The Phosphatase PHLPP2 Plays a Key Role in the Regulation of Pancreatic Beta-Cell Survival

Currently available antidiabetic treatments fail to halt, and may even exacerbate, pancreatic β-cell exhaustion, a key feature of type 2 diabetes pathogenesis; thus, strategies to prevent, or reverse, β-cell failure should be actively sought. The serine threonine kinase Akt has a key role in the regulation of β-cell homeostasis; among Akt modulators, a central role is played by pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) family. Here, taking advantage of an in vitro model of chronic exposure to high glucose, we demonstrated that PHLPPs, particularly the second family member called PHLPP2, are implicated in the ability of pancreatic β cells to deal with glucose toxicity. We observed that INS-1 rat pancreatic β cell line maintained for 12–15 passages at high (30 mM) glucose concentrations (INS-1 HG) showed increased expression of PHLPP2 and PHLPP1 both at mRNA and protein level as compared to INS-1 maintained for the same number of passages in the presence of normal glucose levels (INS-1 NG). These changes were paralleled by decreased phosphorylation of Akt and by increased expression of apoptotic and autophagic markers. To investigate if PHLPPs had a casual role in the alteration of INS-1 homeostasis observed upon chronic exposure to high glucose concentrations, we took advantage of shRNA technology to specifically knock-down PHLPPs. We obtained proof-of-concept evidence that modulating PHLPPs expression may help to restore a healthy β cell mass, as the reduced expression of PHLPP2/1 was accompanied by a recovered balance between pro- and antiapoptotic factor levels. In conclusion, our data provide initial support for future studies aimed to identify pharmacological PHLPPs modulator to treat beta-cell survival impairment. They also contribute to shed some light on β-cell dysfunction, a complex and unsatisfactorily characterized phenomenon that has a central causative role in the pathogenesis of type 2 diabetes.


Introduction
Type 2 diabetes (T2D) is a complex disease, brought about by the combination of abnormalities in both the production and the function of the pancreatic hormone insulin [1]. Although classically these two defects were seen as separate entities, in the last decades, it has become evident that they share common pathogenetic mechanisms, with insulin regulating not only glucose utilization from peripheral target tissues, but also its own synthesis and secretion as well as the maintenance of an adequate β-cell mass [1][2][3]. Notably, while impaired insulin action in peripheral tissue-the socalled "insulin resistance"-remains fairly constant as the disease progresses, β-cell function worsens continuously with time in diabetic patients, as a consequence of the persisting exposure to damaging factors, such as high glucose concentrations (glucose toxicity), increased levels of circulating free fatty acids (lipotoxicity), and proinflammatory cytokines (chronic inflammation) [2][3][4][5]. Furthermore, currently available antidiabetic treatments fail to halt, and may even exacerbate, pancreatic β-cell exhaustion; thus, despite promising observations with molecules belonging to the more recently introduced therapeutic classes [6], strategies to prevent, or reverse, β-cell failure should still be actively sought. ere are two primary components to β-cell dysfunction in T2D: impaired insulin secretion and reduced β-cell mass. In adult humans, the rate of new β-cell formation is low, and the maintenance of an adequate mass is achieved mainly throughout a tight regulation of apoptotic rates [7]. e serine threonine kinase Akt, also known as protein kinase B (PKB), has a key role in the regulation of β-cell homeostasis. Akt exists in three isoforms that are considered indistinguishable in their domain architecture and upstream regulation but are nonredundant in their expression patterns and biological functions [8,9]. Specifically, all three isoforms have been detected in pancreatic β-cells; with studies in knock-out mouse models suggesting that Akt1 regulates mainly β-cell survival, Akt2 is required to modulate the insulin secretory response, while Akt3 loss does not appear to significantly alter either β-cell mass or function [9]. e three Akt isoforms are activated by sequential phosphorylation at two key sites; the phosphorylation of the first residue, located in a segment called the activation loop ( reonine 308/309/307 in Akt1/2/3, respectively), triggers the phosphorylation of a site located in the carboxyl-terminal domain, termed the hydrophobic phosphorylation motif (Serine 473/474/472 in Akt1/2/3 respectively) [8]. We and others have reported decreased Akt activation upon exposure to glucotoxicity, lipotoxicity, and/ or chronic inflammation [4,[9][10][11][12][13][14]. Akt inhibition is mediated by dephosphorylation by two protein phosphatases: protein phosphatase 2A that acts on the threonine residue [15] and pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) family targeting the serine residue [16][17][18]. PHLPP proteins appear to be ubiquitously expressed, with particularly high levels in the brain, and their increased expression has been reported in numerous cancer cell lines, including cancers of the brain, breast, lung, prostate, and ovary. e PHLPP family comprises two members: PHLPP1, which in turn exists in two splice variants, (α and β), and PHLPP2 (also known as PHLPPL). e three isozymes share very similar domain structure but have a certain degree of substrate specificity, with PHLPP1 preferentially targeting Akt2 and 3 and PHLPP2 showing a higher affinity toward Akt1 and 3 [18]. A few years ago, we have shown increased PHLPP1 expression in adipose tissue and skeletal muscle biopsies from obese, insulin-resistant subjects and hypothesized that PHLPPs may represent an additional player in insulin resistance [14]. Here, we investigated if PHLPPs may be implicated in the ability of pancreatic β cells to deal with chronic exposure to high glucose concentrations in an in vitro model. We also aimed to obtain proof-of-concept evidence that modulating PHLPPs expression may help to restore a healthy β cell mass.

