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Dissociation between Fat-Induced in Vivo Insulin Resistance and Proximal Insulin Signaling in Skeletal Muscle in Men at Risk for Type 2 Diabetes

Department of Endocrinology and Clinical Research Unit (H.S., C.B.J., S.M., A.A.V.), Hvidovre Hospital, University of Copenhagen, DK-2650 Hvidovre, Denmark; Department of Clinical Physiology (M.B., X.M.S., J.R.Z.), Karolinska Hospital, Karolinska Institutet, SE 17177 Stockholm, Sweden; and Steno Diabetes Center (A.A.V.), 2820 Gentofte, Denmark

Address all correspondence and requests for reprints to: Heidi Storgaard, Steno Diabetes Center, Niels Steensensvej 2, 2820 Gentofte, Denmark. E-mail: heis{at}steno.dk or hstorgaard{at}dadlnet.dk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The effect of short- (2 h) and long-term (24 h) low-grade Intralipid infusion on whole-body insulin action, cellular glucose metabolism, and proximal components of the insulin signal transduction cascade was studied in seven obese male glucose intolerant first degree relatives of type 2 diabetic patients [impaired glucose tolerance (IGT) relatives] and eight matched control subjects.

Indirect calorimetry and excision of vastus lateralis skeletal muscle biopsies were performed before and during hyperinsulinemic euglycemic clamps combined with 3[³H]glucose. Clamps were performed after 0, 2, or 24 h Intralipid infusion (0.4 ml·kg^-1·min^-1).

Insulin-stimulated glucose disposal decreased approximately 25% after short- and long-term fat infusion in both IGT relatives and controls. Glucose oxidation decreased and lipid oxidation increased after both short- and long-term fat infusion in both groups. Insulin-stimulated glucose oxidation was higher after long-term as compared with short-term fat infusion in control subjects. Short- or long-term infusion did not affect the absolute values of basal or insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation, tyrosine-associated phosphoinositide 3-kinase (PI 3-kinase) activity, insulin receptor substrate-1-associated PI 3-kinase activity, or Akt serine phosphorylation in IGT relatives or matched controls. In fact, a paradoxical increase in both basal and insulin-stimulated PI 3-kinase activity was noted in the total study population after both short- and long-term fat infusion.

Short- and long-term low-grade Intralipid infusion-induced (or enhanced) whole-body insulin resistance and impaired glucose metabolism in IGT relatives and matched control subjects. The fat-induced metabolic changes were not explained by impairment of the proximal insulin signaling transduction in skeletal muscle.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
TYPE 2 DIABETES IS associated with obesity and elevation of plasma free fatty acids (FFAs) (1, 2). Elevated plasma FFA impairs insulin action, affecting oxidative and/or nonoxidative glucose metabolism (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) in a presumably time-dependent manner (3, 5, 8, 10). Randle et al. (15) showed that FFA competes with glucose for substrate oxidation (16) in rat heart muscle and diaphragm. Increased fat oxidation was shown to increase glucose-6-phosphate levels and through this, inhibition of hexokinase II enzyme activity, resulting in decreased membrane glucose uptake. Several human studies have documented the interplay between glucose and fat oxidation as outlined by Randle et al. both at the level of whole body (3) and skeletal muscle glucose metabolism (17). However, recent human in vivo nuclear magnetic resonance studies have challenged this concept, reporting decreased skeletal muscle intracellular glucose-6-phosphate (5, 14) and glucose concentration (11) in response to combined fat and insulin infusion. Thus, defective glucose transport and/or phosphorylation may play a predominant role for the development of fat-induced insulin resistance. To support this, fat infusion decreases glycogen synthase activity in young healthy subjects (3, 10).

The mechanisms underlying the fat-induced changes in insulin-stimulated glucose transport/phosphorylation and glycogen synthesis are unknown but may include impaired insulin signal transduction. Insulin regulates glucose uptake and storage by binding to its specific cell-surface receptor (IR), which results in autophosphorylation and activation of IR tyrosine kinase. IR tyrosine kinase phosphorylate tyrosine residues on IR substrates (IRS). IRS-1 seems to be the major component involved in glucose metabolism in human skeletal muscle (18). Binding of IRS-1 to the regulatory subunit of PI 3-kinase results in activation of the kinase. In skeletal muscle, phosphoinositide 3-kinase (PI 3-kinase) is a key regulator of GLUT4 translocation and glucose transport (19), and the serine/threonine kinase Akt seems to be partly responsible for mediating insulin signaling downstream of PI 3-kinase (20). Akt is known to phosphorylate and inactivate glycogen synthase kinase-3, an inhibitory effector of glycogen synthase (21).

Elevations in FFA appear to impair insulin signaling in skeletal muscle. Five hours supraphysiological fat and heparin exposure (2 mM) reduced insulin-stimulated whole-body glucose disposal and skeletal muscle IRS-1-associated PI 3-kinase activity in young healthy subjects (11). Furthermore, in response to insulin during fat and heparin infusion in vivo in rats, insulin-stimulated glucose disposal and skeletal muscle IRS-1 phosphorylation, as well as IRS-1-associated PI 3-kinase activity is reduced, possibly through increased PKC activity (22). Increased PKC activity correlated with increased serine phosphorylation of IRS-1, and this may account for the reduced ability of IRS-1 to activate PI 3-kinase. However, in these studies, basal biopsies were obtained before fat infusion, and the insulin-stimulated biopsy did not reflect the isolated effect of an elevation of plasma FFA on insulin signaling but rather the combined effect of insulin and fat. Furthermore, the absolute basal and insulin-stimulated values for IRS-1-associated PI 3-kinase activity were not given.

Effects of elevated plasma FFA on insulin action have been determined using gold standard hyperinsulinemic euglycemic clamp, whereby fat infusion was performed for no longer than 2–6 h. Insulin action was only estimated indirectly in other more long-lasting fat infusion studies (23, 24, 25). The effects of elevated plasma FFA on insulin action have been studied in healthy subjects (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) and diabetic patients (4, 13) but not in subjects at high risk for the development of type 2 diabetes. FFA-induced effects on glucose metabolism are largely dose dependent (3, 6), and one concern as to the pathophysiological relevance for type 2 diabetes is that most previous studies used relatively high fat infusion rates (during short time experiments), achieving plasma FFA concentration during insulin stimulation between 1 and 4 mM (4, 5, 8, 11, 12, 13, 14).

