Male mongrel dogs (Antech, Barnhart, MO, USA; n = 16, >1 year old, 30.0 ± 3.5 kg [mean ± SD]) were housed in the Cedars-Sinai Medical Center vivarium under controlled kennel conditions (12:12 h light/dark cycle). Animals were accepted into the study following physical examination and a comprehensive blood panel. Dogs were used for experiments only if judged to be in good health as determined by visual inspection, body temperature and haematocrit. The experimental protocol was approved by the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee (IACUC).
Dogs were fed a control weight-maintenance diet comprising one can of Purina Proplan Puppy Chow (10% protein, 7% fat, 1.5% fibre, 76 moisture; Nestle Purina PetCare Company, St Louis, MO, USA) and 825 g of dry chow (27.7% protein, 29.9% fat, 42.4% carbohydrate, 2.9% fibre; Lab Diet, Purina Mills, St Louis, MO, USA) for at least 4 weeks before the initiation of the study. The control diet consisted of 14,970 kJ/day: 39.2% carbohydrate, 32.5% fat and 28.3% protein. During high-fat feeding, the control diet was supplemented with 6 g/kg of rendered pork fat (lard) to achieve a 52% fat diet consisting of 21,025 kJ/day. Meals were presented for 1 h from 9:00 hours to 10:00 hours, with food weighed before and after presentation.
During weeks 0, 2 and 6, MRI scans were performed to assess total, subcutaneous and intra-abdominal fat mass as previously described . Image analysis was performed using Slice-O-Matic software (4.3 rev 10; Virtual Magic, Montreal, QC, Canada) by a single experienced observer.
In eight dogs, 24 h blood sampling was conducted at weeks 0, 2, 6 and again at week 8 with acute nocturnal NEFA suppression. Catheters attached to vascular access ports (Instech Solomon; Plymouth Meeting, PA, USA) were implanted and maintained as previously described . At 6:00 hours on the day of each experiment, animals were brought to the laboratory for hourly blood sampling from 7:00 hours until 8:00 hours on the following morning, at which time an IVGTT was performed.
Suppression of nocturnal NEFA
Overnight suppression of NEFA after high-fat feeding was achieved by intravenous injections of a selective partial agonist of the A1 adenosine receptor (GS-9667, provided by Gilead Sciences) beginning at 18:00 hours and continuing every 2 h until 8:00 hours. A dose of 10 mg/kg every 2 h was determined to provide maximal inhibition of NEFA. This dose is comparable with that used in clinical studies of NEFA lowering .
Frequently sampled IVGTT
An IVGTT was performed at weeks 0, 2, 6 and 8. In the subset of eight dogs undergoing 24 h sampling, IVGTTs were performed immediately following each 24 h sampling period to test the acute effects of nocturnal NEFA and NEFA lowering on insulin sensitivity and the acute insulin response to glucose. Animals were mildly restrained in a Pavlov sling for this procedure . An intracatheter was inserted into the saphenous or cephalic vein. Blood samples were drawn every 5 min for 15 min (three baseline samples), at which time glucose was given as an intravenous bolus (0.3 g/kg body weight). Blood samples were collected at 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 30, 40, 50, 60, 70, 90, 100, 120, 140, 160 and 180 min. At the 20 min time point, intravenous insulin (0.02 U/kg body weight) was administered.
Sample collection and storage
All samples were collected on ice and centrifuged. Plasma was separated and immediately transferred to storage tubes and placed on ice for the remainder of the experiment. On completion of the experiment, samples were transferred to −80°C freezers for storage until further analysis. Samples for assay of glucose, insulin, cortisol and catecholamines were collected in tubes pre-coated with lithium fluoride and heparin (Brinkmann Instruments, Westbury, NY, USA). Samples for NEFA were collected in non-heparin tubes containing 50 μl EDTA and coated with paraoxon to inhibit lipase activity. Tubes for insulin, glucose and catecholamines also contained 50 μl EDTA. Tubes for catecholamines contained an additional 25 μl of ethylene glycol tetraacetic acid (EGTA).
Glucose was measured by a YSI 2300 or 2700 autoanalyser (Yellow Springs Instruments, Yellow Springs, OH, USA). NEFA was measured using a colorimetric method (Wako Pure Chemical Industries, Richmond, VA, USA). Insulin was measured with an ELISA originally developed for human serum or plasma (Linco Research, St Charles, MO, USA) and adapted for dog plasma. The method is based on two murine monoclonal antibodies that bind to different epitopes of insulin but do not bind proinsulin . Cortisol and catecholamines were measured using commercially available kits (cortisol RIA kit from Diagnostic Products, Los Angeles, CA, USA; catecholamines ELISA from Rocky Mountain Diagnostics, Colorado Springs, CO, USA).
For 24 h hormone levels, the meal was served after the 9:00 hour blood sample, thus the 8 h period following meal presentation (10:00–18:00 hours) was considered the postprandial daytime phase. The nocturnal phase was calculated as the AUC from 18:00 hours until the completion of the study the following morning, as previously described .
For minimal-model variables, insulin sensitivity (SI), acute insulin response to glucose (AIRg) and disposition index (DI) were calculated using the minimal model of glucose kinetics from the IVGTT (MINMOD 6.02, MinMod, Los Angeles, CA, USA). Glucose tolerance was calculated as the slope of the linear regression of the natural log of plasma glucose concentration from 5 to 19 min.
Results are presented as means ± SEM. A linear mixed model was conducted to test the effects of high-fat feeding on IVGTT variables and hormonal profiles using a compound symmetry covariance matrix. Time (week 0, 2, 6 and 8) was specified as a fixed and repeated factor. A mixed-model regression analysis was performed to examine the relationship between NEFA and insulin secretion. Individual values that were greater or less than twice the SD were considered statistical outliers and were excluded from the analyses. Statistical analyses were conducted using SPSS (IBM SPSS Statistics for Windows, version 21.0, Armonk, NY, USA). All differences were considered statistically significant when p < 0.05.