The p66^Shc Protein Mediates Insulin Resistance and Secretory Dysfunction in Pancreatic β-Cells Under Lipotoxic Conditions

The loss of pancreatic β-cell functional mass is a necessary and early event during the development of type 2 diabetes, as well as a potential target for the treatment and potential cure of type 2 diabetes (1). Insulin-secreting β-cells are targeted by insulin itself, which acts in an autocrine manner to promote β-cell viability and function (2). Constitutively secreted insulin is necessary to maintain β-cell glucose sensitivity (3), and alterations in insulin receptor and insulin receptor substrate 1 (IRS-1) result in secretory dysfunction and glucose intolerance (2). However, the physiological relevance of autocrine insulin activity remains somewhat controversial because of the various insulin doses and glucose levels used to assess β-cell secretory function (2,4).

Several metabolic stressors can result in impaired insulin activity or insulin resistance in pancreatic β-cells, as shown in other insulin-targeted tissues. The exposure of pancreatic β-cells to high glucose levels induces the c-Jun N-terminal kinase (JNK)– and extracellular signal-regulated kinase 1/2–mediated inhibitory serine phosphorylation of IRS-1, resulting in inhibition of insulin signaling and consequent impairment of insulin’s ability to regulate its own biosynthesis (5). Similarly, chronic exposure of β-cells to elevated free fatty acid levels, particularly long-chain saturated fatty acids (SFAs), can induce the JNK-mediated phosphorylation of IRS-1/2, inhibiting insulin-induced insulin gene transcription (6).

The Src homology 2 domain-containing transforming protein 1, 66-kDa isoform (p66^Shc), protein is a redox enzyme capable of sensing and generating reactive oxygen species, which also plays a role in metabolic dysfunction. In previous work (7), we described for the first time the proapoptotic role of p66^Shc in INS-1E cells, which depends on its Ser³⁶ phosphorylation. Moreover, p66^Shc expression levels were found to be increased in INS-1E cells exposed to SFAs, in pancreatic islets isolated from mice fed a high-fat diet, and in human pancreatic islets isolated from overweight/obese subjects (7). Previously, p66^Shc has been implicated in the development of obesity-induced insulin resistance through the ribosomal protein S6 kinase (S6K)/IRS-1/protein kinase B (AKT) pathway in endothelial cells and adipose tissue (8,9).

To date, the effects of SFAs and excess body fat on insulin action and signaling in pancreatic β-cells have not been explored, and the role of p66^Shc in this process has not been investigated. In this study, we provide evidence that p66^Shc is crucially involved in lipotoxicity-induced β-cell insulin resistance.

Human islets were isolated from pancreata obtained from multiorgan donors at the Islet Cell Laboratory of the University of Pisa or from pancreatic fragments obtained from patients undergoing pancreatectomy to treat tumors in the ampulla of Vater at the Division of General Surgery of the University Hospital Polyclinic in Bari. Human pancreatic tissues were processed with the approval of the local ethics committee in Pisa and Bari, respectively, after informed consent was obtained. Anonymized patient information is provided in the Human Islets Checklist (Supplementary Material). Twenty male 4–8-week-old C57BL/6 mice were purchased from Charles River Laboratories (Calco, Lecco, Italy). Animal experiments were conducted after obtaining approval from the Ethics Committee of the Genetic Research Centre “Gaetano Salvatore” BioGeM, in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth Edition, and the regulations established in Italy and the European Union for animal experiments. Mouse and human pancreatic islets were isolated and cultured as described in Li et al. (10), Lupi et al. (11), and the Supplementary Methods.

Rat insulin secreting INS-1E cells (passage 15–30) were a gift from C.B. Wollheim (University of Geneva, Geneva, Switzerland). INS-1E cells were cultured as described in the Supplementary Methods.