We determined whether low-grade physiological elevation of plasma FFA levels (within the range observed in type 2 diabetic patients) influenced whole-body insulin action and glucose metabolism in the late prediabetic state defined as glucose intolerant first-degree relatives of type 2 diabetic patients and/or in age- and weight-matched normal glucose-tolerant subjects with no family history of type 2 diabetes. Furthermore, we established whether a potential effect of plasma FFA elevation on insulin action was explained by impaired insulin signal transduction in skeletal muscle. Lastly, we determined whether potential FFA-induced defects exhibited time dependency by performing studies after short- and long-term Intralipid infusion.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Seventy-one men recruited through local newspaper advertisements underwent an oral glucose (75 g) tolerance test (OGTT). Glucose tolerance was diagnosed according to the World Health Organization criteria [i.e. impaired glucose tolerance (IGT): 2 h plasma glucose during OGTT > 7.8 and < 11.1 mM]. We included 15 men; seven were glucose intolerant first-degree relatives of type 2 diabetic patients (IGT relatives) and eight were age- and body mass index-matched control subjects with normal glucose tolerance and no family history of diabetes (controls). The subjects were not undertaking arduous exercise on a regular basis, and they were instructed to avoid excessive physical exercise and alcohol intake for at least 2 and 1 d, respectively, before the clamps. The subjects were also instructed not to change their body weight or eating, drinking, smoking, and exercise habits during their participation in the study. The subjects all participated in an earlier study protocol (26, 27). All subjects agreed to participate after oral and written information. The study was approved by the Copenhagen County Ethical Committee, and the study was conducted according to the principles of the Helsinki Declaration.

Body fat determinations

A dual-energy x-ray absorptiometry scan was performed to measure total body fat. A Norland XR-36 scanner was used (28).

Protocol for in vivo studies

All subjects underwent the following study set-up three times preceded by 0, 2, and 24 h fat infusion, respectively (Fig. 1). Intralipid/saline infusion was continued throughout the iv glucose tolerance test (IVGTT) and clamp studies. All studies commenced at 0800 h after a 10-h overnight fast. A polyethylene catheter was inserted into an antecubital vein for infusion of test substances. Another polyethylene catheter was inserted into a contralateral wrist vein for blood sampling. This hand was kept in a heated Plexiglas box throughout the test to obtain arterialized venous blood (29). Basal samples for insulin, C-peptide, FFA and glucose determination were obtained. Each study was initiated with a basal period (-160 min), when the tracer bolus was given. Indirect calorimetry was performed during the predefined basal steady-state period (-70 to -40 min) for basal glucose oxidation measurements. Thereafter, a basal (noninsulin-stimulated) muscle biopsy was obtained (-40 to -30 min). From -30 to 0 min, an IVGTT was performed to characterize the first-phase insulin response. A bolus of 300 mg glucose/kg body mass (18% glucose) was infused over 1 min. Insulin secretion data are published elsewhere (27).

View larger version (9K): [in this window] [in a new window]   FIG. 1. In vivo study design. Each subject underwent the study three times preceded by 0, 2, and 24 h 20% Intralipid infusion respectively.

Squared priming was performed (0 to +9 min) with an initial high and subsequently stepwise decline in the insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) infusion rate every third minute, reducing the insulin infusion rate from 100 to 80 to 60 to 40 mU·m^-2·min^-1. Thereafter, the insulin infusion rate was fixed at 40 mU·m^-2·min^-1 from +9 to +120 min. Plasma glucose concentration was maintained constant at euglycemia using a variable glucose infusion (180 g·liter^-1) (30). Plasma glucose concentration was monitored every 5 min using an automated glucose oxidase method (Glucose Analyzer 2; Beckman Instruments, Fullerton, CA). Indirect calorimetry was performed during the insulin-stimulated steady-state period (+90 to +120 min) to measure insulin-stimulated glucose oxidation.

Tritiated glucose

One hundred sixty min before the start of insulin (-160 min), an adjusted primed [11 µCi/(fasting plasma glucose concentration/(5 mmol·liter^-1)] continuous (0.11 µCi/min) infusion of 3-[³H]glucose (NEN Life Science Products, Boston, MA) was initiated to ensure isotope equilibrium (31). The tracer infusion was continued throughout the 120-min period of insulin infusion. To obtain constant specific activity during insulin infusion, tritiated glucose was added to the infused glucose solution (54 µCi/500 ml in an 18% glucose solution) (32). Blood samples were drawn in fluoride-treated tubes at the beginning and end of the 30-min basal steady-state period and every 10 min thereafter during the 30-min insulin-stimulated steady-state period for determination of 3-[³H]-glucose activity. Plasma 3-[³H]-glucose was measured every 30 min during the rest of the study. Plasma 3-[³H]-glucose concentration was determined from radioactivity in the evaporated (to avoid 3-[³H]-water) 0.5-ml plasma samples (31). Plasma 3-[³H]-water was determined from the radioactivity in the 0.5-ml plasma sample minus the radioactivity in the same plasma sample after evaporation.

Indirect calorimetry

A ventilated canopy was placed over the subject’s head (Deltatrac, Datex, Helsinki, Finland), and continuous gas exchange measurements were determined. Inspired and expired air was analyzed for oxygen content using a paramagnetic differential oxygen sensor and for carbon dioxide tension using an infrared carbon dioxide sensor. Oxygen consumption and carbon dioxide production were recorded and calculated each minute. After an equilibrium period of 10 min, the average gas exchange over the two 30-min steady-state periods (basal and insulin-stimulated) (-70 min to -40 min and +90 min to +120 min) were used to calculate rates of glucose oxidation as previously described (33, 34).

Muscle biopsy

Muscle biopsies were obtained under local anesthesia, from musculi vastus lateralis using a modified Bergström’s needle (including suction) at -40 (basal) and +120 min (insulin-stimulated). Biopsies were frozen in liquid nitrogen and stored at -80 C for later analysis.

Intralipid infusion

Intralipid is a fat emulsion consisting of triglycerides; 12% palmitic acid (C16:0), 4% stearic acid (C18:0), 21% oleic acid (C18:1 n-9), 53% linoleic acid (C18:2 n-6), 7% -linolenic acid (C18:3 n-3), and 3% others. An intended 10–20% elevation of the fasting plasma FFA concentration was achieved by infusing 20% Intralipid at a rate of 40 (ml·kg^-1·h^-1). The short-term Intralipid infusion was given continuously from the time point -160 to +120 min. At the none fat (control) day, Intralipid was substituted with saline infusion from the time point -160 min to +120 min. For the long-term fat infusion protocol, the subjects were hospitalized and the Intralipid infusion was initiated 24 h before the time point -40 min (at which the basal muscle biopsy was performed) and continued throughout the study period. A polyethylene catheter was inserted into an antecubital vein for infusion of Intralipid. Another polyethylene catheter was inserted into a contra lateral wrist vein for blood sampling. Blood samples were collected at time points -0.10, -0.05, 0.00, 1.45, 2.45, 4.45, 8.30, 10.30, 15.00, and 19.00 h:min. All samples were analyzed for plasma glucose, insulin, C-peptide, and FFA. Standardized meals were served at time points 0.15, 3.15, 9.00, 12.30 h:min. Light exercise (50 W work load) was performed on a bicycle ergometer for 15 min at the time points 2.00 and 5.00 h:min. Short- and long-term Intralipid infusion is referred to as 2 h and 24 h fat infusion, respectively.

