All experiments were approved by the Animal Care Committee at the University Health Network. α-syn knockout mice B6;129 × 1-Snca^{TM 1Rosl}/J were obtained from Jackson Labs (Bar Harbor, ME, United States) (Abeliovich et al., 2000). Both the knockout and control animals are of 50% C57 and 50% 129 background and were age-matched. Mice were characterized at 3 months of age. Human wild type α-syn overexpressing mice of FVB background strain were generated using a hamster prion protein promoter and littermate controls were used (Chen et al., 2013). Mice were characterized at 4 months of age. 3-Month old C57BL6J mice were used for α-syn treatments and for examining α-syn expression. Male mice fed a chow diet were used for all experiments.

Human α-syn monomers were expressed and extracted from bacteria as previously described (Jo et al., 2000). 50 μg of α-syn monomer was injected intraperitoneally in a 100 μL volume at 6 weeks of age, followed by a second injection after 2 weeks. Animals were characterized 4 weeks after second injection at 12 weeks of age.

GTT: Following a 4 h fast, glucose (1.5 g/kg body weight) was given by oral gavage and glucose from tail vein blood was measured at 0, 15, 30, 60, 90, or 120 min using a glucometer. Blood was collected in EDTA-coated microvettes (Sarstedt) at 0 and 15 min and plasma isolated for insulin measurement using an ultrasensitive insulin ELISA (ALPCO Diagnostics). ITT: Following 4 h fast, insulin (0.75 IU/kg body weight) was injected intraperitoneally and plasma glucose was measured at 0, 15, 30, and 60 from tail vein blood using a glucometer.

Mouse islets were isolated by collagenase digestion of the pancreas as described (Wijesekara et al., 2010). Insulin secretion was assessed as reported from isolated islets stimulated for 1 h with 2 mmol/L (basal) or 11 mmol/L glucose (Wijesekara et al., 2010). Secreted insulin was quantified by ELISA and normalized to total DNA. Wild type islets were treated in vitro in culture medium for 24 h with vehicle (20 mM TrisHCl pH 7.4) or 1.68 μg/mL α-syn monomers prior to and during the insulin assay.

L6-GLUT4myc myoblasts were seeded onto 12-well (for protein extraction) or 24-well (for GLUT4 translocation) plates and were differentiated into myotubes in α-MEM supplemented with 2% (vol/vol) FBS and 1% (vol/vol) antibiotics/antimycotics. Cells were allowed to fully differentiate into myotubes and were deprived of serum for 3 h prior to any experimental manipulation. For cell lysate preparation, myotubes were stimulated for 10 min in α-MEM with vehicle (20 mM TrisHCl pH 7.4), 100 nM insulin [Humulin R] (Eli Lilly Canada) or 1.68 μg/mL α-syn monomers. The cells were lysed with 2X Laemmli sample buffer supplemented with 1 mM DTT and protease inhibitors.

Tissue or islet lysates (35 μg total protein) were resolved by 4–10% Tris-Glycine gels and immunoblotted with primary antibody against α-syn [syn-1] (BD Biosciences) [1:1000] and GAPDH (Sigma) [1:1000]. Lysates from myotubes were immunoblotted with primary antibody against phospho-Akt T308 (Cell Signaling) [1:1000] and β-actin (sigma) [1:5000]. Immunoblots were scanned within the linear range and quantified using the computer software NIH Image J. Phospho-Akt is normalized to β-actin and expressed as fold change over vehicle treatment.

Isolated pancreata were fixed in formalin, paraffin-embedded and 5 μm sections were cut at 100 μm intervals. Deparaffinized sections were subjected to antigen retrieval in TE buffer and co-stained with syn-1 (BD Biosciences) [1:250] or insulin (Dako) [1:500] primary antibodies. Images were acquired using a Zeiss confocal microscope using Zen software (Carl Zeiss Canada).

