Dextrose

Animals were sedated with ketamine hydrochloride after overnight fasting (no insulin was administered the night before). Catheters (22-gauge) were placed into bilateral saphenous veins for glucose delivery and blood sampling separately. Fifty percent of dextrose (Hospira Inc., Lake Forest, IL) solution (0.5 g/kg) was injected over a 30-s period at t = 0. Blood samples were taken before dextrose injection and at 1, 3, 5, 10, 15, 20, 30, 60, and 90 min after injection to measure glucose. Serum samples were frozen at −80°C for subsequent analysis. Insulin and C-peptide were measured in duplicate with an ELISA kit (Mercodia AB, Uppsala, Sweden, catalog nos. 10-1132-01 and 10-1141-01).

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed as previously described 49. Briefly, GTTs were performed at 16 weeks of age (1 g/kg of body weight of Dextrose) after an overnight fast on mice fed a NCD or a HFD. Dextrose (Hospira, Inc., Lake Forest, IL) was diluted in injection grade water and administered by oral gavage (Solomon Scientific, San Antonio, TX). ITTs were performed at 17 weeks of age, after a 5 h fast. Humulin R insulin (Eli Lilly and Co., Indianapolis, IN) was diluted in 0.9% saline (Phoenix Pharmaceutical, Inc., Burlingame, CA) and administered by intraperitoneal injection at 0.75 U/kg of body weight and glucose values were measured as using OneTouch Ultra 2 glucometer.

E. chaffeensis Arkansas (49 (link)) was cultured in the canine macrophage cell line DH82 (50 (link)) in Dulbecco’s minimal essential medium (Mediatech, Manassas, VA) or human acute leukemia cell line THP-1 cells (51 (link)) in RPMI 1640 medium supplemented with 5% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% l-glutamine (GIBCO, Grand Island, NY) at 37°C in 5% CO₂ and 95% air in a humidified atmosphere as described previously (51 (link)). E. chaffeensis-infected cells (∼1 × 10⁸ cells, >90% infected, from two T75 flasks) were harvested by centrifugation at 400 × g for 5 min. The pellet was resuspended in culture medium and sonicated on ice for 8 s using a W-380 sonicator (Heat Systems, Newtown, CT) with an output setting of 2. Unbroken cells were removed by centrifugation at 1,000 × g for 5 min. The supernatant was collected after additional centrifugation at 1,700 × g for 5 min, passed through 2.7-μm and 5.0-μm GD/X nylon filters (Whatman, Florham Park, NJ) to remove cell debris, and centrifuged at 9700 × g for 10 min (52 (link)). The resulting bacterial pellet was resuspended in Hanks’ balanced salt solution (HBSS; Sigma-Aldrich) supplemented with 2 mg/ml dextrose (Hospira, Lake Forest, IL) (HBSSd). E. chaffeensis was quantified based on 16S rRNA gene-based quantitative PCR as described below.

A description of the model and methodology has been published previously [24 ]. Briefly, hyperdynamic endotoxemic shock was induced by administration of an escalating dose of lipopolysaccharide (LPS; E coli serotype O55:B5; Sigma-Aldrich, St Louis, USA) up to 4 μg/kg/h over 4 h (total LPS dose 11.25 μg/kg). Adequate endotoxemia was confirmed by the occurrence of systemic hypotension with a mean arterial pressure (MAP) less than 60 mmHg after 3 h of endotoxin infusion. In the final hour of endotoxemia animals received either fluid resuscitation with 40 mLs/kg of 0.9% saline over an hour (FR; n = 8) or commenced protocolised vasopressor support (NFR; n = 8). Noradrenaline was started 60 μg/mL in 5% dextrose (Hospira, Lake Forest, IL, USA) to maintain a MAP between 60 and 65 mmHg. If noradrenaline reached a predetermined 20 μg/min, vasopressin (PPC, Richmond Hill, ON, Canada) was commenced at 0.8 units/h and increased to a maximum of 1.6 units/h if hypotension persisted. The administration protocol was the same for both groups. Post-fluid resuscitation, both groups were monitored for 12 h after the end of endotoxin infusion.