Blood chemistry

Plasma glucose was determined using an automated glucose oxidase method (Glucose Analyzer 2; Beckman Instruments). Tritiated glucose and water activities were measured as previously described (32, 35). Plasma insulin and C-peptide concentrations were determined using the 1234 AutoDELFIA immunoassay system (Wallac Oy, Turku, Finland). Plasma FFAs were quantified using an enzymatic colorimetric method (Wako, Richmond, VA).

Materials and antibodies

Phosphoinositide was from Avanti Polar Lipids (Alabaster, AL). The aluminum-backed Silica Gel 60 thin-layer chromatography plates were from EM Separations (Gibbstown, NJ). Protein A-Sepharose was purchased from Pharmacia (Uppsala, Sweden). All other chemicals were purchased from Sigma (St. Louis, MO) or Merck (Rahway, NJ). Antiphosphotyrosine and p85 antibodies were from Signal Transduction Laboratories (Lexington, KY). IRS-1 polyclonal antibody was from Dr. Ton Maassen (Leiden University, Leiden, Netherlands). Polyclonal antibodies to detect Akt phosphorylation or Akt protein were from New England Biolabs (Beverly, MA).

Tissue processing

Muscle biopsies were homogenized in ice-cold homogenizing buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1 mM Na₃VO₄, 10 mM NaF, 30 mM Na₄P₂O₇, 10% (vol/vol) glycerol, 1 mM benzamidine, 1 mM dithiothreitol (DTT), 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 µM microcystin] using a glass on glass rotating homogenizer, and subjected to centrifugation (12,000 x g for 10 min at 4 C). Protein was determined in the supernatant using a commercial kit (Bio-Rad Laboratories, Richmond, CA).

IRS-1 tyrosine phosphorylation

From the remaining homogenate described above, an aliquot of the supernatant (800 µg) was immunoprecipitated with anti-IRS-1 coupled to protein A-Sepharose. Immunoprecipitates were washed four times in ice-cold homogenizing buffer, resuspended in Laemmli sample buffer with 100 mM DTT, and heated (95 C) for 4 min. Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes incubated with antiphosphotyrosine antibodies, washed, and incubated with appropriate secondary antibodies. The reactions were visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) and quantified by densitometry. Results are presented as arbitrary densitometric units.

PI 3-kinase activity

Muscle specimens were homogenized as described above, and an aliquot of the supernatant (600 µg) was immunoprecipitated overnight (4 C) with antiphosphotyrosine antibody or anti-IRS-1 antibody coupled to protein A-Sepharose. PI 3-kinase activity was assessed directly on the protein A-Sepharose beads as previously reported (36). Reaction products were resolved by thin-layer chromatography and quantitated using a phosphor imager (Bio-Rad Laboratories). Results are presented as arbitrary phosphor imager units.

Akt phosphorylation

Aliquots of muscle homogenate (40 µg) were solubilized in Laemmli sample buffer with 100 mM DTT, and heated (95 C) for 6 min. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted as described above, using a phospho-specific antibody that recognizes Akt when phosphorylated at Ser 473 (New England Biolabs). Phosphorylated Akt was visualized by ECL and quantified by densitometric scanning.

Protein expression

For protein expression of IRS-1, p85 subunit of PI 3-kinase, and Akt, an aliquot (40 µg) of supernatant was resuspended in sample buffer and heated (95 C) for 6 min. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and blocked as described above. Membranes were incubated with the appropriate primary antibodies as indicated above, washed, and incubated with appropriate secondary antibodies as recommended by the supplier (Amersham). Proteins were visualized by ECL and quantified by densitometry.

Calculations

Glucose turnover rates. Glucose appearance rates (Ra), glucose disposal rates (Rd), and endogenous glucose production rates (EGPs) were calculated during the steady-state periods using Steele’s nonsteady-state equation (37). During the insulin-stimulated steady-state period, Rd and EGP were calculated at 10-min intervals. In these calculations the distribution volume of glucose was taken as 200 ml/kg body weight and the pool fraction of 0.65 (37). In the calculations of exogenous glycolytic flux (GF) from appearance rate of tritiated water (35, 38, 39), total water was estimated as 93% of total plasma volumen. EGP during the basal steady-state period was equal to the rate of appearance of 3-[³H]-glucose (Ra), whereas EGP during the clamp steady-state period was calculated as the difference between Ra and the exogenous glucose infusion rate. Exogenous glucose storage (EGS) (exogenous is used to emphasize that the calculated turnover rates are based on flux of the infused glucose tracer) was calculated as Rd - GF; nonoxidative glucose metabolism (NOGM) as Rd - glucose oxidation (GOX). Glucose metabolism data were expressed as milligrams·fat-free body mass^-1·min^-1.

Insulin clearance

During the hyperinsulinemic euglycemic clamp, insulin clearance was calculated as insulin infusion rate divided by plasma insulin concentration.

Basal values

Basal values (except basal Rd) were calculated from mean values derived from two blood samples collected at the time points -70 and -40 (in the beginning and the end of the basal steady-state period) for the test days with 0, 2, and 24 h fat infusion, respectively.

Statistical analysis

The results are presented as mean ± SEM. The effects of fat infusion and subgroup status (IGT relative or control subject) were analyzed by two-way ANOVA with subjects as a random factor. The effect of fat infusion was analyzed as an effect of duration, i.e. the contrast corresponding to the difference between the 2- and 24-h infusion and as an overall effect of fat, compared with saline infusion, i.e. the contrast corresponding to the difference between fat and saline infusion. The latter analysis was considered appropriate only if the effect of duration was not statistically significant. The ANOVA also provided a test for similarity of the effects of fat infusion in the two groups, i.e. an interaction effect. Only if this effect was insignificant will it be appropriate to perform the test of an overall difference between the two groups. Differences between mean values in the two groups were compared by Mann-Whitney rank-sum test. Differences within groups were compared by Wilcoxon rank-sum test. Statistical significance was accepted at P < 0.05.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical characteristics

The 2-h plasma glucose concentration after the OGTT was, as expected, higher in the IGT relatives (Table 1). Fasting plasma glucose concentration during the basal steady-state period was higher in IGT relatives (Table 2).

