Abstract
BackgroundMAP kinase phosphatase 3 (MKP-3) is known to attenuate the ERK signaling pathway. It has been recently demonstrated that MKP-3 is also a player in promoting hepatic glucose output in obese state by interacting and activating FOXO1. Reduction of hepatic MKP-3 expression is sufficient to reduce blood glucose levels in both diet-induced and genetically obese mice.Methodology/Principal FindingsIn current study, the mechanism of MKP-3/FOXO1 interaction and the effects on transcription of gluconeogenic gene and glucose output was investigated in Fao hepatoma cells by using mutated MKP-3 and FOXO1 adenoviral constructs. The results indicate that MKP-3 phosphatase activity is not required for MKP-3/FOXO1 interaction but is essential for FOXO1 nuclear translocation and MKP-3 promoted gluconeogenesis. Compared to GFP control (1±0.38), MKP-3 increased G6Pase gene expression by 242% (3.42±0.62) while inactive MKP-3 does not change G6Pase expression (0.98±0.17). The residues 200–260 of MKP-3 and the residues 360–456 of FOXO1 are essential for mediating MKP-3/FOXO1 interaction. Interestingly, ERK phosphorylation deficient but not Akt phosphorylation deficient FOXO1 mutant lost interaction with MKP-3. Furthermore, in vivo experiments showed that Akt phosphorylation resistant FOXO1 3A mutant is sufficient to rescue the hypoglycemia caused by MKP-3 knock down in the liver of lean mice (from 141±6.78 to 209±14.64 mg/dL).Conclusions/Significance1) Critical residues mediating MKP-3/FOXO1 interaction have been identified; 2) ERK phosphorylation deficient FOXO1 mutant is as potent as Akt phosphorylation deficient FOXO1 mutant in activating transcription of gluconeogenic genes; 3) Constitutively active FOXO1 can rescue the hypoglycemic effect caused by reduced hepatic MKP-3 expression in vivo.
Highlights
Obesity is becoming a public health burden and poses as a risk factor for development of many life threatening diseases, insulin resistance and type 2 diabetes [1,2]
Phosphorylation levels on Ser256 of FOXO1 were used to examine activities of MAP kinase phosphatase 3 (MKP-3) since we previously reported that the insulin antagonizing effect of MKP-3 on gluconeogenesis was at least partially attributed to dephosphorylation of FOXO1 on Ser256
MKP-3 wild type and MKP-3 S159AS197A effectively prevented insulin stimulated phosphorylation on Ser256 of FOXO1 while MKP-3 153–381 and MKP-3 C293S did not have any effect on dephosphorylating Ser256 (Figure 1A, the bottom two panels)
Summary
Obesity is becoming a public health burden and poses as a risk factor for development of many life threatening diseases, insulin resistance and type 2 diabetes [1,2]. Impaired insulin action in obese state is reflected by increased glucose output from the liver and decreased glucose utilization by adipose tissue and muscle [3,4,5]. Dysregulation of hepatic glucose homeostasis in obesity is mainly caused by increased gluconeogenesis. Suppression of hepatic glucose output has been shown to be an effective therapeutic approach for controlling hyperglycemia in type 2 diabetes. Insulin is an important hormone for suppressing liver gluconeogenesis mainly through Akt mediated phosphorylation and inactivation of FOXO1, a transcription factor that stimulates expression of gluconeogenic genes by directly binding to the promoters [6,7,8,9]. It has been recently demonstrated that MKP-3 is a player in promoting hepatic glucose output in obese state by interacting and activating FOXO1. Reduction of hepatic MKP-3 expression is sufficient to reduce blood glucose levels in both diet-induced and genetically obese mice
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