Rabbit liver glycogen

Rabbit liver glycogen and pancreatic α-amylase were from Sigma. Microcystin-LR was from Enzo Life Sciences. NADH was from Apollo Scientific. Antibodies against pS8 GYS1 and pS8/S11 GYS1 were kindly donated by D. Grahame Hardie (University of Dundee). pS641 GYS1 (#3891) and total GYS1 (#3893) antibodies were from Cell Signaling Technologies. Total GYS1 (sc-81173) and PP1c (sc-7182) antibodies were from Santa Cruz Biotechnology. pS641/645 GYS1 (S486A, 3rd bleed) and GN1 (S197C, 1st bleed) antibodies were generated by the Division of Signal Transduction Therapy (DSTT) at the University of Dundee as previously described [4] (link). Peroxidase conjugated secondary antibodies were from Jackson Immunoresearch. Glutathione Sepharose 4B, NHS-Sepharose FF, Superdex 200 3.2/300, Q-Sepharose HP and Enhanced Chemiluminescent (ECL) reagent were from GE Healthcare. Gel filtration standards were from Bio-Rad (151-1901). All other reagents, unless otherwise indicated, were from Sigma.

Formalin fixed-paraffin embedded placental tissue was sectioned at 5 μm through the mid-line using a Leitz 1512 rotary microtome (Leica). Placental sections were stained with haematoxylin and eosin to assess gross morphological structure and layer proportions. Placental glycogen was stained using a periodic acid Schiff (PAS) stain. Briefly, sections were dewaxed and rehydrated before incubation with 0.5% periodic acid (Sigma, UK) for 90 min. Sections were then washed and incubated in Schiff's reagent (Sigma, UK) for 15 min, before dehydrating and mounting in DPX. The percentage of positive magenta staining was measured by a blinded operator using Image J software.
Total placental glycogen content was determined using the protocols of Lo et al. (1970) and Rampon et al. (2008) with some modifications [24 ,25 (link)]. Briefly, 10 mg of placental tissue was homogenised in 30% KOH saturated with Na₂SO₄ (Sigma, UK) and incubated at 100 °C for 30 min, followed by cooling on ice. Glycogen was precipitated in 100% ethanol and separated by centrifugation at 1,000×g for 30 min. Glycogen standards were prepared using Rabbit Liver Glycogen (G8876; Sigma, UK), with concentrations ranging from 400 μg/ml to 12.5 μg/ml. Samples and standards were combined with 5% phenol with H₂SO₄ (Sigma, UK) to initiate a colorimetric change. Absorbance was read at 490 nm on Benchmark Bio-Rad microplate reader.

Cyanobacterial glycogens, rabbit-liver glycogen (Sigma-Aldrich Co., St. Louis, MO) and waxy maize starch (Cargill, Minneapolis, MN) were used for production of β-limit dextrins. Samples (50 mg) were pre-wetted with water (0.5 mL), and then 4.5 mL of DMSO was added. The resulting mixtures were heated in a boiling water bath for 1 h and were precipitated with 5 volumes of absolute ethanol. The air-dried pellets were dissolved in 50 mM acetate buffer (pH 4.8), and boiled for another 0.5 h. After cooling down the dispersions to ambient temperature, sweet potato β-amylase (10 units mg⁻¹ glucan, Sigma-Aldrich Co., St. Louis, MO) was added. The hydrolysis reaction proceeded at 37°C for 24 h, and then another 10 unit mg⁻¹ of β-amylase was added to completely hydrolyze the external α-1,4 linkages for 24 h. The resulting samples were precipitated with 2 volumes of ethanol, washed twice with 75% (v/v) ethanol, and then once with 100% ethanol. The air-dried pellets, β-limit dextrins, were used for further analysis.