Basal plasma FFA concentrations increased from 0 to 2- and 24-h fat infusion in the IGT relatives but not in the controls (Table 2). Nevertheless, basal plasma FFA concentrations did not differ significantly between IGT relatives and controls with or without prior short- or long-term fat infusion (Table 2). Basal plasma triglyceride concentrations increased significantly after 2-and 24-h fat infusion in both IGT relatives and controls (Table 2) and did not differ between the two study groups. Basal plasma glucose, insulin, and C-peptide concentrations were unaffected by 2-h fat infusion in both study groups (Table 2) and increased after 24-h fat infusion in the control subjects but not in the IGT relatives (Table 2). The IGT relatives had higher basal plasma glucose and C-peptide levels than the controls (Table 2).

Clamp values for plasma FFA, triglyceride, glucose, insulin, and C-peptide

Plasma FFA and triglyceride concentrations were significantly elevated during Intralipid infusion as compared with saline infusion, with no difference after 2- as compared with 24-h fat infusion (Table 2). Although the mean clamp plasma glucose concentration was slightly higher after 2- and 24-h fat infusion in IGT relatives, plasma glucose concentrations during the clamps were not different between the groups (Table 2). Clamp plasma insulin concentrations were similar in the two study groups during all three different study protocols. Insulin clearance was not affected by fat infusion in any of the groups. However, clamp plasma C-peptide increased significantly after both 2- and 24-h fat infusion in both groups (Table 2).

Metabolic measures during basal and insulin-stimulated steady-state

Tracer equilibrium [constant specific activity (SA)] was reached in both steady-state periods both with and without fat infusion in both groups (Table 2). The SAs increased slightly from the basal to the insulin stimulated steady-state period for 0, 2-, and 24-h fat infusion in both groups (Table 2). However, small deviations in plasma SA (less than 20% from baseline levels) are not associated with major errors in the final calculations based on the tracer data (40). A linear increase in plasma-tritiated water was observed during both steady-state periods both with and without fat infusion (clamp steady-state period, total study population; 0 fat: r² = 0.59 ± 0.09; 2-h fat: r² = 0.50 ± 0.07; 24-h fat: r² = 0.61 ± 0.08).

Rd. Short- and long-term fat infusion did not interfere with the basal Rd in any of the groups (Table 3). During short- and long-term fat infusion, the insulin-stimulated Rd decreased approximately 25% in both groups. IGT relatives had significantly lower insulin-stimulated Rd than controls, both with and without fat infusion (Table 3).

GOX. Basal and insulin-stimulated GOX rates were decreased after both 2- and 24-h fat infusion in IGT relatives and insulin-stimulated GOX rates decreased after both 2- and 24-h fat infusion in control subjects (Table 3). Interestingly, fat infusion had a transient effect on both basal and insulin-stimulated GOX rates in control subjects. Thus, both basal and insulin-stimulated GOX rate decreased after 2-h fat infusion, whereas a somewhat paradoxical significant increase and normalization of the GOX rate was observed in the controls after 24-h fat infusion (Table 3).

NOGM. Basal NOGM was unaffected by fat infusion in IGT relatives and control subjects (Table 3). Insulin-stimulated NOGM was unaffected by fat infusion in IGT relatives, whereas both 2- and 24-h fat infusion decreased insulin-stimulated NOGM in control subjects (Table 3). Insulin- stimulated NOGM was significantly lower in IGT relatives, compared with controls both with and without fat infusion.

GF. Both basal and insulin-stimulated GF was unaffected by fat infusion in IGT relatives and control subjects (Table 3).

EGS. Short- and long-term fat infusion did not interfere with the basal EGS in IGT relatives or control subjects (Table 3) or with insulin-stimulated EGS in IGT relatives. However, insulin-stimulated EGS decreased significantly after both 2- and 24-h fat infusion in the controls. IGT relatives had lower insulin-stimulated EGS than control subjects without fat infusion, but no differences were found after short- or long-term fat infusion.

Lipid oxidation (LIPOX). Basal and insulin-stimulated LIPOX increased after both 2- and 24-h fat infusion in both groups (Table 3). IGT relatives had lower insulin-stimulated LIPOX, compared with control subjects both with and without fat infusion (Table 3).

Insulin signal transduction

IRS-1 tyrosine phosphorylation. Short- and long-term fat infusion did not decrease the absolute basal, insulin-stimulated, or incremental (insulin-stimulated-basal value) IRS-1 tyrosine phosphorylation in either IGT relatives or control subjects (Figs. 2A, 3A, and 4). In fact, the insulin-stimulated and incremental IRS-1 tyrosine phosphorylation paradoxically increased after 2-h fat infusion in the IGT relatives. Insulin failed to stimulate IRS-1 tyrosine phosphorylation in IGT relatives with and without fat infusion, whereas a significant increase was observed in control subjects without fat infusion. The IGT relatives had significantly lower incremental IRS-1 tyrosine phosphorylation than controls without fat infusion but not after 2- and 24-h fat infusion. IRS-1 protein expression was unaffected by fat infusion in both study groups (0, 2, and 24 h: 1.2 ± 0.2 vs. 1.0 ± 0.2 vs. 1.0 ± 0.2 arbitrary units, NS).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Insulin action on IRS-1 tyrosine phosphorylation (A), phospho-tyrosine-associated PI 3-kinase activity (B), IRS-1-associated PI 3-kinase activity (C), and Akt serine phosphorylation (D) in skeletal muscle from control subjects. Muscle biopsies were obtained under basal and insulin-stimulated conditions as described in Subjects and Methods after 0 (), 2 (), and 24 () h fat infusion. Data are from n = 6–8. , P < 0.05 vs. 2 and 24 h fat. *, P < 0.05 vs. basal.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Insulin action on IRS-1 tyrosine phosphorylation (A), phospho-tyrosine-associated PI 3-kinase activity (B), IRS-1-associated PI 3-kinase activity (C), and Akt serine phosphorylation (D) in skeletal muscle from IGT first-degree relatives of type 2 diabetic patient. Muscle biopsies were obtained under basal and insulin-stimulated conditions as described in Subjects and Methods after 0 (), 2 (), and 24 () h fat infusion. Data are from n = 5–7. ^, P < 0.05 vs. 0 fat. *, P < 0.05 vs. basal.

View larger version (43K): [in this window] [in a new window]   FIG. 4. Basal and insulin-stimulated IRS-1 tyrosine phosphorylation and Akt serine phosphorylation after 0, 2, and 24 h Intralipid infusion in skeletal muscle from control subjects and IGT first-degree relatives of type 2 diabetic patients. Muscle biopsies were prepared as described in Subjects and Methods. Representative immunoblots are shown. Quantified data are presented in Figs. 2, A and D, and 3, A and D.