Human (preparation 13–17 in the Human Islets Checklist [Supplementary Material]) and mouse islets and INS-1E cells were treated with 0.5 mmol/L palmitate or oleate solution (Sigma-Aldrich Inc., St. Louis, MO) or with the same volume of a 10% w/v fatty acid–free BSA solution as a control. After 24 h, islets and cells were stimulated with 10–100 nmol/L human recombinant insulin (Roche Diagnostics, Mannheim, Germany) for different times. To achieve p66^Shc knockdown, INS-1E cells were transfected with 100 nmol/L p66^Shc-targeted siRNA (QIAGEN, Hilden, Germany) using Lipofectamine RNAiMAX reagent and Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA) for 48 h (7). The overexpression of p66^Shc was achieved by transducing INS-1E cells with recombinant adenoviruses carrying a construct encoding the p66^Shc protein (7). Where indicated, INS-1E cells were transfected with an adenoviral p66^Shc construct harboring a Ser³⁶ to Ala³⁶ mutation. An empty adenovirus vector was used as control (mock). See the Supplementary Methods for further details.

After a wash with PBS 1×, murine and human pancreatic islets and INS-1E cells were incubated in Krebs-Ringer bicarbonate HEPES buffer (KRBH) (0.1% w/v BSA, 3 mmol/L glucose, 114 mmol/L NaCl, 4.4 mmol/L KCl, 1 mmol/L MgSO₄, 29.5 mmol/L NaHCO₃, 1.28 mmol/L CaCl₂, and 10 mmol/L HEPES, pH 7.4, all from Sigma-Aldrich) (12) for 100 min, followed by stimulation with insulin (10–100 mol/L) diluted in fresh KRBH solution for 40 min. To evaluate insulin content, cells were washed twice with PBS 1×, then mechanically lysed in a nondenaturing lysis buffer (13). The level of C-peptide released in the medium, as a measure of endogenously produced insulin, and insulin content were measured using ELISAs specific for mouse (Shibayagi Co., Ltd., Ishihara, Japan), humans, and rats (Mercodia AB, Uppsala, Sweden).

Cell lysates were obtained and analyzed by immunoblotting assays (13) (Supplementary Methods). Supplementary Table 1 lists the antibodies used.

RNA isolation, cDNA synthesis, and quantitative real-time PCR were conducted as previously reported (13) (Supplementary Methods). Supplementary Table 2 lists the primer sequences.

Data are presented as the mean ± SEM. Statistical analysis was performed using the two-tailed unpaired Student t test. Significance was set at P < 0.05.

The data sets generated and/or analyzed during the study are available from the corresponding author upon reasonable request. No applicable resources were generated or analyzed during this study.

Insulin stimulation increased insulin content and C-peptide secreted levels in INS-1E cells (Fig. 1A and B) and human (Fig. 1C and D) and murine (Fig. 1E and F) pancreatic islets. This was associated with increased gene expression levels of insulin 1, but not insulin 2, in INS-1E cells (Supplementary Fig. 1).

Prior exposure of cells and islets to the SFA palmitate impaired the stimulatory effects of insulin on its own cellular content (Fig. 1A, C, and E) and C-peptide secretion (Fig. 1B, D, and F). This occurred also in INS-1E cells exposed to the unsaturated fatty acid oleate (Supplementary Fig. 2A and B).

The p66^Shc silencing approach resulted in a 40–50% reduction of p66^Shc protein levels in all experimental conditions compared with control cells treated with vehicle only (Fig. 2A). Under these conditions, the palmitate-induced impairment of the insulin stimulatory effect on its own cellular content was largely prevented (Fig. 2B). In addition, p66^Shc knockdown resulted in increased insulin-induced C-peptide secretion in both the absence and presence of palmitate compared with control cells (Fig. 2C).

Conversely, p66^Shc overexpression in INS-1E cells resulted in a marked increase of p66^Shc protein levels (Fig. 2D) and in the inhibition of the ability of insulin to augment insulin content (Fig. 2E) and secreted C-peptide levels (Fig. 2F) in cells not exposed to palmitate. In the presence of palmitate, these effects of insulin were impaired further (Fig. 2E and F).

Following overexpression of the p66^ShcAla³⁶ mutant protein (Fig. 2D), which is unable to undergo phosphorylation at the key Ser³⁶ site (7), the palmitate-induced impairment of the effects of insulin to enhance its own content (Fig. 2E) and C-peptide secretion (Fig. 2F), respectively, were no longer observed.

