Prion protein modulates glucose homeostasis by altering intracellular iron

The prion protein (PrPC), a mainly neuronal protein, is known to modulate glucose homeostasis in mouse models. We explored the underlying mechanism in mouse models and the human pancreatic β-cell line 1.1B4. We report expression of PrPC on mouse pancreatic β-cells, where it promoted uptake of iron through divalent-metal-transporters. Accordingly, pancreatic iron stores in PrP knockout mice (PrP−/−) were significantly lower than wild type (PrP+/+) controls. Silencing of PrPC in 1.1B4 cells resulted in significant depletion of intracellular (IC) iron, and remarkably, upregulation of glucose transporter GLUT2 and insulin. Iron overloading, on the other hand, resulted in downregulation of GLUT2 and insulin in a PrPC-dependent manner. Similar observations were noted in the brain, liver, and neuroretina of iron overloaded PrP+/+ but not PrP−/− mice, indicating PrPC-mediated modulation of insulin and glucose homeostasis through iron. Peripheral challenge with glucose and insulin revealed blunting of the response in iron-overloaded PrP+/+ relative to PrP−/− mice, suggesting that PrPC-mediated modulation of IC iron influences both secretion and sensitivity of peripheral organs to insulin. These observations have implications for Alzheimer’s disease and diabetic retinopathy, known complications of type-2-diabetes associated with brain and ocular iron-dyshomeostasis.


Most of the PrP C in pancreatic β-cells is cleaved at the β-site.
Under physiological conditions, PrP C expressed on neuronal cells recycles from the plasma membrane and undergoes α-cleavage in an endocytic compartment, resulting in the generation of C1 27 . Cleavage at the β-site is typical of disease-associated PrP-scrapie (PrP Sc ) 14,[29][30][31] , and is triggered by exposure to reactive oxygen species 32 .
To evaluate whether post-translational processing of PrP C on pancreatic β-cells differs from neurons 27,33 , pancreatic lysates from C6 mice and human brain (as a representative tissue for neurons) were deglycosylated and probed with 8H4. Surprisingly, pancreatic lysates from C6 PrP +/+ samples revealed barely detectable FL PrP C , most of which was detected as C2 and C1 fragments. Human brain sample, on the other hand, showed equivalent amounts of FL and C1, and C6 PrP −/− samples did not show any reactivity as expected (Fig. 2a, lanes 1-3).
Similar results were obtained from deglycosylated pancreatic lysates from Tg40 PrP mice probed with 3F4. Majority of PrP C in the pancreas exhibited the mobility of C2. Systemic iron overload induced by injecting 36 µg/22 g mouse weight ferric ammonium citrate (FAC) upregulated FL PrP C to some extent, but the major difference was in the intensity of C2. (Fig. 2b, lanes 1-6; Fig. 2c). Pancreatic lysates from PrP −/− mice showed no reactivity with 3F4, while brain lysates revealed mainly FL PrP C and minimal reactivity for C2 (Fig. 2b, lanes 7-9; Fig. 2c). The small increase in C2 in iron over-loaded pancreas (Fig. 2c) indicated iron-mediated cleavage of PrP in vivo, a phenomenon that was investigated further in the 1.1 B4 cells in vitro. Similar results were obtained from probing the membrane with 8H4 (see Supplementary Fig. S2).
Accordingly, 1.1B4 cells expressing green-fluorescent protein (GFP) tagged PrP C (PrP C-GFP ) were exposed to FAC, and live cells were imaged every 5 min for up to 20 min. Since the GFP tag is inserted between N-terminal residues 37 and 38 of PrP 20,34 , the premise was that the tag would be lost following αor β-cleavage of PrP C . Representative images from time zero (T-0) and 20 min (T-20) time point are shown (Fig. 2d, panels 1-4). In control cells exposed to vehicle, the signal from PrP C-GFP was prominent at the plasma membrane, peri-nuclear Golgi region, and endocytic vesicles at T-0 and T-20 (Fig. 2d, panels 1 & 2). In FAC exposed cells, the distribution of PrP C-GFP at T-0 was similar to controls (Fig. 2d, panels 1 & 3). However, at T-20 most of the GFP signal was lost from the plasma membrane, though peri-nuclear and endosomal signal remained (Fig. 2d, panel 4).
(The intensity of green fluorescence in the inset was increased 5-fold in panels 3 and 4). Thus, the GFP carrying N-terminus of PrP C is lost within 20 min of exposure to FAC. Based on Western blot results in Fig. 2b and published reports 32,35 , it is likely that FAC induces β-cleavage of PrP C .