IRS-1-associated PI 3-kinase activity. In a subset of IGT relatives (n = 4) and control subjects (n = 5), sufficient material was available to assess IRS-1-associated PI 3-kinase in basal and insulin-stimulated muscle (Figs. 2C and 3C). Basal IRS-1-associated PI 3-kinase activity increased from 0 to 2 h fat infusion, whereas insulin-stimulated IRS-1-associated PI 3-kinase activity was not significantly altered by fat infusion. Short- and long-term fat infusion did not affect basal, insulin-stimulated, or incremental IRS-1-associated PI 3-kinase activity in control subjects.

Akt phosphorylation (41). Basal Akt phosphorylation increased after 24 h fat infusion in the IGT relatives but not in the controls (Figs. 2D, 3D, and 4). Insulin-stimulated or incremental Akt phosphorylation was not affected by short- or long-term fat infusion in any of the study groups. Akt phosphorylation was stimulated by insulin both with and without fat infusion in IGT relatives and control subjects. Akt protein expression was not altered by fat infusion in the total study population (0, 2, and 24 h: 2.5 ± 0.6 vs. 2.0 ± 0.3 vs. 3.0 ± 0.5 arbitrary units, NS).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the present study, individuals with a high a priori risk for developing type 2 diabetes, defined as glucose intolerant first-degree relatives of type 2 diabetic patients, and an age-, body mass index-, and sex-matched control group underwent Intralipid infusion 2 and 24 h before, and continuously throughout, hyperinsulinemic euglycemic clamps. In contrast to most previous studies (4, 5, 8, 11, 12, 13, 14), we intended to mimic FFA levels observed in type 2 diabetic patients by performing low-grade Intralipid infusion. Skeletal muscle biopsies were performed during basal and insulin-stimulated conditions. Interestingly, we found that even low-grade Intralipid infusion reduced whole-body glucose disposal by approximately 25% in both study groups after both short- and long-term fat infusion. Insulin resistance was observed in both the oxidative and nonoxidative pathway in the control group, whereas only the oxidative pathway was impaired in the IGT relatives. In fact, a time-dependent increase in glucose oxidation was found in both groups. Furthermore, we found that the IGT-relatives had lower lipid oxidation during insulin stimulation compared with the control subjects. Finally, we did not find that the fat-induced insulin resistance was explained by impairment of proximal components in the insulin signaling transduction cascade in skeletal muscle.

Whole-body Rd was reduced around 25% in both study groups after both short- and long-term fat infusion. Several previous studies consistently found that fat infusion decreases glucose oxidation within 1–2 h of fat exposure (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 25), whereas a decrease in whole-body glucose uptake as well as in nonoxidative glucose metabolism, or storage, was not seen until after 3–6 h combined insulin and fat infusion (3, 8, 10, 14). In this study, applying a low-grade physiological iv fat exposure for 2 and 24 h, and measuring insulin action using the gold standard hyperinsulinemic euglycemic clamp technique, we did not find any evidence supporting the notion of any time dependency of the detrimental effect of intravenous fat infusion on in vivo insulin action and/or muscle insulin signal transduction in man. In fact, we found significant impairment(s) of both total Rd, GOX, and EGS rates in the control subjects after only 2 h low-grade fat exposure before the clamp, and only 2 h combined fat and insulin infusion. The discrepancy between the present and published data may be ascribed to the fact that in our study, subjects were older and more obese and therefore perhaps more sensitive to the deleterious effects of FFAs. Alternatively, the presumed time-dependent effect of FFAs on insulin action might simply be related to the effect of performing a prolonged (4–6.5 h) supraphysiological fat infusion (2 mM during insulin infusion) in combination with prolonged hyperinsulinemia (400–800 pM) (6, 9).

GOX

Basal GOX was reduced after both short- and long-term fat infusion in IGT relatives, whereas only short-term fat infusion caused reduction of basal GOX in the control subjects. Insulin-stimulated GOX was reduced after both short- and long-term fat infusion in IGT relatives and control subjects. Basal and insulin-stimulated LIPOX was correspondingly increased in both groups, indicating substrate competition and operation of the glucose fatty acid cycle as described previously in other studies (3, 4, 5, 6, 8, 9, 10, 11, 13, 15). However, instead of a further impairment of GOX after long-, compared with short-term, fat infusion, we found a somewhat paradoxical significant increase in both basal and insulin-stimulated GOX from 2- to 24-h fat infusion in the controls, indicating that the impairment of GOX by fat infusion to some extent may be transient. The same trend was found during insulin stimulation in the IGT relatives, although this did not reach statistical significance. To our knowledge this compensatory increase in glucose oxidation after long-, compared with short-term, fat infusion has not been observed previously, and the mechanism responsible for this potentially important phenomenon is unknown. Theoretically a compensatory increase in transcription of enzymes involved in glucose oxidation may occur. Nevertheless, the finding of a time-dependent increase in GOX may account for the minor (or no) defects in oxidative, as compared with nonoxidative glucose metabolism in some nondiabetic insulin-resistant states, with a putative influence of elevated FFAs such as nondiabetic first-degree relatives of type 2 diabetic patients (42).

LIPOX/lipid clearance

Despite similar basal LIPOX rates in IGT relatives and controls, IGT relatives had significantly lower LIPOX during insulin stimulation when studied with and without prior fat infusion. Furthermore, IGT relatives tended to have higher plasma FFA concentrations during insulin stimulation. This is consistent with the report of increased postprandial plasma triglyceride concentrations in glucose-tolerant first-degree relatives, compared with matched controls (43). Another study found normal average FFA levels in the face of elevated insulin concentrations in a 24-h study simulating daily living in first-degree relatives (44). Furthermore, diminished plasma FFA uptake and oxidation has been observed in males with impaired glucose tolerance and type 2 diabetic patients, as compared with healthy controls (45). The explanation for the reduced fatty acid uptake, oxidation, and clearance rates is unknown, but its presence certainly adds to the hazards associated with fat intake in prediabetic as well as in diabetic subjects.

EGS (Rd-GF)

Insulin-stimulated EGS was unaffected by fat infusion in the IGT relatives, whereas after both short- and long-term fat exposure, insulin-stimulated EGS was reduced in controls to a comparable level as observed in the IGT relatives. Thus, the IGT relatives displayed severe impairment of insulin stimulated EGS even without fat infusion. Short-term fat exposure decreased EGS in young, healthy subjects (5, 8, 10), whereas FFAs either had no effect on (13) or impaired EGS in type 2 diabetic patients (4). In the present study, the low-grade fat infusion was not an adequate stimulus for further impairment of EGS in IGT relatives. This is consistent with the hypothesis that elevated plasma FFAs might affect EGS at a very early stage in the development of overt type 2 diabetes, perhaps even contributing to insulin resistance in younger relatives of type 2 diabetic patients with fully normal glucose tolerance (46).

