Genetic ablation of PRAS40 improves glucose homeostasis via linking the AKT and mTOR pathways
Graphical abstract
Introduction
In the United States, 29.1 million people or 9.3% of the population suffer from diagnosed or undiagnosed diabetes [1]. Diabetes is characterized by hyperglycemia mainly due to defects in insulin secretion and/or action [2], [3], [4]. Various studies have reported the identification of signaling molecules that mediate diabetic responses, however, only few have confirmed it in in vivo to be critical for normal glucose homeostasis [5], [6], [7]. Binding of insulin to its receptor on insulin responsive tissues such as skeletal muscle, fat, and liver, stimulates phosphorylation and activation of the intrinsic tyrosine kinases, leading to the recruitment and phosphorylation of members of the insulin receptor substrate (IRS) protein family (IRS1–4) [8]. Once activated, these docking proteins such as IRS1 activate downstream signaling cascades, like the phosphatidylinositol-3-kinase (PI3K) pathway. Products of PI3K attract serine kinases to the plasma membrane, including phosphoinositide-dependent kinase (PDK1). PDK1 can then access and phosphorylate the T308 residue of the serine–threonine protein kinase AKT (or protein kinase B, or PKB) [9], providing a mechanism for insulin to control glucose uptake in various tissues [10], [11]. Activation of both sites of AKT/PKB (T308 and S473) has been implicated in glucose homeostasis via alterations in glucose transporters [12] including the insulin responsive glucose transporter-4 (GLUT4) [13], [14]. As well, insulin-mediated AKT phosphorylation of downstream substrates has been found to be impaired in diabetic patients [15], [16].
The AKT1S1 or proline-rich AKT substrate of 40 kDa (hereafter referred as PRAS40) protein was first reported by the Roth laboratory as a 14-3-3 binding protein and a direct substrate of AKT in lysates from insulin treated hepatoma cells [17]. Subsequently, PRAS40 was identified as a substrate, inhibitor, and negative regulator of the mammalian target of rapamycin complex 1 (mTORC1) [18], [19], [20]. The mTORC1 modulates cell growth and protein synthesis by phosphorylating p70 ribosomal S6 kinase (p70S6K) and the eukaryotic initiation factor 4E-binding protein 1 (4EBP1) [18], [21]. However, PRAS40 knockdown and overexpression studies in different tissues or cell-types have reported contrasting findings on the role of PRAS40 in regulating mTORC1 [22], [23]. In support of PRAS40 as a negative regulator of mTORC1, PRAS40 knockdown was shown to increase insulin-mediated phosphorylation of the mTORC1-regulated p70S6K and 4EBP1 proteins preventing apoptotic cell injury in SH-SY5Y neuroblastoma cells [24]. In contrast, studies in other tissues report that silencing PRAS40 down-regulated mTORC1 activity by decreasing phosphorylation of p70S6K and 4EBP1 [20], [25], [26].
Since both the AKT and mTORC1 pathways have been implicated in regulating glucose homeostasis [27], [28], we hypothesized that PRAS40 may play a plausible role in diabetes by increasing blood glucose levels. Therefore, in this study, we examined glucose homeostasis in PRAS40 knockout (PRAS40−/−) mice and then determined whether disruption of PRAS40 would prevent hyperglycemia in the streptozotocin (STZ)-induced diabetes mouse model. STZ is a commonly used chemical method to spontaneously induce type 1 and type 2 diabetes models that mimic human pathological conditions [29]. By directly disrupting β-cells, the STZ-induced model is ideal for this study as it does not act through alterations in insulin signaling. Results presented here demonstrate for the first time that genetic ablation of PRAS40 in mice improves overall body glucose homeostasis, reducing glycated hemoglobin (HbA1C), activating hepatic p-AKT (T308) and mTORC1 (p-p70S6K) signaling and altering expression of glucose transporters (GLUTs).
Section snippets
Creation of PRAS40−/− mice and genotyping
PRAS40−/− mice were kindly provided by Dr. Richard Roth, Department of Chemical and Systems Biology, Stanford University and were previously described [26]. Since C57BL/6 PRAS40−/− mice were crossbred with an FVB mouse model obtained from Jackson Laboratories (Farmington, USA), the presence and copy numbers of the PRAS40 transgene in the offspring were identified by polymerase chain reaction (PCR) analysis. In brief, genomic DNA was extracted from the tail of mice using a DNeasy Blood and
Loss of PRAS40 improves glucose metabolism in mice
The genotypes of all animal subjects were confirmed by PCR. Western blots of protein extracts prepared from liver, muscle, and fat tissues of PRAS40−/− as compared to PRAS40+/+ mice confirmed PRAS40 deletion in these tissues (Fig. 1A). Besides minor non-significant differences in body weight, PRAS40−/− mice show no detectable phenotype differences such as growth, shape, size, hair color, or litter size. Moreover, PRAS40−/− mice from heterozygous matings are born at the expected Mendelian ratio,
Discussion
This study demonstrates for the first time that PRAS40 deletion in a whole mouse model improves body glucose homeostasis. Mechanistically, it was observed that this was due to increased activation of hepatic AKT and mTORC1 signaling through differential expression of glucose transporters (GLUTs). Although not statistically significant, PRAS40−/− mice exhibit mildly lower random blood glucose levels despite lower serum insulin levels (Fig. 1B and D). As well, PRAS40−/− mice exhibit a slightly
Grants
This study was supported by the T. J. Long School of Pharmacy & Health Sciences, University of the Pacific as well as by a Scholarly/Artistic Activity Grant from the University of the Pacific.
Disclosures
No conflicts of interest, financial or otherwise, are declared by the authors.
Acknowledgements
The authors thank Dr. Richard A. Roth (Department of Physiology and Pharmacology, Stanford University) for providing the PRAS40−/− mice and valuable discussion. Dr. Roshanak Rahimian (Department of Physiology and Pharmacology, University of the Pacific, T.J. Long School of Pharmacy and Health Sciences) for her esteemed diabetes expertise. The authors gratefully acknowledge Dr. Joan Lin-Cereghino, Dr. Geoff Lin-Cereghino, Dr. Lisa Wrischnik (Department of Biological Sciences, University of the
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