To evaluate whether β-cleavage of PrP C is coupled with iron transport through divalent metal transporter-1 (DMT-1) or members of the Zrt Irt-like protein (ZIP) family, in particular ZIP14 20,36,37 , 1.1B4 cells transfected with GFP-tagged DMT-1, ZIP8, and ZIP14 were exposed to FAC and imaged at T-0 and T-20. Unlike PrP C-GFP (Fig. 2d), there was no difference in the intensity or localization of DMT-1-GFP, ZIP8-GFP, or ZIP14-GFP following exposure to exogenous FAC (Fig. 2e, panels 1-6). However, overexpression of these transporters in 1.1B4 cells resulted in increased β-cleavage of PrP C in cells transfected with DMT-1 and ZIP14 (Fig. 2f, lanes 6 & 8; Fig. 2g). Since ZIP14 is known to mediate uptake of non-transferrin bound iron (NTBI) by pancreatic β-cells 38 (see Supplementary Fig. S1), these results suggest that PrP C functions as a FR partner for ZIP14, and is cleaved at the β-site during this process.

PrP C mediates uptake of iron by pancreatic β-cells in vitro and in vivo.
To confirm the facilitative role of PrP C in iron uptake by the pancreas 39-41 , 1.1B4 cells were transfected with siRNA to silence PrP C , and exposed to FAC. Following an incubation of 16 h, control and experimental cell lysates were processed for Western blotting and probed for ferritin. Silencing of PrP C resulted in significant downregulation of ferritin relative to non-transfected and scrambled siRNA transfected controls (Fig. 3a, lanes 1-3; Fig. 3b). Exposure to FAC caused significant upregulation of ferritin in controls as expected, but had minimal effect in the absence of PrP C (Fig. 3a, lanes 4-6); Fig. 3b).   Values are mean ± SEM of the indicated n. *Represents change in ferritin relative to untreated, non-transfected control. ## Represents change in ferritin relative to FAC exposed non-transfected control. *p < 0.05, ## p < 0.01. (c) Western blotting shows upregulation of PrP in iron-overloaded C6 PrP +/+ , no signal in C6 PrP −/− samples, and the expected glycoforms in human brain sample (lane 9). Ferritin is significantly higher in iron-overloaded relative to untreated C6 PrP +/+ samples and matched C6 PrP −/− samples. There is no change in ferritin iron overloaded C6 PrP −/− samples relative to untreated controls. Probing for TfR shows significant reduction in iron-overloaded relative to untreated C6 PrP +/+ samples and matched C6 PrP −/− samples. There is minimal change in TfR expression in iron-overloaded C6 PrP −/− samples relative to untreated controls. The above observations were substantiated in C6 PrP +/+ and C6 PrP −/− mice injected with iron to create systemic iron overload (Fig. 3c-e). Evaluation of pancreatic lysates by Western blotting revealed upregulation of PrP C in iron-overloaded C6 PrP +/+ mice as in Fig. 2b above (Fig. 3c, lanes 1, 2, 5, 6; Fig. 3d). Probing for ferritin revealed significantly less ferritin in C6 PrP −/− relative to C6 PrP +/+ controls. Overloading with iron caused significant upregulation of ferritin in C6 PrP +/+ , but not in C6 PrP −/− samples (Fig. 3c, lanes 1-8; Fig. 3d). Expression of transferrin receptor (TfR) was reduced in iron overloaded C6 PrP +/+ mice, but showed minimal change in similarly treated C6 PrP −/− mice (Fig. 3c, lanes 1-8; Fig. 3d). Similar results were observed in the Tg40 PrP and PrP −/− mice (see Supplementary Fig. S4).
Together, the above results leave little doubt that PrP C mediates iron uptake in pancreatic β-cells. Since increased systemic iron is associated with the risk of type-2-diabetes, further studies were directed at whether PrP-mediated change in pancreatic β-cell iron influences insulin production and/or secretion and blood glucose levels.
PrP C -mediated increase in intracellular iron downregulates glucose transporters in the pancreas, liver, and retina. Pancreatic β-cells sense blood glucose levels through GLUT2, a bidirectional glucose transporter, and release insulin to maintain glucose concentrations within a defined range 22,23 . To evaluate whether PrP C -mediated increase in β-cell iron alters the expression of GLUT2 (Fig. 4) and insulin (Fig. 6), pancreas from iron over-loaded Tg40 PrP and corresponding PrP −/− mice were subjected to Western blotting and immunohistochemistry ( Fig. 4a-c).