GF

The inhibition of total GOX in the face of a total lack of inhibition of GF by short and/or long term fat infusion in both study groups is consistent with previous findings of a predominant inhibition of GOX from endogenous depots (i.e. glycogenolysis), as compared with glucose derived from the extracellular pool (47).

EGP

In some previous studies, FFA infusion partially reversed the insulin-induced suppression of EGP (3, 4, 7, 12), whereas no effect of fat on EGP was found in at least one other study besides the present study (48). Differences between study designs, fat infusion rates, and/or study subjects, ours being elderly obese subjects with and without IGT and family history of type 2 diabetes, may explain the different results. We found that insulin suppressed EGP approximately 50% in IGT relatives, whereas insulin did not suppress EGP in the controls. This phenomenon might, to some extent, be explained by our study design. We speculate that the hyperinsulinemia and hyperglycemia during the IVGTT might have increased glucose storage in the liver (i.e. glycogen synthesis), resulting in less insulin-induced suppression of glycogenolysis during the clamp. Wise et al. (49) found that a relatively high hepatic glycogen content increased both the absolute rate of EGP and glycogenolysis (the higher glycogen content, the higher EGP) and decreased percent contribution of gluconeogenesis to EGP but did not alter hepatic insulin action.

The IGT relatives tended to have lower EGP than the controls during the clamp, which might be explained by lower hepatic glycogen content in the IGT relatives facilitating insulin suppression of EGP. In support of this notion, ¹³C nuclear magnetic resonance spectroscopy revealed 50% lower hepatic glycogen concentrations and decreased glycogenolysis in poorly controlled type 2 diabetic patients, compared with matched control subjects 4 h after ingesting a meal (50).

Insulin signal transduction

The absolute values of basal or insulin-stimulated IRS-1 tyrosine phosphorylation, PI 3-kinase activity, IRS-1-associated PI 3-kinase activity, or Akt serine phosphorylation in skeletal muscle were not different between IGT relatives or control subjects after short- or long-term low-grade fat infusion. In contrast, basal and insulin-stimulated PI 3-kinase activity increased significantly after 2- and 24-h fat infusion in the total study population. This is in contrast to previous reports of decreased IRS-1-associated PI 3-kinase activity and PI 3-kinase activity in young healthy subjects (11, 51). However, in contrast to our study, higher plasma FFA concentrations and longer-lasting insulin infusions were applied in considerably younger and leaner subjects in former studies (11, 51).

The effect of low-grade fat exposure (0.5 mM) on a more proximal step in the insulin signaling transduction cascade (i.e. insulin receptor tyrosine autophosphorylation) was determined in healthy young subjects (52). Whereas insulin-stimulated glucose uptake was reduced 15% after low-grade fat infusion, insulin receptor autophosphorylation was normal. Our finding of increased basal and insulin-stimulated PI 3-kinase activity might be a compensatory phenomenon, with the aim to overcome other fat-induced defects of signal transduction, glucose transport or glycogen synthesis. The exact mechanism responsible for this compensation is unknown, although subtle fat-induced increments of plasma glucose and insulin concentrations might play a role.

Insulin stimulation did not alter IRS-1 tyrosine phosphorylation, PI 3-kinase, or IRS-1-associated PI 3-kinase activity in skeletal muscle from IGT relatives without fat infusion. Conversely, in skeletal muscle from control subjects, insulin increased IRS-1 tyrosine phosphorylation, PI 3-kinase, and IRS-1-associated PI 3-kinase activity without fat infusion, whereas insulin infusion did not alter phosphorylation/ activity of any of these signal transduction parameters in control subjects when studies were performed with short- and long-term fat infusion. Finally, insulin increased Akt serine phosphorylation in IGT relatives and control subjects both with and without fat infusion. The fat-induced attenuation of insulin signaling at the proximal steps in the insulin signal cascade in skeletal muscle from control subjects was not explained by significantly decreased absolute values of insulin-stimulated IRS-1 tyrosine phosphorylation, tyrosine-associated PI 3-kinase, or IRS-1-associated PI 3-kinase activity but rather as discussed above by a tendency toward a fat-induced increase of basal signal transduction. Interestingly, low-grade fat-infusion-induced alterations of proximal signal transduction in skeletal muscle from control subjects were similar to impairments found in skeletal muscle from IGT relatives without fat infusion (26). Low-grade fat infusion did not further impair insulin signaling (i.e. incremental values) in IGT relatives. We cannot exclude the possibility that this relative defect of insulin responsiveness may contribute to the fat-induced in vivo insulin resistance in this group. Moreover, exposure to higher plasma FFA concentrations and/or FFA exposure for a longer period of time might cause primary impairments of the insulin signaling transduction cascade. The lack of insulin responsiveness in the insulin signaling parameters after fat infusion in the controls may only be of relatively minor importance for the fat-induced insulin resistance even in this group. In our study of low-grade fat infusion in middle-aged obese subjects at increased risk for type 2 diabetes, we interpret these findings to indicate that changes in the insulin signaling parameters are not the primary cause of the fat-induced whole-body insulin resistance. Instead, distal or parallel insulin signaling defects or even altered spatial intracellular organization of signaling parameters (53, 54) may play a quantitatively greater role for the fat-induced insulin resistance.

In summary, using the euglycemic clamp technique in combination with tritiated glucose tracer infusion, indirect calorimetry and studies in skeletal muscle biopsies, we demonstrated that low-grade short- and long-term fat infusion induces (or enhances) in vivo insulin resistance in middle-aged obese subjects with IGT and a family history of diabetes and in age- and weight-matched controls. Whereas short- and long-term low-grade fat infusion caused quantitatively similar relative impairments of insulin-stimulated glucose uptake in the two study groups, our results provide evidence to suggest that several potentially important time- and hereditary-dependent differences have an impact on cellular total GOX, GF, glucose storage, and LIPOX. Importantly, low-grade fat-induced insulin resistance was not fully explained by significant impairments of proximal insulin signal transduction parameters in either IGT relatives or control subjects.

	Acknowledgments

 
We thank all the subjects who participated in the study for their cooperation; Susanne Reimer and Sussi Polmann (Hvidovre Hospital) for excellent technical assistance; and lector, Ph.D. Huiling Mu for performing lipid analyses of the Intralipid emulsion.

