Effects of free fatty acids on glucose transport and IRS-1–associated phosphatidylinositol 3-kinase activity (original) (raw)

Subjects.

Fourteen healthy volunteers (thirteen males, one female; age range: 18–33 years; body wt: 75.8 ± 2.9 kg; body mass index: 23.8 ± 0.5 kg/m2) without family history of diabetes mellitus, dyslipidemia, or bleeding disorders were put on an isocaloric diet (30 kcal per kg/day; carbohydrate/protein/fat: 60%/20%/20%) for 3 days. They were admitted to the Yale/New Haven Hospital General Clinical Research Center the evening before the study and fasted for 12 h before participating in either an NMR or muscle biopsy study. On the morning of the study, Teflon catheters were inserted in the antecubital veins of the right and left arm for blood sampling and glucose/lipid/hormone infusions, respectively. Each subject was studied twice (basal plasma FFA concentrations [glycerol/heparin infusion] or increased plasma FFA concentrations [lipid/heparin infusion]) in either an NMR protocol (n = 7) or muscle biopsy protocol (n = 7). Informed and written consent was obtained from each volunteer. All protocols were approved by the Yale University Human Investigation Committee.

Lipid/glycerol infusions.

To examine the effect of increased plasma FFA concentrations on insulin-stimulated muscle glucose metabolism, plasma concentrations of FFAs were increased by intravenous infusion of a triglyceride emulsion (1.5 ml/min; Liposyn II, Abbott Laboratories, North Chicago, Illinois, USA) combined with a prime (200 IU)–continuous (0.2 IU per kg/min) infusion of heparin to activate lipoprotein lipase. During the control experiments, glycerol (0.7 mg per kg/min) was substituted for the lipid infusion. Subjects were randomized with respect to the order in which they received the two infusions. Studies were performed within 2–4 weeks of each other.

Hyperinsulinemic-euglycemic clamp studies.

After 5 h of the lipid or glycerol infusion, a hyperinsulinemic-euglycemic clamp using [1-13C]glucose (20g/dl, 40%–60% carbon-13 enriched; Cambridge Isotopes, Cambridge, Massachusetts, USA) was begun (t = 0 minutes). The subjects were also infused with [1-13C]mannitol (10g/dl, 99% carbon-13 enriched; Cambridge Isotopes) (prime [3.8 g]–continuous [1.4 g/min]) to assess intracellular glucose concentration, as previously described (21). Insulin (Humulin-Regular; Eli Lilly and Co., Indianapolis, Indiana, USA) was administered as a prime (18 mU/kg)–continuous (1 mU per kg/min) infusion to create conditions of hyperinsulinemia (∼400 pM) for 240 min. Plasma concentrations of glucose were maintained at ∼5 mM by varying the infusion rate of the [1-13C]glucose. Both the lipid and glycerol infusions were continued throughout the clamp for both the NMR and biopsy studies.

NMR studies.

In vivo carbon-13/phosphorous-31 NMR spectroscopy was performed on the right calf muscle to assess glycogen, glucose-6-phosphate, and intracellular glucose concentrations. Subjects remained in the supine position inside a 2.1 T Biospec NMR spectrometer system (Bruker Instruments Inc., Billerica, Massachusetts, USA), and phosphorous-31 NMR spectra were acquired (22) to measure intracellular glucose-6-phosphate concentration before the clamp (t = –40 to 0 min) and from 20 to 60 min during the clamp. Carbon-13 NMR spectra for glycogen concentrations were acquired before the clamp (t = –60 to –40 min) and from 120 to 180 min during the clamp to measure rates of muscle glycogen synthesis as previously described (23, 24). Intracellular glucose concentrations were measured by carbon-13 NMR spectroscopy from 180 to 240 min as described previously (21). The radio frequency (RF) coil assembly consisted of two circular hydrogen-1 coil loops (13-cm diameter each) arranged spatially to generate a quadrature field and an 8.5-cm diameter circular surface coil for carbon-13 detection (25). Unlocalized shimming was performed using FASTERMAP (26). The B2 field was calibrated using a stimulated echo y column profile that generated a 180° null at the gradient isocenter. Heteronuclear nuclear Overhauser enhancement was achieved by a train of inversion pulses to the hydrogen-1 nuclei during the relaxation delay. A numerically optimized adiabatic half-passage pulse was used for carbon-13 excitation (27) followed by WALTZ-16 hydrogen-1 decoupling. The repetition rate was 2 sec per scan. The creatine (Cr) signal observed in the carbon-13 NMR spectrum is from both creatine and phosphocreatine (PCr). Assuming the total creatine (Cr + PCr) signal is compartmentalized to the same intracellular muscle space and is natural abundance carbon-13, we calculate the extracellular to intracellular volumes from the in vivo NMR signal intensity of Crtotal to mannitol as described previously (21).