Immunohistochemistry of pancreas mirrored the Western blot results. Iron overloading decreased GLUT2 reactivity in Tg40 PrP, but had minimal effect on PrP −/− samples ( To evaluate whether PrP C -mediated increase in IC iron alters glucose transporters on neuronal cells, in particular glucose transporter 3 (GLUT3) 42 , brain lysates from Tg40 PrP and PrP −/− mice were subjected to Western blotting and probed for GLUT3. As noted in pancreatic β-cells, expression of GLUT3 was upregulated in PrP −/− relative to Tg40 PrP samples (Fig. 5a, lanes 1-4; Fig. 5b).
Further confirmation of this phenomenon was obtained in M17 cells, a neuroblastoma cell line transfected to over-expressing PrP C and the respective vector control. Immunoreaction of fixed, permeabilized cells for GLUT3 revealed significantly less reactivity in PrP C -expressing cells relative to vector controls (Fig. 5c, panels 1 & 2). Exposure to exogenous iron decreased GLUT3 reactivity in both cell lines, but significantly more in PrP C -expressing cells relative to vector controls (Fig. 5c, panels 3 & 4). These results were confirmed by Western blotting of similarly treated cell lysates. Probing for GLUT3 revealed significantly lower expression in PrP C -expressing cells relative to vector controls (Fig. 5d, lanes 2 & 3; Fig. 5e). Exposure to exogenous iron downregulated GLUT3 in both cell lines, but significantly more in PrP C -expressing cells relative to vector controls (Fig. 5d, lanes 1 & 4; Fig. 5e).
Evaluation of samples from the liver and neuroretina of Tg40 PrP and PrP −/− mice mimicked the results obtained in the pancreas, the brain, and neuronal cells. Western blotting showed downregulation of glucose transporter 1 (GLUT1) in the neuroretina and GLUT2 in the liver of Tg40 PrP relative to PrP −/− samples ( Fig. 5f and h,  lanes 1 & 3). Iron overloading resulted in the downregulation of GLUT1 and GLUT2 in Tg40 PrP mice, but not in PrP −/− mice ( Fig. 5f and h, lanes 2 & 4; Fig. 5g and i).
All the above investigation was also carried out in the C6 mice in both the strains; C6 PrP +/+ and C6 PrP −/− , with similar results in all 4 tissues (see Supplementary Fig. S5).

PrP-mediated increase in β-cell iron downregulates insulin.
To evaluate whether expression of GLUT2 influences insulin levels in β-cells, pancreatic lysates from control and iron overloaded Tg40 PrP mice were subjected to Western blotting and probed for insulin with two different antibodies 43,44 . Essentially the same observations were noted as for GLUT2 (Fig. 4). Reactivity for insulin was significantly higher in PrP −/− relative to Tg40 PrP samples. Iron overloading decreased insulin in Tg40 PrP, but had minimal effect on PrP −/− samples (Fig. 6a, lanes 1-8, upper and lower panels; Fig. 6b). This experiment was also conducted in C6 mice in both the strains; C6 PrP +/+ and C6 PrP −/− , with similar results (see Supplementary Fig. S6).
samples. *p < 0.05. Full-length blots are provided in Supplementary File (Raw data). (e) Immunoreaction of fixed pancreatic sections from the above mice for ferritin shows a positive reaction in mainly β-cell rich endocrine islets (panels 1-4). Iron over-loading increases ferritin reactivity in C6 PrP +/+ sections, but shows minimal change in C6 PrP −/− samples (panels 3 & 4). Reactivity for TfR is also localized to the endocrine islets (panels 5-8). Iron-overloading down-regulates TfR expression in C6 PrP +/+ samples (panel 6 vs. 5), but has minimal effect on C6 PrP −/− samples (panels 7 & 8). Scale bar 20 μm. Scientific  Immunoreaction of fixed pancreas for insulin showed reactivity on β-cells as expected. The intensity of insulin reactivity was higher in PrP −/− relative to Tg40 PrP samples, and excess iron reduced the reaction in Tg40 PrP but had minimal effect on PrP −/− samples, consistent with Western blot results in panel A (Fig. 6c, panels 1-4). A similar analysis for glucagon showed increased reactivity in iron overloaded Tg40 PrP samples, indicating proliferation of glucagon producing α-cells by this treatment 45 . PrP −/− samples, on the other hand, showed minimal reactivity in untreated and iron overloaded samples (Fig. 6c, panels 5-8).