	Footnotes

 
This work was supported by grants from the Novo-Nordisk Foundation; Danish Research Council; Danish Diabetes Association; Association of Danish Female Doctors; Grosserer C. P. Frederiksens Foundation, Beckett Foundation; Direktør E. Danielsen and Wife’s Foundation; Eli Lilly’s Diabetes Research Foundation; Handelsgartner Ove V. B. Olsen’s and Edith B. Olsen’s Foundation; Ebba Celinders Foundation; Christian the 3rd Foundation; H:S Research Foundation; the Swedish Medical Research Council; and the Swedish Diabetes Association. H.S. was granted a research fellowship scholarship from the Faculty of Medicine, University of Copenhagen, Denmark.

Abbreviations: DTT, Dithiothreitol; ECL, enhanced chemiluminescence; EGP, endogenous glucose production; EGS, exogenous glucose storage; FFA, free fatty acid; GF, glycolytic flux; GOX, glucose oxidation; IGT, impaired glucose tolerance; IR, insulin receptor; IRS, IR substrate; IVGTT, intravenous glucose tolerance test; LIPOX, lipid oxidation; NOGM, nonoxidative glucose metabolism; OGTT, oral glucose tolerance test; PI 3-kinase, phosphoinositide 3-kinase; PVDF, polyvinylidene difluoride; Ra, glucose appearance rate; Rd, glucose disposal rate; SA, specific activity.

Received July 17, 2003.