The amount of cell surface GLUT4myc was determined by an antibody-coupled colorimetric absorbance assay, as was previously described (Wijesekara et al., 2006). Briefly, cells were stimulated for 20 min in a-MEM with vehicle or 1.68 μg/mL α-syn monomers in the presence or absence of 100 nM insulin. Cells were then exposed to polyclonal anti-myc antibody (1:100) for 60 min, fixed with 4% paraformaldehyde (PFA) for 10 min, and incubated with peroxidase-conjugated goat anti-rabbit IgG (1:1,000) for 1 h. Cells were washed six times and 1 ml of OPD reagent (0.4 mg/ml o-phenylenediamine dihydrochloride and 0.4 mg/ml urea hydrogen peroxide) was added for 30 min at room temperature. The reaction was stopped with 0.25 ml of 3 N HCl. Optical absorbance of the supernatant was measured at 492 nm. Background, as measured in samples incubated in peroxidase-conjugated anti-rabbit IgG alone (without primary antibody), was subtracted from all values.

The rate of GLUT4myc recycling was determined as described (Wijesekara et al., 2006). Briefly, at 37°C, cells were incubated in the absence or presence of α-syn or insulin while being exposed to polyclonal anti-myc antibody for the times indicated. At each time point, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonX-100 for 15 min, and reacted with peroxidase-conjugated goat anti-rabbit IgG (1:1000) for 1 h at 4°C to determine the amount of anti-myc antibody bound at the cell surface as well as that which had become labeled at the surface and subsequently internalized during the incubation time. 1 mL of OPD reagent was added for 30 min at room temperature. The reaction was stopped with 0.25 mL of 3 M HCl. Absorbance of the supernatant was measured at 492 nm. Total amount of GLUT4myc was determined by incubating the cells with the anti-myc antibody following permeabilization. The amount of GLUT4myc labeled at each time point was expressed as a percent of total cellular GLUT4myc content.

GLUT4myc internalization was measured as was previously described (Wijesekara et al., 2006). Briefly, cells were stimulated with or without α-syn or insulin at 37°C for 20 min. Cells were rinsed 3 times with ice-cold PBS and reacted with the polyclonal anti-myc antibody (1:200) at 4°C for 1 h. After washing 2 times with ice-cold PBS, the surface labeled GLUT4myc was allowed to internalize by re-warming the cells to 37°C in the presence or absence of α-syn or insulin. At the indicated times, cell plates were placed on ice, washed with ice-cold PBS, fixed with 4% paraformaldehyde for 10 min and incubated with peroxidase-conjugated goat anti-rabbit IgG (1:1000) for 1 h at 4°C. 1 mL of OPD reagent was added for 30 min at room temperature. The reaction was stopped with 0.25 mL of 3 M HCl. Absorbance of the supernatant was measured at 492 nm. The amount of GLUT4myc remaining on the cell surface at any time point after re-warming was expressed as a percentage of the cell surface GLUT4myc level at 0 min of endocytosis.

Data are expressed as mean ± SEM. Significance was determined using Student’s t-test or one-way or two-way ANOVA with Tukey or Bonfferoni post hoc test. p < 0.05 was considered statistically significant.

We observed α-syn expression by immunoblot analysis in total lysates of pancreas, skeletal muscle and isolated pancreatic islets from wild type mice (Figure 1A). Brain and islet lysates from α-syn global knockout (α-synKO) mice were used as negative controls. α-syn was observed to partially co-localize with insulin, confirming its expression in pancreatic beta cells (Figure 1B).