To confirm a causal relationship between β-cell iron levels and insulin, 1.1B4 cells were exposed to FAC for 2 and 4 hours, and immunostained for insulin. Reactivity for insulin decreased after 2 h, and was negligible after 4 h. Nuclear staining revealed normal nuclear morphology with no obvious signs of toxicity (Fig. 6d, panels 1-3). Downregulation of PrP C by siRNA, however, reversed the negative effect of iron on insulin reactivity (Fig. 6e, panels 1-4).
Together, the above observations demonstrate that increase in β-cell iron results in downregulation of GLUT2 and insulin, and PrP C plays a significant role in this process by facilitating iron uptake.
PrP C -mediated dysregulation of blood glucose is exacerbated by iron. To evaluate whether decreased reactivity for insulin in PrP C -expressing β-cells and exaggeration of this phenotype by excess iron is due to decreased synthesis or rapid release from cells, control and iron overloaded C6 PrP +/+ and C6 PrP −/− mice were  injected with glucose or insulin according to guidelines for conducting glucose tolerance test (GTT) and insulin tolerance test (ITT) in laboratory mice 46 . Blood glucose was measured and recorded at the indicated times (Fig. 7). As expected, both mouse lines showed a spike in blood glucose after 15 min (T-15), and a gradual decline to basal levels (T-0) after 180 min (Fig. 7a). It is notable that blood glucose was significantly higher in C6 PrP +/+ samples at T-60 relative to C6 PrP −/− samples (Fig. 7a). This difference was more widespread and exaggerated following over-loading, and the blood glucose of C6 PrP +/+ mice was significantly higher than C6 PrP −/− at all the time-points tested (Fig. 7b). The results from ITT were more dramatic. Following an injection of insulin, the blood in C6 PrP +/+ mice remained significantly higher than C6 PrP −/− samples at all the time points tested regardless of iron overloading (Fig. 7c & d).
Relatively poor glucose tolerance in C6 PrP +/+ mice especially after iron overloading suggests reduced levels of circulating insulin in response to glucose, consistent with decreased levels of GLUT2 and insulin in pancreatic β-cells observed above. Impaired response of C6 PrP +/+ mice to injected insulin regardless of iron overload suggests peripheral resistance to insulin that increases after iron overload.
Together, the above observations indicate that C6 PrP +/+ mice display a phenotype of type-2-diabetes, i.e. impaired synthesis of insulin and peripheral resistance to available insulin 47 , exacerbated further by iron overload. C6 PrP −/− mice, on the other hand, are relatively resistant to iron-mediated fluctuations in blood glucose. These experiments were also conducted on Tg40 PrP and PrP −/− mice with similar trend in GTT and ITT results.

Discussion
Our data demonstrate a direct correlation between PrP C and IC iron, and a converse relationship between IC iron and glucose uptake in pancreatic β-cells, neuronal cells, hepatocytes, and the retina. In pancreatic β-cells, PrP C -mediated increase in IC iron and downregulation of GLUT2 reduced the synthesis and release of insulin. In peripheral organs, iron overloading increased peripheral resistance to insulin in a PrP C -dependent manner, suggesting a dual role of PrP C -mediated increase in systemic iron on glucose homeostasis. Experimental conditions show that stimulated iron uptake by the pancreas resulted in β-cleavage of PrP C , indicating regulation of iron uptake through this process. Together, these observations explain the mechanism underlying PrP C -mediated modulation of blood glucose 17 , and confirm the positive correlation between iron and type-2-diabetes 2 (Fig. 8). Since GLUT3 is downregulated in AD and prion disease affected brains 48,49 , conditions associated with brain iron dyshomeostasis 50 , it is likely that PrP C induces toxicity by the combined effect of oxidative stress and glucose deprivation 51 .
It was surprising that PrP C had a significant effect on IC iron in pancreatic β-cells despite relatively low expression 17 . PrP C was localized to insulin-producing β-cells in mouse pancreas, and showed the expected glycoforms as in neuronal cells. However, unlike neuronal cells, most of the PrP C on pancreatic β-cells and 1.1B4 cells was cleaved at the β-site, a processing event that was triggered by exogenous iron and over-expression of divalent metal transporters DMT-1 and ZIP14. The concomitant increase in cellular ferritin suggested that β-cleavage of PrP C is coupled with iron uptake 32 , and proteolytic cleavage of PrP C could serve to regulate iron uptake 35 . Further exploration is necessary to understand this phenomenon. The expression and functional activity of ZIP14 and DMT-1 on human and mouse pancreatic β-cells has been described recently 38,52 , and supports the above assumption.