Accepted December 1, 2003.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD 1988 Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020–1024[Abstract]
2. McGarry JD 1992 What if Minkowski had been ageusic? An alternative angle on diabetes. Science 258:766–770[Abstract/Free Full Text]
3. Boden G, Chen X, Ruiz J, White JV, Rossetti L 1994 Mechanisms of fatty acid-induced inhibition of glucose uptake. J Clin Invest 93:2438–2446[Medline]
4. Boden G, Chen X 1995 Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest 96:1261–1268[Medline]
5. Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW, Shulman GI 1996 Mechanism of free fatty acid-induced insulin resistance in humans. J Clin Invest 97:2859–2865[Medline]
6. Thiebaud D, DeFronzo RA, Jacot E, Golay A, Acheson K, Maeder E, Jequier E, Felber JP 1982 Effect of long chain triglyceride infusion on glucose metabolism in man. Metabolism 31:1128–1136[CrossRef][Medline]
7. Bevilacqua S, Bonadonna R, Buzzigoli G, Boni C, Ciociaro D, Maccari F, Giorico MA, Ferrannini E 1987A cute elevation of free fatty acid levels leads to hepatic insulin resistance in obese subjects. Metabolism 36:502–506
8. Bonadonna RC, Zych K, Boni C, Ferrannini E, DeFronzo RA 1989 Time dependence of the interaction between lipid and glucose in humans. Am J Physiol 257(1 Pt 1):E49–E56
9. Felley CP, Felley EM, van Melle GD, Frascarolo P, Jequier E, Felber JP 1989 Impairment of glucose disposal by infusion of triglycerides in humans: role of glycemia. Am J Physiol 256(6 Pt 1):E747–E752
10. Boden G, Jadali F, White J, Liang Y, Mozzoli M, Chen X, Coleman E, Smith C 1991 Effects of fat on insulin-stimulated carbohydrate metabolism in normal men. J Clin Invest 88:960–966[Medline]
11. Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI 1999 Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 103:253–259[Medline]
12. Ferrannini E, Barrett EJ, Bevilacqua S, DeFronzo RA 1983 Effect of fatty acids on glucose production and utilization in man. J Clin Invest 72:1737–1747[Medline]
13. Bevilacqua S, Buzzigoli G, Bonadonna R, Bevilacqua S, Buzzigoli G, Bonadonna R, Brandi LS, Oleggini M, Boni C, Geloni M, Ferrannini E 1990 Operation of Randle’s cycle in patients with NIDDM. Diabetes 39:383–389[Abstract]
14. Roden M, Krssak M, Stingl H, Gruber S, Hofer A, Furnsinn C, Moser E, Waldhausl W 1999 Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans. Diabetes 48:358–364[Abstract]
15. Randle PJ, Garland PB, Hales CN, Newsholme EA 1963 The glucose fatty-acid cycle: its role in insulin sensitivity and the metabolism disturbances of diabetes mellitus. Lancet 1:785–789[Medline]
16. Randle PJ, Garland PB, Newsholme EA, Hales CN 1965 The glucose fatty acid cycle in obesity and maturity onset diabetes mellitus. Ann NY Acad Sci 131:324–333[Medline]
17. Vaag AA, Handberg A, Skott P, Richter EA, Beck-Nielsen H 1994 Glucose-fatty acid cycle operates in humans at the levels of both whole body and skeletal muscle during low and high physiological plasma insulin concentrations. Eur J Endocrinol 130:70–79[Abstract/Free Full Text]
18. Krook A, Björnholm M, Galuska D, Jiang XJ, Fahlman R, Myers Jr MG, Wallberg-Henriksson H, Zierath JR 2000 Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes 49:284–292[Abstract]
19. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem 269:3568–3573[Abstract/Free Full Text]
20. Coffer PJ, Jin J, Woodgett JR 1998 Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335(Pt 1):1–13
21. Schmitz-Peiffer C 2000 Signalling aspects of insulin resistance in skeletal muscle: mechanisms induced by lipid oversupply. Cell Signal 12:583–594[CrossRef][Medline]
22. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI 1999 Free fatty acid-induced insulin resistance is associated with activation of protein kinase C and alterations in the insulin signaling cascade. Diabetes 48:1270–1274[Abstract]
23. Boden G, Chen X, Rosner J, Barton M 1995 Effects of a 48-h fat infusion on insulin secretion and glucose utilization. Diabetes 44:1239–1242[Abstract]
24. Carpentier A, Mittelman SD, Bergman RN, Giacca A, Lewis GF 2000 Prolonged elevation of plasma free fatty acids impairs pancreatic ß-cell function in obese nondiabetic humans but not in individuals with type 2 diabetes. Diabetes 49:399–408[Abstract]
25. Paolisso G, Gambardella A, Amato L, Tortoriello R, D’Amore A, Varricchio M, D’Onofrio F 1995 Opposite effects of short- and long-term fatty acid infusion on insulin secretion in healthy subjects. Diabetologia 38:1295–1299[Medline]
26. Storgaard H, Song XM, Jensen CB, Madsbad S, Björnholm M, Vaag A, Zierath JR 2001 Insulin signal transduction in skeletal muscle from glucose intolerant relatives of type 2 diabetic patients. Diabetes 50:2770–2778[Abstract/Free Full Text]
27. Storgaard H, Jensen CB, Vaag AA, Vølund A, Madsbad S 2003 Insulin secretion after short- and long-term low-grade free fatty acid infusion in men with increased risk of developing type 2 diabetes. Metabolism 52:885–894[CrossRef][Medline]
28. Gotfredsen A, Baeksgaard L, Hilsted J 1997 Body composition analysis by DEXA by using dynamically changing samarium filtration. J Appl Physiol 82:1200–1209[Abstract/Free Full Text]
29. McGuire EA, Helderman JH, Tobin JD, Andres R, Berman M 1976 Effects of arterial versus venous sampling on analysis of glucose kinetics in man. J Appl Physiol 41:565–573[Abstract/Free Full Text]
30. DeFronzo RA, Tobin JD, Andres R 1979 Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol (Endocrinol Metab) 237:E214–E223
31. Hother-Nielsen O, Beck-Nielsen H 1990 On the determination of basal glucose production rate in patients with type 2 (non-insulin-dependent) diabetes mellitus using primed-continuous 3–3H-glucose infusion. Diabetologia 33:603–610[CrossRef][Medline]
32. Molina JM, Baron AD, Edelman SV, Brechtel G, Wallace P, Olefsky JM 1990 Use of a variable tracer infusion method to determine glucose turnover in humans. Am J Physiol 258(1 Pt 1):E16–E23
33. Frayn KN 1983 Calculation of substrate oxidation rates in vivo from gaseous exchange. J Appl Physiol 55:628–634[Abstract/Free Full Text]
34. Ferrannini E 1988 The theoretical bases of indirect calorimetry: a review. Metabolism 37:287–301[CrossRef][Medline]
35. Vaag A, Alford F, Henriksen FL, Christopher M, Beck-Nielsen H 1995 Multiple defects of both hepatic and peripheral intracellular glucose processing contribute to the hyperglycaemia of NIDDM. Diabetologia 38:326–336[Medline]
36. Krook A, Whitehead JP, Dobson SP, Griffiths MR, Ouwens M, Baker C, Hayward AC, Sen SK, Maassen JA, Siddle K, Tavare JM, O’Rahilly S 1997 Two naturally occurring insulin receptor tyrosine kinase domain mutants provide evidence that phosphoinositide 3-kinase activation alone is not sufficient for the mediation of insulin’s metabolic and mitogenic effects. J Biol Chem 272:30208–30214[Abstract/Free Full Text]
37. Steele R 1959 Influence of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Sci 82:420–430[Medline]
38. Del Prato S, Bonadonna RC, Bonora E, Gulli G, Solini A, Shank M, DeFronzo RA 1993 Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus. J Clin Invest 91:484–494[Medline]
39. Rossetti L, Giaccari A 1990 Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake. A dose-response euglycemic clamp study in normal and diabetic rats. J Clin Invest 85:1785–1792[Medline]
40. Hother-Nielsen O, Henriksen JE, Staehr P, Beck-Nielsen H 1995 Labelled glucose infusate technique in clamp studies. Is precise matching of glucose specific activity important? Endocrinol Metab 2:275–287
41. Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727–736[CrossRef][Medline]
42. Vaag A, Henriksen JE, Beck-Nielsen H 1992 Decreased insulin activation of glycogen synthase in skeletal muscles in young nonobese Caucasian first-degree relatives of patients with non-insulin-dependent diabetes mellitus. J Clin Invest 89:782–788[Medline]
43. Axelsen M, Smith U, Eriksson JW, Taskinen MR, Jansson PA 1999 Postprandial hypertriglyceridemia and insulin resistance in normoglycemic first-degree relatives of patients with type 2 diabetes. Ann Intern Med 131:27–31[Abstract/Free Full Text]
44. Nyholm B, Walker M, Gravholt CH, Shearing PA, Sturis J, Alberti KG, Holst JJ, Schmitz O 1999 Twenty-four-hour insulin secretion rates, circulating concentrations of fuel substrates and gut incretin hormones in healthy offspring of type II (non-insulin-dependent) diabetic parents: evidence of several aberrations. Diabetologia 42:1314–1323[CrossRef][Medline]
45. Mensink M, Blaak EE, van Baak MA, Wagenmakers AJ, Saris WH 2001 Plasma free fatty acid uptake and oxidation are already diminished in subjects at high risk for developing type 2 diabetes. Diabetes 50:2548–2554[Abstract/Free Full Text]
46. Perseghin G, Ghosh S, Gerow K, Shulman GI 1997 Metabolic defects in lean nondiabetic offspring of NIDDM parents: a cross-sectional study. Diabetes 46:1001–1009[Abstract]
47. Wolfe BM, Klein S, Peters EJ, Schmidt BF, Wolfe RR 1988 Effect of elevated free fatty acids on glucose oxidation in normal humans. Metabolism 37:323–329[CrossRef][Medline]
48. Groop LC, Bonadonna RC, Shank M, Petrides AS, DeFronzo RA 1991 Role of free fatty acids and insulin in determining free fatty acid and lipid oxidation in man. J Clin Invest 87:83–89[Medline]
49. Wise S, Nielsen M, Rizza R 1997 Effects of hepatic glycogen content on hepatic insulin action in humans: alteration in the relative contributions of glycogenolysis and gluconeogenesis to endogenous glucose production. J Clin Endocrinol Metab 82:1828–1833[Abstract/Free Full Text]
50. Magnusson I, Rothman DL, Katz LD, Shulman RG, Shulman GI 1992 Increased rate of gluconeogenesis in type II diabetes mellitus. A 13C nuclear magnetic resonance study. J Clin Invest 90:1323–1327[Medline]
51. Kruszynska YT, Worrall DS, Ofrecio J, Frias JP, Macaraeg G, Olefsky JM 2002 Fatty acid-induced insulin resistance: decreased muscle PI3K activation but unchanged Akt phosphorylation. J Clin Endocrinol Metab 87:226–234[Abstract/Free Full Text]
52. Gumbiner B, Mucha JF, Lindstrom JE, Rekhi I, Livingston JN 1996 Differential effects of acute hypertriglyceridemia on insulin action and insulin receptor autophosphorylation. Am J Physiol 270(3 Pt 1):E424–E429
53. Clark SF, Martin S, Carozzi AJ, Hill MM, James DE 1998 Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton. J Cell Biol 140:1211–1225[Abstract/Free Full Text]
54. Inoue G, Cheatham B, Emkey R, Kahn CR 1998 Dynamics of insulin signaling in 3T3–L1 adipocytes. Differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2. J Biol Chem 273:11548–11555[Abstract/Free Full Text]

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