. Indinavir, ritonavir, nelfinavir, and amprenavir were obtained from Merck, Abbott, Agouron, and GlaxoSmithKline, respectively. Rodents were purchased from Charles River Labs (Wilmington, MA). Blood glucose levels were determined using a Glucometer Elite XL (Bayer, Tarrytown, NY). BCA protein assay reagents were obtained from Pierce (Rockford, IL). Rat insulin radioimmunoassay kits were obtained from Linco (St. Charles, MO). RPMI and Dulbecco's modified Eagle medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). [5-3
H]glucose was obtained from American Radiolabeled (St. Louis, MO). MIN6 cells were a gift from Dr. Y. Oka (Yamaguchi University, Yamaguchi, Japan) (12
). All other reagents were obtained from Sigma Chemical (St. Louis, MO).
Isolation and culturing of pancreatic islets and β-cells. Anesthetized mice (C57/Bl × CBA, aged 4-12 months) were killed by cervical dislocation. Pancreata were immediately removed and injected with Hank's solution containing type XI collagenase (138 mmol/l NaCl, 5.6 mmol/l KCl, 4.2 mmol/l NaHCO3, 1.3 mmol/l CaCl2, 0.44 mmol/l KH2PO4, 0.4 mmol/l MgSO4, 0.3 mmol/l NaHPO4, 1 mmol/l EGTA, 1 mg/ml BSA, 5.6 mmol/l D-(+)-glucose, and 1 mg/ml collagenase [pH 7.4]). Pancreata were digested for 7 min at 37°C and then hand-shaken and washed three times in ice-cold Hank's solution. Islets were isolated by hand under dissecting microscope and pooled. Cells were maintained at 37°C in a humidified incubator in RPMI medium (11 mmol/l glucose) supplemented with 10% calf serum and penicillin (100 units/ml) and streptomycin (100 μg/ml). Insulinoma MIN6 cells were cultured in complete RPMI medium (10% FBS). MIN6 cells used in the present study were harvested at passages 30-40.
Insulin release experiments. Batch incubation was performed in duplicate on pancreatic islets (10/well) or MIN6 cells (1 × 106 cells/well) incubated in glucose-free DMEM supplemented with D-(+)-glucose (1, 7, or 16.7 mmol/l). PIs were added immediately before the glucose stimulus. Control cells were incubated with appropriate water or ethanol vehicle. Cells were incubated for 1hat37°C unless indicated, and medium was removed and assayed for insulin content by radioimmunoassay. For reversibility experiments, islets were assayed for insulin release as described but with the addition of either the sulfonylurea glibenclamide (1 μmol/l) or a mix of glutamine and leucine (10 mmol/l each).
2-Deoxyglucose uptake experiments. MIN6 cells grown to 50-70% confluence were washed three times with Krebs-Ringer phosphate buffer, pH 7.0 (KRP; 100 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl2, 1 mmol/l NaH2PO4, and 1 mmol/l CaCl2). The cells were then incubated in the presence of [3H]-2-deoxyglucose (50 μmol/l) for 6 min at 37°C. Reactions were terminated by washing the cells three times with ice-cold KRP buffer containing 0.1 mol/l phloretin. After air drying for 15 min, the cells were permeabilized with 1% Triton X-100 and aliquots were removed for radioactivity determination by liquid scintillation counting and protein determination (BCA protein assay). PIs were added to the reaction 2 min before initiation of the transport assay. Nonspecific uptake was measured in the presence of 40 μmol/l cytochalasin B and subtracted from the experimental values. Uptake in this assay was determined to be linear over the 6-min assay interval. Data were analyzed as picomoles of 2-deoxyglucose incorporated per milligram of protein. All experiments were performed in triplicate with the data reported as a percentage of the uptake from control plates incubated in the absence of PI.