A positive correlation between systemic iron and type-2-diabetes is well-established, and several studies have addressed the underlying mechanism 2,4 . The increase in intracellular (IC) iron down-regulates hypoxia inducible factors 1 (HIF1α) 53 , resulting in the downregulation of GLUTs. The inverse scenario where hyperglycemia results in iron dysregulation has also been studied 54 . Our data strengthen these observations, and provide a novel mechanism of glucose modulation through PrP C , a mainly neuronal protein, thereby linking both peripheral and neuronal glucose homeostasis to IC iron. Given the relatively low expression of PrP C on pancreatic β-cells in comparison to neurons, reduced expression of GLUT2 and insulin in the pancreas of Tg40 PrP relative to PrP −/− mice was intriguing, and suggested that even minor changes in β-cell IC iron are sufficient to alter glucose homeostasis. Systemic iron overload amplified this effect and increased peripheral resistance to insulin 47 , simulating the phenotype of type-2-diabetes. PrP −/− mice, on the other hand, remained unaffected, demonstrating a direct correlation between iron, GLUT2, and insulin. The dose of iron used in our study was within the recommended range for the treatment of anemia, making it unlikely that the observed effects are due to iron-induced β-cell toxicity 4 . Knock-out of ZIP14 in mouse pancreas has been reported to induce hyperinsulinemia and hypoglycemia due to iron deficiency as in PrP −/− mice, lending support to our observations 55 . Similar observations were noted in the liver of Tg40 PrP and PrP −/− mice, indicating that the inverse correlation between IC iron and glucose transport is not limited to the pancreas, and relatively low expression of PrP C is sufficient to induce this change. Our results differ from a previous report indicating impaired or delayed response of PrP −/− mice to hyperglycemia 17 , a discrepancy that is difficult to explain from our data. However, diverse observations regarding PrP C and glucose homeostasis have been reported in the literature [56][57][58][59][60][61][62] requiring further exploration on this subject.
Reduced expression of GLUT3 in brain homogenates and GLUT1 in the neuroretina of Tg40 PrP relative to PrP −/− suggests similar regulation of IC iron and glucose transporters by PrP C as in pancreatic β-cells. These observations have significant implications for neuronal cells that express high levels of PrP C and require glucose for their high metabolic demands. Since several neurodegenerative diseases including AD and Creutzfeldt-Jakob disease (sCJD) are associated with neuronal iron dyshomeostasis 12 , it is likely that in pathological conditions such as these, PrP C accentuates neuronal injury by the combined effect of iron-mediated oxidative stress and glucose deprivation. It is notable that GLUT3 is downregulated in scrapie infected animal brains 49 and human cases of CJD 63 . A similar downregulation of GLUT3 has been observed in AD brains 48 , a known long-term complication of type-2-diabetes 10 . Whether PrP C is the principal regulator of GLUT3 expression in neurons is not clear from our data, and remains an open question. However, considering our observations on pancreatic β-cells and the similarities between GLUT2 and GLUT3 21 , it is tempting to speculate that PrP C plays a similar role in neurons, and is likely to influence neuronal health under normal and pathological conditions by modulating iron and glucose uptake 24 . Likewise, PrP C is expressed widely in the neuroretina, including retinal pigment epithelial cells in the outer blood retinal barrier where it mediates uptake of iron by the neuroretina. PrP C is therefore likely to modulate both iron and glucose homeostasis in the retina, that, like the brain, has a high metabolic rate and depends on glucose for normal function.
In conclusion, our observations reveal a novel function of PrP C in regulating blood glucose through iron, and reaffirm the correlation between systemic iron and type-2-diabetes. The relatively high expression of PrP C in the brain and the neuroretina underscores its significance as a key protein that regulates iron and glucose homeostasis, vital processes critical for the functioning of these metabolically active tissues. The correlation between PrP C , iron, and glucose homeostasis is likely to provide new, untapped therapeutic opportunities for type-2-diabetes, AD, and other neurodegenerative conditions associated with iron dyshomeostasis.

Materials and Methods
Ethics statement. All

Mouse strains.