Total RNA Extraction and Real-Time Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR).
Total RNA was extracted from INS-1 NG and INS-1 HG using Trizol (Life Technologies, Gaithersburg, MD), reverse transcribed and analyzed by RT-qPCR using a Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Results were normalized to β-actin levels according to the Livak method, as previously described [21][22][23]. Primers sequences are available upon request.

Insulin Stimulation and Western Blot Analysis.
To assess insulin-stimulated protein phosphorylation, INS-1 were serum-starved for 48 hrs with FBS-free medium containing bovine serum albumin and glucose at the appropriate concentrations; human insulin (10 − 7 M) was then added, when indicated, for 7 minutes before cell lysis in a buffer containing 1.5% NP-40. Cell lysates were processed and analyzed by Western Blot, according to previously established methods [14,20]. A home-made primary antibody generated and validated by our research group [14] was employed to detect PHLPP1 levels; anti-PHLPP2 antibody was obtained from Abcam (Cambridge, United Kingdom). e following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-Bad, anti-Bcl-xL, anti-cleaved caspase-3, anti-LC3-II, anti-total and phospho Akt, anti-total and phospho FoxO1, and anti-total and phospho mTor. Equal protein loading was confirmed by reblotting the membranes with monoclonal antibody against β-actin (Sigma-Aldrich; Milan, Italy); p-Akt/Akt, p-FoxO1/ FoxO1, and pmTor/mTor ratios were calculated to analyze the relative phosphorylation levels. Densitometric analysis was performed using a ImageJ software (NIH, USA).

Insulin Secretion
Assay. INS-1 cells were seeded in 24multiwell plates at a density of 10 5 cells/well in growth medium containing 11.1 mM or 30 mM glucose, as appropriate. Twenty hours before the insulin secretion assay, cells were switched to a medium containing 5 mM glucose; the medium was then replaced with a glucose-free Krebs phosphate buffer for 2 hrs. INS-1 was then incubated in the presence of increasing concentrations of glucose in fresh Krebs buffer for 20 minutes. Cell media were removed and diluted to assess insulin concentration with a specific rat insulin Elisa assay (Mercodia, Uppsala, Sweden).

Statistical Analysis.
All results were calculated as mean fold variation (±SD) over the appropriate control point. Statistical differences were assessed by Student's t test or ANOVA as indicated. A p value ≤0.05 was considered statistically significant. Analyses were performed with GraphPad Prism version 8.2.0 software (San Diego, CA, USA).

Chronic Exposure to High Glucose Concentrations Results in a Significant Increase of PHLPP2 and PHLPP1 Expression.
To mimic chronic exposure to high glucose levels, we cultured INS-1 rat pancreatic β-cells at 30 mM glucose for . is glucose concentration has been previously shown to efficaciously induce glucose toxicity in pancreatic β-cell lines that require culture media containing 11.2 mM glucose for their normal growth [24]. Since PHLPP dephosphorylates and inactivates Akt kinase [16][17][18], we assessed Akt phosphorylation on the activation loop residue, Ser473, which is directly targeted by

Infection with shRNA Constructs Against PHLPP2/1 Resulted in a Significant Knock-Down of the Expression of Both
Isoenzymes. To investigate if PHLPPs had a casual role in the alteration of INS-1 homeostasis observed upon chronic exposure to high glucose concentrations, we took advantage of shRNA technology to specifically knock-down PHLPPs, employing a previously validated protocol [20]. We choose to concurrently downregulate both PHLPP family member, since the expression of both appeared increased upon chronic exposure to high glucose concentrations, and specifically reducing only one isoenzyme may have induced compensatory overexpression of the cognate protein, confounding the data interpretation. To this end, we employed a mixture containing two adenoviral vectors encoding for a shRNA against PHLPP1 or against PHLPP2 in a 1 : 1 ratio. We initially performed a dose-response curve and observed that a dose of at least 4.5 × 10 5 PFU was required to consistently reduce PHLPP2 levels; doubling this dose, we obtained an almost complete PHLPP2 knock-down (Figure 3(a), p � 0.0001, as compared to mock-infected INS- 1 HG cells). We then tested if these two concentrations resulted in PHLPP1 expression inhibition and observed a 15% reduction of PHLPP1 levels with the 4.5 × 10 5 PFU (p � 0.04) and a 85% decrease with the 9 × 10 5 PFU dose (p � 0.0001) (Figure 3(b)). Higher viral doses resulted in a notable and rapid decrease in cell viability; all subsequent  experiments were thus carried out with the above-men-