In order to understand the role of α-syn in glucose metabolism, glucose and insulin tolerance tests were performed in α-synKO mice (Figure 2A). At 3 months of age, α-synKO mice were both glucose intolerant and insulin resistant compared to controls (Figures 2B,C). Fasting plasma insulin was elevated, likely as a response to the insulin resistance, but showed impaired GSIS response, suggesting the presence of beta cell dysfunction in the absence of α-syn (Figure 2D). α-synKO mice maintained body weights (26.1 ± 1.2 vs. 27.3 ± 1.7 g) similar to wild type mice and displayed comparable fasting blood glucose (Figure 2E). This suggests that α-syn is required for proper mainenance of glucose tolerance and insulin senstivity. To confirm that the observed effects are directly linked to α-syn in beta cells, GSIS was measured in isolated islets from α-synKO and wild type mice. In vitro glucose-stimulation of α-synKO islets showed a 25% reduction in insulin secretion compared to wild type controls (Figure 2F). These data are consistent with a functional role of α-syn in insulin secretion by pancreatic beta cells.

In contrast to α-syn knockout, trangenic mice overexpressing human α-syn at 4 months of age showed improved glucose tolerance and insulin sensitivity, with body weight (24.5 ± 0.5 vs. 24.3 ± 0.5 g), fasting blood glucose and fasting plasma insulin similar to non-transgenic controls (Figure 3A–E). GSIS was significantly elevated in the α-syn overexpressing mice (Figure 3C). In order to further understand the impact of α-syn, wild type mice were subjected to two intraperitoneal injections of recombinant, purified α-syn monomers at 2 week intervals, starting at 6 weeks of age. Four weeks after the second injection (i.e., 12-weeks of age), α-syn injected animals exhibited improvements in glucose tolerance and insulin sensitivity compared to vehicle only injected mice (Figures 3F,G). Body weight (25.8 ± 0.7 vs. 24.8 ± 1.1 g), fasting blood glucose and fasting plasma insulin were unaffected (Figures 3H,I). This suggests that both overexpression and acute elevation of peripheral α-syn has a positive effect on glucose homeostasis. In vitro treatment of wild type isolated islets with α-syn monomers increased GSIS (Figure 3J), further confirming the ability of α-syn to directly stimulate insulin secretion from pancreatic beta cells.

Glucose uptake in skeletal muscle is mediated by GLUT4 glucose transporters. L6 myotubes stably expressing myc-tagged GLUT4 (GLUT4myc) are a cellular model used for studying GLUT4 traffic (Wang et al., 1998). Treatment of these cells with α-syn monomers resulted in a gain in GLUT4myc trafficked to the plasma membrane, which was not additive to the insulin response (Figure 4A). A previous report demonstrated that 3T3L1 preadipocytes exhibited a modest increase in Akt phosphorylation following treatment with α-syn, however, this was not observed in the current study (Rodriguez-Araujo et al., 2013; Figure 4B). The current findings indicate that α-syn in myotubes directly increases GLUT4 at the plasma membrane, independent of any change in Akt signaling. The impact of α-syn on GLUT4 trafficking was assessed further to determine if α-syn increases cell surface GLUT4 by enhancing GLUT4 exocytosis, reducing endocytosis or potentially via both of these pathways. We examined whether α-syn led to a reduction in GLUT4 endocytosis by measuring the surface GLUT4myc remaining at different times after internalization of labeled GLUT4myc. In both vehicle treated and insulin-stimulated states, GLUT4myc were internalized rapidly and to a similar extent under the two conditions (Figure 4C). In the presence of α-syn, GLUT4myc was largely retained at the cell surface, so that 64 ± 7% of the amount labeled remained associated with the surface after 20 min, compared with 31 ± 6% in the basal state. Conversely, to determine whether α-syn can boost the rate of GLUT4myc exocytosis, the time course of GLUT4myc appearance on the cell surface was detected in a live-cell recycling assay. The results showed a significant reduction at 180 min in cells stimulated with α-syn, while as previously reported, insulin increased GLUT4 exocytosis (Wijesekara et al., 2006; Figure 4D). Therefore, the α-syn-induced increase in surface GLUT4 levels is prominently associated with a reduction in GLUT4 endocytosis, while exocytosis is eventually reduced may be as a response to the impaired endocytosis. Cumulatively these data suggest that α-syn is also a key regulator of glucose uptake in skeletal muscle.