In islets (preparation 1–12 in the Human Islets Checklist [Supplementary Material]) obtained from overweight/obese subjects, p66^Shc mRNA levels were higher than in islets from lean subjects (Fig. 2G). In addition, the effects of insulin to increase insulin content (Fig. 2H) and secreted C-peptide (Fig. 2I) were blunted.

The inhibition of JNK (Fig. 3A) and tumor suppressor protein 53 (p53) protein activity (Fig. 3E) reduced palmitate-induced p66^Shc phosphorylation at Ser³⁶ (Fig. 3B) and p66^Shc protein expression (Fig. 3F), respectively, and prevented the palmitate effects on insulin-induced insulin content (Fig. 3C and G) and C-peptide secretion (Fig. 3D and H). Of note, the reduction in reactive oxygen species levels impaired insulin effects under both basal and palmitate-stimulated conditions (Supplementary Fig. 3).

Palmitate treatment increased S6K Thr³⁸⁹ phosphorylation (Fig. 4A) and IRS-1 Ser³⁰⁷ phosphorylation, resulting in both reduced IRS-1 protein levels (Fig. 4B) and impaired ability of insulin to stimulate AKT Ser⁴⁷³ phosphorylation (Fig. 4C). However, in β-cells with p66^Shc knockdown, the effects of palmitate on phosphorylation of S6K (Fig. 4A) and IRS-1 (Fig. 4B) proteins were abrogated, and this enhanced insulin-stimulated AKT Ser⁴⁷³ phosphorylation (Fig. 4C). Conversely, p66^Shc overexpression did not alter S6K Thr³⁸⁹ phosphorylation (Fig. 4D) and exacerbated the palmitate-mediated IRS-1 Ser³⁰⁷ phosphorylation and reduction of IRS-1 protein levels (Fig. 4E), leading to complete abrogation of insulin-stimulated AKT Ser⁴⁷³ phosphorylation (Fig. 4F). Of note, p66^Shc overexpression also increased IRS-1 Ser³⁰⁷ phosphorylation, reduced IRS-1 protein levels (Fig. 4E), and impaired insulin-stimulated AKT Ser⁴⁷³ phosphorylation (Fig. 4F) in cells not exposed to palmitate.

We show that acute insulin stimulation increased cellular insulin content and C-peptide release in rat INS-1E cells and human and murine pancreatic islets. This occurred independently of glucose interference, as all experiments were conducted under low-glucose conditions. Moreover, both exposure to palmitate in vitro and presence of excess body fat in vivo resulted in the impaired ability of insulin to stimulate its biosynthesis and secretion in β-cells and murine and human pancreatic islets in vitro and in human pancreatic islets ex vivo, respectively. These results mirror and corroborate the previous observation of blunted insulin-induced insulin gene transcription following exposure of mouse pancreatic islets to palmitate (6). Our results in INS-1E cells indicate that palmitate alters the insulin signaling pathway by activating the phosphorylation of Thr³⁸⁹ in the S6K protein and the inhibitory phosphorylation of Ser³⁰⁷ in IRS-1, which are known to result in reduced IRS-1 protein levels and impairment of insulin-induced AKT activation (9,14). These results support the emerging concept that lipotoxicity is capable of inducing an insulin resistant state in pancreatic β-cells similar to other classical insulin target tissues.

Importantly, we show for the first time that p66^Shc plays a key role in this lipotoxicity-mediated insulin resistance in pancreatic β-cells, since p66^Shc knockdown and overexpression respectively prevented and worsened palmitate-induced insulin resistance by reducing and boosting palmitate-induced activation of the S6K/IRS-1/AKT pathway (Fig. 4G). These results resemble the effects of lipotoxicity in endothelial and adipose cells (8,9). Experiments with overexpression of the wild-type and mutant (Ala³⁶) forms of p66^Shc suggested that p66^Shc phosphorylation at Ser³⁶ is required to mediate the effects of palmitate on insulin action. Furthermore, we found that palmitate-induced insulin resistance in INS-1E cells is mediated by p53 and JNK proteins. These results resemble the mechanisms already demonstrated for palmitate-induced β-cell apoptosis (7).