Conclusions
In the present study, we report that the altered pancreatic β-cell homeostasis observed upon chronic exposure to 30 mM glucose is paralleled by increased expression of PHLPPs, with a consequent reduction of the phosphorylation levels of their primary target, the serine threonine kinase Akt. Interestingly, knocking-down PHLPPs, throughout adenoviral-mediated shRNA delivery, we were able to restore a prosurvival profile in INS-1 HG cells chronically exposed to high glucose levels. Specifically, the ratio between the proapoptotic factor Bad and its prosurvival counterpart Bcl-xL went back to the value measured in healthy INS-1 NG cells. Bad levels have been suggested to be directly regulated by Akt pathway [25], and indeed, we observed significantly increased phosphorylation of Akt and of its major antiapoptotic effector FoxO1 [26] in INS-1 HG infected with the adenoviral constructs encoding for specific shRNA sequences against PHLPP2 and PHLPP1. e insulin-stimulated activation of another Akt substrate mTor was also significantly improved when PHLPPs expression was knocked down. mTor senses nutrient availability and regulates cell homeostasis, and it has been suggested that its loss impairs β-cells homeostasis as well as insulin sensitivity in peripheral tissues [27]. Among the intracellular processes controlled by mTor, autophagy as gained attention as a possible player in the survival of pancreatic β-cells with conflicting data showing, in different experimental models, pro-or antiapoptotic effects of an increased activation of autophagic pathways [28][29][30]. In our model, the recovered prosurvival profile was paralleled by decreased expression of the autophagic marker LC3-II, supporting the hypothesis of a negative impact of a disproportionated activation of autophagic pathways on cell survival.
At odds with the restored cell survival profile, efficient glucose-stimulated insulin secretion and synthesis were not recovered by INS-1 HG infected with adenoviral vectors carrying the shRNA sequences against PHLPP2/1. It has been reported that Akt isoforms have different roles in pancreatic β-cells, with Akt1 mainly controlling cell survival and Akt2 mostly involved in the regulation of insulin secretion [9], and it has also been suggested that PHLPP family members possess a selective preferences toward Akt isoforms, as PHLPP2 seems to prefer Akt1 and PHLPP1 favors Akt2, even if this may depend on the predominantly expressed substrate isoform [17,18]. Since we obtained an almost complete silencing of PHLPP2 in INS-1 cells infected with the highest adenoviral concentration (INS-1 HG AV-HD), while the maximal reduction of PHLPP1 achieved was around 65%, it may be possible to 8 International Journal of Endocrinology hypothesize that Akt2 function, and consequently insulin secretion and synthesis, was less efficiently restored. However, the observation that no significant difference was observed when comparing INS-1 HG AV-HD, expressing 45% of PHLPP1 with respect to mock-infected INS-1 HG, with cells infected with a lower adenoviral titer with PHLPP1 levels around 85% of those observed in mock INS-1 HG (INS-1 HG AV-LD), renders this explanation quite unlikely and rather points to the possibility that glucose toxicity may more profoundly damage β-cell function than β-cell survival, causing a depletion of insulin deposits that may not be restored with a short-term improvement of Akt activation [31,32]. Indeed, we believe that any attempt to restore β-cell homeostasis in a in vivo setting should not overlook the important difference among the mechanisms regulating β-cell mass and those regulating specific β-cell functions such as insulin synthesis and secretion. e lack of significant differences between INS-1 HG AV-LD and INS-1 HG AV-HD cells for the majority of functional and molecular read-out analyzed also suggests that PHLPP2 may play a more pivotal role than the cognate protein, PHLPP1, in the dysregulation of beta-cell survival. Our data thus contribute a small piece of knowledge to the comprehension of the specific functions of PHLPP family members and to the less explored mechanisms regulating their own expressions [18,33]. Interestingly, a few years ago, we and others showed a specific increase of PHLPP1, with unaltered PHLPP2 levels, in adipose tissue and skeletal muscle biopsies of obese, insulin-resistant individuals [14,34]; these data have been more recently confirmed by Behera et al. in a high-fat fed animal model [35]. Here, we report a significant increase of both isoenzymes upon exposure to high glucose concentrations; however, PHLPP2 showed the larger and more statistical sound changes. e results of the shRNA-mediated inhibition experiments confirm a preeminent role of PHLPP2 in the regulation of pancreatic β-cells homeostasis.
Furthermore, in the older study, we did not observe any direct correlation between PHLPP1 expression and glucose levels, while the agent of the increased expression appeared to be insulin levels. In contrast in the present study, PHLPPs levels were increased in response to high glucose concentration, even if our data do not allow to establish if glucose is able to directly promote PHLPP transcription or transduction or if the increase in PHLPP protein levels was mediated by indirect mechanisms [14]. Nonetheless our results underlie that PHLPPs possess a specificity that has not been fully explored to date and may be differentially regulated in different tissue. Clarifying this point is mandatory in order to exploit these phosphatases as possible pharmacological targets.
In conclusion, our data provide initial support for future studies aimed to identify pharmacological PHLPPs modulator to treat beta-cell survival impairment.
ey also contribute to shed some light on β-cell dysfunction, a complex and unsatisfactorily characterized phenomenon that has a central causative role in the pathogenesis of type 2 diabetes.

Data Availability
All data used to support the findings of the study are included within the manuscript.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.