≤0.05 was considered as statistically significant. An asterisk “*” denotes significance between WT and Mrp4^−/− mice.

Figure 2-. Lack of Mrp4 promotes the development of insulin resistance.

Mrp4 deficiency impairs glucose metabolism and hormone levels in mice (n=5 mice per group). A) Plasma leptin, insulin and GLP-1 levels; B) Blood glucose; C) Glucose tolerance test; and D) Insulin tolerance test. Data are presented as mean ± SEM and p≤0.05 was considered as statistically significant. An asterisk “*” denotes significance between WT and Mrp4^−/− mice.

Figure 3-. Lack of Mrp4 alters energy metabolism in mice.

The metabolic phenotype of WT and Mrp4^−/− female mice (n=4–5 mice per group) was characterized using metabolic cages. Indirect calorimetry measurements such as volumetric oxygen and carbon dioxide (A), food consumption (B), respiratory exchange ratio (RER) (C), energy expenditure (EE) (D), ambulatory activity (E) and wheel counts (F) were measured using the CLAMS system. Data are presented as mean ± SEM and p≤0.05 was considered as statistically significant. An asterisk “*” denotes significance between WT and Mrp4^−/− mice.

Figure 4-. Mrp4 deficiency alters adipose tissue gene expression.

Adipose tissue gene expression was determined using RNA-Seq analysis along with DEG seq analysis in male mice. A) Volcano plot identifying up-regulated and down-regulated genes. B) Plot showing KEGG pathway functional enrichment of DEGs. C) Number of altered DEGs in enriched pathways. D) Fold change of some of the genes that were identified in RNA-seq analysis. E) Gene expression analysis verified using RT-qPCR analysis. F) Adipose tissue Mrp1 and 5 mRNA levels. Data are presented as mean ± SEM and p≤0.05 was considered as statistically significant. An asterisk “*” denotes significance between WT and Mrp4^−/− mice.

Figure 5-. Inhibition of Mrp4 promotes adipogenesis in in vitro models.

Decreased Mrp4 activity increased adipogenesis in 3T3-L1 fibroblasts and human pre-adipocytes. A) 3T3-L1 cells were treated with either DMSO or Mrp4 inhibitors MK-571 and C1 at 50 μM concentration during the initial three days of differentiation. mRNA levels of adipogenesis marker genes were analyzed using RT-qPCR analysis in differentiated adipocytes (day-8). B) ORO staining of differentiated adipocytes (images taken at 200X magnification) for quantification of adipogenesis in 3T3-L1 cells treated with Mrp4 inhibitor at 50 μM concentration. C) Gene expression analysis of adipogenesis markers such as Fabp4, Pparγ and C/ebpβ expression during the initial phase (day-0, 1 and 2) of differentiation in 3T3-L1 fibroblasts cells treated with Mrp4 inhibitors (50μM). D) Gene expression analysis of adipogenic genes in undifferentiated and differentiated human pre-adipocytes (day-14) treated with either DMSO or C1 (50 μM). E) ORO staining (images taken at 200X magnification) for quantification of adipogenesis in human pre-adipocyte cells (Day-14) treated with either DMSO or C1 (50 μM). Data are presented as mean ± SEM and p≤0.05 was considered as statistically significant. An asterisk “*” denotes significance between DMSO and Mrp4 inhibitors.

Figure 6. Transport of cAMP and prostaglandins by Mrp4 plays an important role in the regulation of adipogenesis.

The deficiency of Mrp4 function alters cAMP and PGE levels in intra- and extracellular compartment, respectively. A) Intracellular cAMP levels in 3T3-L1 cells treated with either DMSO or forskolin or Mrp4 inhibitors at 50 μM concertation. B) Western blot analysis of phospo- and total isoform of Creb protein levels in 3T3-L1 cells treated with Mrp4 inhibitors (50 μM) for 1 and 3 hrs in adipogenic differentiation media. C) Adipose tissue phosphor-Creb and total-Creb, and Mrp4 protein expression in male WT and Mrp4^−/− mice. D) Plasma PGE metabolite (PGEM) levels in male WT and Mrp4^−/− mice. E) Extra- and intracellular PGE₂ levels in 3T3-L1 cells treated with either DMSO or Mrp4 inhibitors (50 μM). F) Pictorial representation of molecular mechanisms through which lack of Mrp4 increases the risk of development of metabolic diseases such as obesity and diabetes. Loss of Mrp4 function either through pharmacological inhibition or genetic ablation increases intracellular cAMP levels, which is known to increase Creb activity in the fibroblasts/ pre-adipocytes. These increases in Creb activation results in increased adipogenesis. Lack of Mrp4 also decreases extracellular PGE₂ and its metabolite levels. These decreases in PGE will further promote adipogenesis in the fibroblasts/ pre-adipocytes. Data are presented as mean ± SEM and p≤0.05 was considered as statistically significant. An asterisk “*” denotes significance due to lack of Mrp4 function.