Four mouse strains were used to improve the validity of this study; PrP-knock out (PrP −/− ) mice deposited by 25 to Jackson Laboratories (cat # 129-Prnptm2Edin/J Stock No: 012938) and crossed with C57BL/6 wild-type mice for 10 generations. F2 generation of wild type (C6 PrP +/+ ) and corresponding PrP −/− (C6 PrP −/− ) were used for these studies. FVB/NJ Tg40 (Tg40 PrP) that express 2 × human PrP were generated from FVB/Prnp 00 mice 26 kindly provided by the Prusiner laboratory and used as corresponding controls for Tg40 PrP mice 64 . All mouse lines were fed regular chow (Prolab Isopro RMH 3000 from www.labdiet.com) and maintained under similar conditions. All experiments were conducted on 6-8 week old male mice since females show less pronounced phenotype of glucose intolerance 17 , and carried out at the same time of the day.
Cell lines, transfection, and RNAi knockdown. Insulin producing human pancreatic β-cell line 1.1B4 was obtained from Sigma Aldrich (Cat. No: 10012801), USA and cultured as described 66 . This is a PANC-1 hybrid human pancreatic beta cell line that secretes insulin. Cells were cultured in RPMI-1640 supplemented with 2 mM Glutamine and 10% heat inactivated FBS. Cells were passaged every fourth day. For silencing PrP C , 1.1B4 cells were transfected with siRNA against PrP C or scrambled control using Lipofectamine RNAimax (Invitrogen). After 72 h, cells were exposed to 16.7 mM of glucose for 1 h and processed for Western blotting. Human neuroblastoma cells (M17) were purchased from ATCC. Cells expressing vector or PrP C were generated described in previous reports 67,68 , and cultured in DMEM supplemented with 10% FBS. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 .
Antibodies. PrP C -specific antibodies 3F4 and 8H4 were from Signet laboratories (Dedham, MA) and Sigma Aldrich respectively. Other antibodies were obtained from the following sources: ferritin specific for heavy and light chain (F5012) from Sigma Aldrich, USA, GLUT-1 (NB110-39113) from Novus Biologicals, GLUT-2 (ab54460), and GLUT-3 (ab41525) from Abcam, USA, TfR (13-6800) from Invitrogen, USA, insulin from Santa Cruz Biotechnology Inc. (sc-9168) and Novus Biologicals (NBP2-34260), USA (recognize insulin and a 51-amino acid polypeptide composed of A and B chains connected through the C-peptide), glucagon (sc-13091) from Santa Cruz Biotechnology Inc, USA, and β-actin (MAB1501) from Millipore, USA. HRP-conjugated secondary antibodies (anti-mouse, NA931V, anti-rabbit, NA934V) were from GE Healthcare, UK. Iron treatment. 1.1B4 and M17 cells cultured in complete medium were exposed to vehicle or 30 μM of ferric ammonium citrate (FAC) for 16 h at 37 °C before processing for immunostaining and Western blot as described 69 . To create systemic iron overload, age and sex-matched C6 PrP +/+ , C6 PrP −/− , Tg40 PrP and PrP −/− mice were injected intraperitoneally with 36 µg/22 g mouse weight of FAC and euthanized after 24 h for further analysis 37 . The dose of iron used (1.6 mg/kg body weight) is well below the recommended ~10 mg/kg/day used for the treatment of anemia in an average adult weighing 75 kg 70 . Western blotting and Immunostaining. Western blotting and immunostaining were carried out as described 69 . In short, cells and tissues were lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.5% deoxycholate), boiled in reducing gel-loading buffer for 5 min at 100 °C, and fractionated by SDS-PAGE. Fractionated proteins were transferred to PVDF membranes and probed for specific proteins. Quantification of protein bands was performed by densitometry using UN-SCAN-IT gels (version6.1) software (Silk Scientific) and analyzed graphically using GraphPad Prism (Version 5.0) software (GraphPad Software Inc.).
Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT). GTT and ITT were performed in age and sex-matched mice at the same time of day as described 46 . For GTT, the mice were fasted overnight with ad libidum access to water, and 1 g glucose/kg body weight was injected intraperitoneally. Blood glucose was monitored at 0, 15, 30, 60, 120, and 180 min post-injection with a glucometer (EasyMax-Diabetic Promotions, USA). For ITT, the mice had ad libidum access to food and water because PrP −/− mice went into hypoglycemic shock after insulin injection. Accordingly, non-fasted animals were injected with 0.75 U insulin/kg body weight intraperitoneally, and blood glucose was monitored as above at 0, 15, 30, 45, 60 and 120 min post-injection.
Statistical analysis. Data were analyzed using GraphPad Prism5 (GraphPad Software, Inc., La Jolla, CA) and presented as Mean ± SEM. Level of significance was calculated by Two-way ANOVA between the control and experimental groups.