The ex vivo experiments performed in human pancreatic islets isolated from donors without diabetes support the involvement of p66^Shc in lipotoxicity-induced β-cell insulin resistance in human obesity, as both elevated p66^Shc mRNA expression and impaired insulin effects were observed in islets obtained from overweight/obese subjects compared with islets obtained from lean subjects. Overall, p66^Shc can be identified as a crucial mediator of lipotoxicity-promoted β-cell failure in the context of human obesity. Interestingly, elevated p66^Shc levels have also been found in the liver of patients with alcoholic and nonalcoholic steatohepatitis (15,16), in the heart in response to cardiac dysfunction (17) and hyperglycemia (18), and in endothelial cells exposed to dyslipidemia (19), hyperglycemia (20), and proinflammatory cytokines (21). Taken together, this evidence suggests that p66^Shc is ubiquitously involved in multiorgan damage in response to metabolic stressors.

The physiological significance of the effects of autocrine basal insulin in vivo are still debated (2,4,22–24), even though under low-glucose conditions insulin could regulate its own content and release, representing a physiological mechanism to replenish insulin stores and make β-cells prone to rapidly satisfy insulin demand in response to secretagogues (3,22,25–27). Of note, insulin signaling in β-cells can be triggered by insulin binding at high concentrations to insulin receptor, IGF-I receptor, or insulin receptor/IGF-I receptor hybrids (28). It has been suggested that insulin promotes its own biosynthesis and secretion mainly through insulin receptor/IRS-1 signaling (29–31). On the other hand, insulin binding to IGF-I receptor mainly leads to the activation of IRS-2, which could play a major role in the regulation of β-cell mass (32,33).

In conclusion, p66^Shc mediates lipotoxicity-induced β-cell insulin resistance, an emerging mechanism for β-cell failure in obesity and type 2 diabetes. Of interest, glucagon-like peptide 1 receptor agonists, which reportedly inhibit palmitate-induced apoptosis by preventing JNK phosphorylation (34), also inhibited p66^Shc phosphorylation on Ser³⁶ (7) and may thus use this mechanism as well to enhance insulin secretion. Since the loss of pancreatic β-cell functional mass is a necessary and early event in type 2 diabetes (1), the combined effects of p66^Shc on β-cell secretory function and survival suggest that this protein may represent a potential target for the prevention or treatment type 2 diabetes onset or progression.

Funding. This work was supported by COMPETITIVE–Claims of Olive Oil to Improve the Market Value of the Product (AGER 2 Project) grant 2016-0174), the Ministero dell’Università e della Ricerca of Italy (Progetti di Rilevante Interesse Nazionale, 2015), European Union (European Social Fund, Programma Operativo Nazionale “Ricerca e Innovazione” 2014–2020, Attrazione e Mobilità Internazionale dei Ricercatori) grant 1810057, and Fondazione per la Ricerca Biomedica Saverio e Isabella Cianciola.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. G.B., N.M., L.D., A.B., M.R., R.D., V.A.G., and C.C. contributed to the formal analysis and investigation. G.B., N.M., L.D., A.N., and F.G. contributed to the data curation. I.P., P.M., and L.V. contributed resources. A.C., S.P., and L.L. contributed to the visualization. L.L., F.G., and A.N. provided supervision. F.G. and A.N. contributed to the conceptualization and project administration. F.G. reviewed and edited the manuscript. A.N. wrote the draft of the manuscript. All of authors contributed to conception and design; acquisition, analysis, and interpretation of data; critical revision of the manuscript for important intellectual content; and final approval of the version to be published. F.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the 79th Scientific Sessions of the American Diabetes Association, San Francisco, CA, 7–11 June 2019; 40th National Meeting of the Italian Society of Endocrinology, Rome, Italy, 29 May–1 June 2019; 27th National Meeting of the Italian Society of Diabetology, Rimini, Italy, 16–19 May 2018; 53rd Annual Meeting of the European Association for the Study of Diabetes, Lisbon, Portugal, 11–15 September 2017; International Symposium on Insulin Receptor and Insulin Action, Nice, France, 20–22  April 2017; and 39th National Meeting of the Italian Society of Endocrinology, Rome, Italy, 21–24 June 2017.