Muscle biopsy studies.

After the subjects were supine and resting quietly for 60 min, the right vastus lateralis muscle was sterilely prepared with betadine, and 1% lidocaine was given subcutaneously. A 2-cm incision was made using a scalpel, and a 5-mm Bergstrom biopsy cannula (Warsaw, Indiana, USA) was used to perform a baseline punch muscle biopsy. The muscle tissue was blotted and snap frozen in liquid nitrogen. After this, a 5-h infusion of lipid/heparin or glycerol/heparin was given. At the fifth hour, while the infusions continued, a hyperinsulinemic-euglycemic clamp was begun as described above. After 30 min of hyperinsulinemia, a repeat punch muscle biopsy was performed. All samples were stored at –80°C until assay.

Materials.

Phosphatidylinositol was purchased from Avanti Polar Lipids (Arlington, Alabama, USA) and phosphatidylinositol 4-phosphate from Sigma Chemical Co. (St. Louis, Missouri, USA). Reagents for the detection of Western blots by enhanced chemiluminescence, rainbow-colored molecular weight markers for SDS-PAGE, and [γ-32P]ATP (6000 Ci/mmol) were purchased from Amersham Life Sciences Inc. (Arlington Heights, Illinois, USA). Protein GPLUS/Protein A-Agarose immunoprecipitation reagent was purchased from Calbiochem (Cambridge, Massachusetts, USA). Antibodies against IRS-1CT (rabbit polyclonal) and PI 3-kinase (p85 subunit, rabbit polyclonal) were a gift from M.F. White (Joslin Diabetes Center, Boston, Massachusetts, USA). Horseradish peroxidase–labeled anti–mouse and anti–rabbit secondary antibodies were obtained from Rockland Inc. (Gilbertsville, Pennsylvania, USA).

Muscle preparation for insulin signaling studies.

Muscle extracts were made from the frozen specimens. Muscles were first powdered under liquid nitrogen with a mortar and pestle and then homogenized in ice-cold buffer (20 mM HEPES, pH 7.4, 50 mM β-glycerol phosphate, 2 mM DTT, 1 mM Na3VO4, 2 mM EDTA, 1 mM PMSF, 1% Triton X-100, 10% glycerol, 10 μM Leupeptin, 3 mM Benzamidine, 5 μM Pepstatin A, 10 μg/ml Aprotinin, 200 μg/ml soybean trypsin inhibitor). The homogenate was solubilized on a rotating mixer at 4°C for 30–60min and centrifuged at 15,000 rpm for 60 min in a 70.1 Ti rotor in a Beckman Ultracentrifuge (Fullerton, California, USA). The supernatant was collected and assayed for total protein content using the Bio-Rad Laboratories Inc. (Hercules, California, USA) protein assay kit.

PI 3-kinase activity measurements.

IRS-1–associated PI 3-kinase activity was measured in immunoprecipitates obtained with antibodies to IRS-1 as described previously, with some modifications (28, 29). A 1-mg aliquot of muscle extract (total protein) was added to the immune complex composed of protein A/G agarose and anti–IRS-1 antibody and allowed to incubate overnight. The immunocomplexes were collected by centrifugation, washed twice with PBS containing 1% NP-40/100 μM Na3VO4, twice with 100 mM Tris (pH 7.5) containing 500 mM LiCl2/100 μM Na3VO4, and twice with Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA/100 μM Na3VO4. The pellets were then resuspended in 50 μl of the final wash buffer and 12 mM MgCl2, and 20 μg phosphatidylinositol was added. To start the PI 3-kinase reaction, 10 μl of 440 μM ATP containing 30 μCi [32P]ATP was added to the pellets at room temperature. After 10 min, 20 μl of 8 M HCL was added to stop the reaction, followed by 160 μl of CHCl3/CH3OHOH (1:1). The phases were separated by centrifugation, and 50 μl of the lower organic phase was spotted onto a glass-backed silicon TLC plate. The lipids were resolved by TLC in CH3OHOH/CHCl3/H2O/NH4OH (60:47:11.3:2) and visualized by autoradiography. The radioactivity that comigrated with the PI 4-phosphate standard was scraped from the TLC plate and quantified by scintillation counting.

