Independent role of PP2A and mTORc1 in palmitate induced podocyte death

Highlights

• Palmitate induced insulin resistance and podocyte death is associated with PP2A and mTORc1 activation.

• Palmitate induced cell death involves alteration in inhibition of insulin induced podocin rearrangement, SIRT 1 activity and p53 degradation.

• Both mTORc1 and PP2A are independently activated under palmitate induced insulin resistance.

• The best therapeutic approach for treatment of diabetic kidney disease should involve manipulating phosphorylation of both PP2A and mTORc1.

Abstract

Molecular mechanism behind palmitate associated insulin resistance (IR) and podocyte death is not yet fully understood. The present study shows that palmitate treatment induces IR, in human urine derived podocyte-like epithelial cells (HUPECs), which is characterised by decrease in insulin-induced p-AKT, p-GSK3 β and p-ERK1/2. This impairment in insulin signalling prevents insulin induced SIRT 1 expression and deacetylation of p53. Further, palmitate treatment prevents insulin induced phosphorylation of PP2A and FOXO1 but it potentiates the phosphorylation of mTOR at Ser 2448. Interestingly, selective inhibition of PP2A, by Okadaic acid at 5 nM, restored insulin induced phosphorylation of AKT, FOXO1, SIRT1 activity and p53 degradation. However, PP2A inhibition had no effect on mTOR phosphorylation at Ser 2448. On the other hand, partial inhibition of mTORc1, by low dose of Rapamycin (1 nM) also restored phosphorylation of AKT and SIRT1 activity, whereas no significant changes were observed in insulin induced phosphorylation of PP2A after mTORc1 inhibition. To the best of our knowledge this is the first report suggesting independent role of PP2A and mTORc1 in palmitate induced IR and associated podocyte death. Therefore, the best therapeutic approach for treatment of diabetic kidney disease should involve manipulating phosphorylation of both PP2A and mTORc1.

Introduction

One of the major cause of chronic kidney disease is insulin resistance (IR) which has been shown to be associated with early stages of diabetic nephropathy [1]. Insulin resistance is marked by impaired insulin signalling and reduced glucose uptake. Lipotoxicity due to the elevated levels of free fatty acid is one of the key reasons behind this altered signalling [2], [3]. Though all the three components of glomerulus, fenestered endothelium, glomerular basement membrane and podocytes, are required for maintaining filtration barrier but recently much attention has been paid to podocyte injury which contributes to the progression of diabetic nephropathy. Podocytes form an extensive network of interdigitated foot processes forming junctions called slit diaphragms. The slit diaphragm is composed of membrane and cytoskeletal proteins like podocin, nephrin, synaptopodin, alpha-actinin-4, podoplanin and CD2AP in addition to signalling molecules and is important for the maintenance of proper filtration barrier [4], [5]. The loss in function or reduced expression of these important molecules may trigger chronic kidney disease (CKD) [6].

Like in other insulin responsive cells, GLUT1 and GLUT 4 mediate glucose uptake in podocytes. Slit diaphragm protein such as nephrin regulates glucose uptake by allowing GLUT1 and GLUT4 vesicles to fuse with plasma memberane [7]. Various mechanisms have been suggested to explain the role of hyperglycemia in podocyte death and subsequent loss. These mechanisms include production of reactive oxygen species, intracellular accumulation of sorbitol and activation of protein kinase C pathway [8], [9]. However, little is known regarding podocyte function in state of insulin resistance. Palmitate is the prevalent free fatty acid in the blood circulation and plays an important role in the development of insulin resistance in various cell types including podocytes [3], [10], [11], [12]. A strong correlation between the circulating palmitic acid and disruption of glomerular filtration barrier has been reported [12]. Sieber et al; suggested an interesting mechanism for podocyte death via endoplasmic reticulum stress [13]. However, they did not address insulin signalling and its role in podocyte death.

Recently, the involvement of protein phosphatase PP2A has been shown in the development of insulin resistance [14]. Further, in experimental models of gluco-lipotoxicity and diabetes, hyperactivity of PP2A has been observed [15]. Pharmacological inhibition of PP2A by okadaic acid improves insulin sensitivity in hepatocytes [14]. However, there are no reports regarding role of PP2A in podocyte death. An interesting report provided evidence that mammalian target of rapamycin (mTOR) is the strongest determinant of podocyte function and also of diabetic nephropathy and it has also been found that reduction of mTORc1 activity is important for both lowering ER stress and maintaining intact localization of the slit diaphragm proteins [16]. mTOR forms two functionally distinct complexes, mTORc1 and mTORc2 which favours insulin resistance and insulin sensitivity respectively [17]. mTORc1 phosphorylates and activates the ribosomal S6 kinases (S6K1 and S6K2), which are required for the translation of a group of mRNAs possessing a 5′ terminal oligopyrimidine tract (5′TOP). In addition, mTOR phosphorylates and inactivates the binding protein of eukaryotic translation initiation factor 4E (4E/BP) which leads to 4E-mediated translation of mRNA species possessing a 5' 'cap. mTOR may have a pleiotropic function in the regulation of cell death. Serine 2448 phosphorylation of mTOR indicates mTORc1 activity and serine phosphorylation at serine 2441 indicates mTORc2 activity [18]. However, role of mTORc1 is poorly characterised in podocyte death which is induced by insulin resistance.

Taken together, in the present study we have addressed two major questions: (i) Whether palmitate induced insulin resistance promotes podocyte death (ii) and what is the role of PP2A and mTOR in insulin resistance induced podocyte cell death.