3 hydroxybutyrate

Administration of a vehicle (saline, 0.25 ml/30s, n = 5), saline solution of BA (Sodium butyrate, Sigma-Aldrich, Poznan, Poland) at a dose of 1.4 (n = 5), 2.8 (n = 5) and 5.6 mmol/kg (n = 5) and ANT at a dose of 5.6 mmol/kg (n = 5).

Administration of BA at a dose of 5.6 mmol/kg after the pretreatment with the ANT (3-hydroxybutyrate, Sigma-Aldrich, Poznan, Poland) at a dose of 5.6 mmol/kg (n = 5).

Administration of BA at a dose of 5.6 mmol/kg after the subphrenical vagotomy (n = 5), administration of BA at a dose of 5.6 mmol/kg after the sham procedure (n = 5) and vagotomy alone (n = 5).

Administration of BA at a dose of 5.6 mmol/kg during the intravenous treatment with hexamethonium, an autonomic ganglia blocker (Sigma-Aldrich, Poznan, Poland, 15 mg/kg bolus followed by continuous infusion at a rate of 1.5 mg/kg/min, n = 5) or after the intravenous treatment with atropine (Atropinum Sulfuricum WZF, Polfa Warszawa, Warsaw, Poland) at a dose of 1 mg/kg (n = 5).

After hemodynamic studies, rats were killed by decapitation.

Cell growth was assessed utilizing a UV-Vis spectrophotometer (Biochrom, UK) at an optical density of 600 nm (OD₆₀₀) after apt dilution to ensure that the initial OD₆₀₀ was maintained between 0.3 and 0.8. Samples were collected from the culture every 2 hr, and the residual glucose concentration was immediately determined using a glucose analyzer (SBA-40E biosensor, Shandong Academy of Sciences, China). The DCW and PHA content of the culture were analyzed by sampling every 4 hr.
Harvest 30 mL of cell culture from shaker flasks or bioreactors, use a centrifuge (USA Thermo) at room temperature (25 °C) at 8000 rpm for 10 min. After centrifugation, wash the cells once with distilled water. Freeze the washed cell pellet at −80 °C for 24 hr, and then freeze dry it in a freeze dryer for 32–40 hr (USA GOLD SIM). Use the lyophilized biomass to measure the DCW.
To determine PHA content, lyse 30–40 mg of lyophilized cell powder at 100 °C in a decane solution composed of 2 mL of chloroform and 2 mL of methanolysis solution (97 wt% methanol, 3 wt% H₂SO₄, and 1 g/L benzoic acid) for 4 hr. Then, after cooling to room temperature (25 °C), add 1 mL of water to extract and separate phases. The PHA content in the denser phase was analyzed by gas chromatography (Agilent, USA) with 35 mg of high-purity 3-hydroxybutyrate from Sigma–Aldrich as the standard (Tan et al., 2011 (link)).