Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and I[kappa]B-[alpha]

Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and I[kappa]B-[alpha] – Rapid Publication

Samar I. Itani

Work by several laboratories the over past 10 years has provided evidence that elevated plasma free fatty acid (FFA) levels are responsible for much of the insulin resistance present in obese subjects. The evidence can be summarized as follows: 1) most obese people have elevated plasma levels of FFA (1,2); 2) acute elevations of plasma FFA produce insulin resistance dose-dependently in diabetic and nondiabetic individuals (3-6); and 3) chronically elevated plasma FFA levels cause insulin resistance, as demonstrated by the finding that lowering elevated plasma FFA levels overnight (from ~600 to ~300 [micro]mol/l) normalized insulin sensitivity in obese nondiabetic subjects and significantly improved it in obese diabetic patients (7). The mechanisms by which elevated levels of FFA produce insulin resistance are not well understood. However, the observation that acutely increasing plasma FFAs decreased insulin-stimulated glucose uptake, glycogen synthesis (8), and phosphatidylinositol (PI) 3-kinase activity in skeletal muscle (9) suggested that FFAs interfered with insulin signaling.

Interestingly, it takes 2-4 h for insulin resistance to develop after an acute elevation in plasma FFA and an equally long time for insulin resistance to disappear after plasma FFA levels return to normal (3). The long (2-4 h) delay suggested an indirect rather than direct effect of FFAs. In support of this notion, acute increases in plasma FFAs have been shown to cause increases in intramyocellular triglyceride (IMCL-TG) content in soleus muscle of healthy volunteers. The increase in IMCL-TG occurred several hours after the elevation of plasma FFA levels and coincided with the development of insulin resistance (10), suggesting that an insulin resistance–causing signal was generated during the synthesis (or the breakdown) of IMCL-TG. We hypothesize that diacylglycerol (DAG) may be such a signal because an increase in its synthesis would coincide with that of triglycerides (11) and because it is a well-known allosteric activator of protein kinase C (PKC) (12-14), an enzyme that has been linked to insulin resistance in muscle in a wide variety of rodent models (11,15,16), including rats infused with lipid (17) and massively obese humans (18,19). To evaluate this possibility, DAG mass and PKC activity and distribution were assayed in skeletal muscle biopsies obtained from normal human volunteers in whom insulin resistance was produced by raising plasma FFA levels during a euglycemic-hyperinsulinemic clamp. Because PKC is known to activate NF-[kappa]B (20,21) and because NF-[kappa]B has recently been linked to fatty acid–induced impairment of insulin action in muscle in rodents (22,23), we have also examined the possibility that the development of insulin resistance in these individuals was related to alterations in the IKK/I[kappa]B/NF[kappa]B pathway. In addition, the accumulation of ceramide, another lipid metabolite linked to insulin resistance, was evaluated (24).


Subjects. Twelve healthy men participated in these studies. Each subject’s age, weight, height, and body composition are shown in Table 1. The two groups differed modestly (mid nonsignificantly) with respect to age and BMI; however, far greater differences altered neither the time of onset nor the magnitude of FFA-induced insulin resistance (G.B., unpublished observations). None of the participants had a family history of diabetes or other endocrine disorders or were taking medications. Their body weights were stable for at least 2 months, and their diets contained a minimum of 250 g/day of carbohydrate for at least 2 days before the studies. Informed written consent was obtained from all subjects after explanation of the nature, purpose, and potential risks of these studies. The study protocol was approved by the Institutional Review Board of Temple University Hospital.

Experimental design. All subjects were admitted to the Temple University Hospital General Clinical Research Center the day before the studies. At 6:00 P.M. they ingested a meal of 14 kcal/kg body wt consisting of 53% carbohydrate, 15% protein, and 32% fat. The studies began at 8:00 A.M. the following day, with the subjects reclining in bed. A short polyethylene catheter was inserted into an antecubital vein for infusion of isotopes. Another catheter was placed in a contralateral forearm vein for blood sampling. Tiffs arm was wrapped with a heating blanket (~79[degrees]C) to arterialize venous blood. The following studies were performed:

Study 1 was a 6-h euglycemic-hyperinsulinemic clamp during which plasma FFA levels decreased to very low levels.

Study 2 was a 6-h euglycemic-hyperinsulinemic clamp with simultaneous intravenous infusion of lipid plus heparin. Plasma FFA levels rose because lipolysis of the infused fat exceeded insulin mediated antilipolysis.