Respiratory exchange measurements.

During the biopsy studies continuous indirect calorimetry was performed from 95 to 115 min during the hyperinsulinemic-euglycemic clamp to determine rates of whole-body glucose oxidation. From these data and from the amount of nitrogen excreted in the urine, rates of nonoxidative metabolism were calculated.

Analytical procedures.

Plasma glucose concentrations were measured by the glucose oxidase method (Glucose Analyzer II; Beckman Instruments Inc., Fullerton, California, USA). Plasma concentrations of FFAs were determined using microfluorimetric methods. Plasma immunoreactive insulin was determined by a double antibody RIA (Diagnostic Systems Laboratories Inc., Webster, Texas, USA). Plasma [1-13C]glucose and mannitol (as acetate derivatives) atom percent excesses were measured using gas chromatography–mass spectrometry with a Hewlett-Packard (Palo Alto, California, USA) 5890 gas chromatograph (HP-1 capillary column, 12-m × 0.2-mm × 0.33-mm film thickness) interfaced to a Hewlett-Packard 5971 MSD (methane chemical ionization) (21, 23).

Calculations and data analyses

Increments in muscle glycogen concentration were determined from the change in [1-13C]glycogen concentration and the plasma [1-13C]glucose atom percent excess as described previously (24). Rates of glycogen synthesis were then calculated from the slope of the least-square linear fit to the glycogen concentration curve during the given time periods as described previously (24). The intracellular glucose concentration was calculated as described previously (21). All data are presented as mean ± SEM. Statistical comparisons between control and lipid infusion experiments were performed by using the paired Student's t test, except for the PI 3-kinase data, which were analyzed using analysis of variance followed by the Student-Newman Keuls post-hoc test.

Hypothetical intracellular glucose concentration resulting from reduced hexokinase flux with normal glucose transporter kinetics. We can simplify the metabolic steps regulating glycogen synthesis flux in muscle during steady state as: Δ[Glcin ]/Δ_t_ = _V_1 – _V_–1 – V_HK = 0, where Δ[Glcin ]/Δ_t is the change in the intracellular glucose concentration per change in time, _V_1 is the rate of glucose transport influx into the cell, _V_-1 is the rate of glucose efflux from the cell, and _V_HK is the rate of hexokinase flux. Assuming Michaelis-Menten kinetics yields: _V_1 = _V_1max [Glcex]/(_K_m1 +[Glcex]) and _V_-1= _V_-1max [Glcin]/(_K_m–1 + [Glcin]). If we assume (a) symmetrical transport of glucose by Glut4 (_V_1max = _V_-1max, _K_m1 = _K_m–1), (b) a _K_m for Glut4 of 5 mM (37), and (c) that _V_HK is approximately equal to the glycogen synthetic rate as measured in the glycerol infusion studies (∼0.10 mmol per liter muscle/min), then given an extracellular glucose concentration of 5 mM and an intracellular glucose concentration of 0.1 mM, substituting into equation 1 yields: _V_1max [Glcex]/(_K_m1+[Glcex]) – _V_–1max [Glcin]/(_K_m–1 + [Glcin]) ≈ _V_glycogen synthetic rate and _V_1max [5/(5+5)] – _V_–1max [0.1/(5+0.1)] = 0.1 mmol per liter muscle/min) and _V_1max = 0.21 mmol per liter muscle/min. Assuming _V_HK is decreased exclusively by FFA and that it is approximately equal to the glycogen synthetic rate in the lipid-infused subjects (–0.05 mmol per liter muscle/min) then repeating this calculation and substituting _V_1max = 0.21 mmol per liter muscle/min yields: 0.21 [5/(5+5)] – 0.21 [Gin] / ([Gin] + 5) ] = 0.05, [Gin ] = 1.8 mM. This value is more than 40 times higher than that measured in the lipid-infused subjects.