. Glucokinase activity in MIN6 cell extracts was assayed using a spectrophotometric enzyme-coupled assay as described previously (13
). Briefly, MIN6 cells were harvested in 20 mmol/l phosphate buffer, pH 7.4, containing 0.02% BSA, 0.5 mmol/l EDTA, 5 mmol/l β-mercaptoethanol, 25% glycerol, and 0.5% Triton X-100. The cells were homogenized in a glass-Teflon homogenizer, and glucose phosphorylating activity was determined by adding the cell extract to a cuvette containing 100 mmol/l Tris-HCl, 7.5 mmol/l MgCl2
, 5 mmol/l ATP, 1 mmol/l NAD+
, pH 7.4, with glucose-6-phosphate dehydrogenase and indinavir as noted. The rate of NADH formation was monitored by measuring the change in absorbance at 340 nm. Hexokinase and glucokinase activities were determined including 0.5 mmol/l and 20 mmol/l glucose, respectively. Glucokinase activity (activity at 20 mmol/l glucose minus hexokinase activity) represented ~75% of all glucose phosphorylating activity in MIN6 cell extracts.
3-O-methylglucose uptake experiments
. The uptake of 3-O-methylglucose (3-MG) into MIN6 cells was measured using the oil stop-method described by Johnson et al. (14
). Briefly, MIN6 cells were harvested with PBS containing 0.5 mmol/l EDTA for 5 min and incubated with PBS containing14
C-labeled urea (2 mmol/l, 0.5 mCi/mmol) as an intracellular space marker at 37°C for 20 min, followed by incubation at 22°C for 10 min. Assay tubes were prepared by placing a reaction stop mixture containing 0.1% SDS, 1 mol/l glucose, and 10 mmol/l EDTA into microcentrifuge tubes. This was overlaid with a 4:1 mixture of dibutyl phthalate and dinonyl phthalate. An assay mixture containing PBS, 14
C-labeled urea, and3
H-labeled 3-MG (20 mmol/l) was placed above the oil layer. Reactions were initiated by adding aliquots of cell suspension ± 20 μmol/l indinavir to the assay tube and terminated by sedimentation through oil layer. Tritiated sugar incorporation was calculated as micromoles of 3-MG per liter of islet cell space, determined from14
C radioactivity, and expressed as a ratio of the radioactivity at equilibrium. Incorporation of nontransportable L-glucose under identical conditions is shown as negative control and represents background counts.
. Glucose utilization in MIN-6 cells was assessed using the method of Ishihara et al. (12
). Briefly, MIN6 cells were harvested with PBS containing 0.5 mmol/l EDTA for 5 min and incubated with glucose-free DMEM supplemented with 16.7 mmol/l glucose. Indinavir was added to a 1.5-ml microtube containing 250 μl (1 × 106
cells) 5 min before the addition of [5-3
H] glucose. Aliquots of the assay mixture for insulin assay determination were removed immediately before the termination of the 1-h incubation by addition of 3 N HCl:ethanol (1:4 [vol/vol]). The unstoppered tubes were incubated in sealed 20-ml glass scintillation vials that contained 500 μl of unlabeled water at 37°C for 16 h, and the radioactivity of the equilibrated water in the scintillation vial was determined by liquid scintillation counting. Background radioactivity was determined in identically treated samples incubated in the absence of MIN6 cells (representing <10% of total counts) and was subtracted from control and indinavir-treated samples. From tritiated water controls, the efficiency of water equilibration was determined to be ~90% under these conditions.
. All animal procedures were approved by the animal studies committee at Washington University School of Medicine. Rats and mice were housed in the animal facility at Washington University and fed a standard diet and water ad libitum. In clamp experiments (outlined below), catheters were inserted into the left internal carotid artery and right jugular vein under methohexital anesthesia as previously described (15
). Rats were allowed to recover from surgery for at least 4 days before experiments were performed, and rats were fasted overnight before each experiment.
Hyperglycemic clamp experiments
. Clamps were performed on 200- to 300-g male Wistar rats as previously described (7
) with noted differences. A constant infusion of indinavir (0.5 mg • kg-1
) or water (for control animals) was initiated through the venous catheter 30 min before the start of the clamp procedure. Blood indinavir reached 25-30 μmol/l during the period of drug infusion, as measured by HPLC assay (16
). After determination of fasting glucose concentrations, dextrose (50%) was infused through the venous catheter at a rate sufficient to maintain a plasma glucose level of 200 mg/dl during the assay period. Blood (20 μl) was sampled directly from the arterial catheter at times shown for determination of both blood glucose and insulin levels.