Muscle biopsies were performed before, during (~2 h), and at the end (~6 h) of the clamps.

Materials. The PKC assay kit was purchased from Amersham Life Science (Arlington Heights, IL). PKC antibodies (polyclonal) were purchased from Santa Cruz technologies (Santa Cruz, CA). Unless otherwise specified, all other reagents were purchased from either Sigma Chemical (St. Louis, MO) or Fisher Scientific (Springfield, NJ).

Euglycemic-hyperinsulinemic clamping with and without lipid/heparin. Regular human insulin (Humulin R; Elli Lilly, Indianapolis, IN) was infused intravenously at a rate of 7 pmol * [kg.sup.-1] * [min.sup.-1] for 6 h and plasma glucose concentrations were clamped at ~5 mmol by a feedback-controlled variable glucose infusion (studies 1 and 2). Glycerol (2.14 g/100 ml) was co-infused with insulin in study 1 to match the glycerol content of the Liposyn II infused in study 2. In study 2, euglycemic-hyperinsulinemic clamping was performed as described above. In addition, Liposyn II (Abbott Laboratories, North Chicago, IL), a 20% triglyceride emulsion (10% safflower and 10% soybean oil) containing 2.14 g/100 ml glycerol plus heparin (0.4 units * [kg.sup-1] * [min.sup.-1]) was infused at a rate of 1.5 ml/min for 6 h. Serial measurements of rates of glucose turnover and substrate and hormone analyses were obtained before and during the clamps.

Muscle biopsies. Biopsies were obtained from the lateral aspect of the vastus lateralis muscle ~15 cm above the patella from all subjects as described (3). The excised muscle (~150 mg) was dropped immediately into isopentane kept at its freezing point (-160[degrees]C) by liquid nitrogen. The frozen muscle was stored at -80[degrees]C until it was aliquotted for measurement of PKC bioactivity and isoform distribution, DAG and ceramide mass, and I[Kappa]B[alpha] a abundance.

Glucose turnover. Glucose turnover was determined with 3-[[sup 3]H]glucose, which was infused intravenously for 8 h, starting with a bolus of 40 [micro]Ci followed by a continuous infusion of 0.4 [micro]Ci/min. This produced steady-state tracer specific activities within 120 min. Glucose was isolated from blood for determination of 3-[[sup.3]H]glucose specific activity as described (25). Rates of total body glucose appearance ([G.sub.Ra]) and disappearance ([G.sub.Rd]) were calculated using Steele’s equation for non-steady-state conditions (26). Rates of endogenous glucose production were obtained by subtracting rates of glucose infused to maintain euglycemia from [G.sub.Ra].

Body composition. Body composition was determined by bioelectrical impedance analysis (27).

Substrate and hormone analyses. Plasma glucose was measured with a glucose analyzer (YSI, Yellow Springs, OH). Insulin was determined by radioimmunoassay using an antiserum with minimal (0.2%) cross-reactivity with proinsulin (Linco, St. Charles, MO) Plasma FFA concentrations were determined with a kit from Wako Pure Chemical (Richmond, VA).

Western analysis of PKC and I[kappa]B-[alpha]. PKC western analysis was performed as described elsewhere (19) with some modifications. In brief, frozen muscle tissue was homogenized in 20 mmol/l Tris, pH 7.4, containing 10 mmol/l EDTA, 2 mmol/l EGTA, 100 mmol/l [beta]-glycerophosphate, 0.05 mg/ml phenylmethylsuffonyl fluoride, 1 mg/ml leupeptin, and 0.1 mg/ml aprotinin. Samples were centrifuged in a Beckman air fuge at 160,000g for 20 min to separate the cytosolic fraction. The pellet was resuspended with the same homogenization buffer with 0.2% Triton added, sonicated (Heat systems sonicator, microtip), and left on ice for 1 h. The homogenates were then recentrifuged and the resultant supernatant labeled as the membrane (particulate) fraction. Samples were loaded at a protein concentration of 50 [micro]g/lane onto an 8 and 12% SDS-polyacrylamide gel for PKC and I[kappa]B-[alpha], respectively. PKC was determined in the cytosolic and membrane fractions, whereas I[kappa]B-[alpha] was determined in the cytosolic fraction only. Proteins were electrotransferred onto a polyvinylidene fluoride microporous membrane and isoform-specific polyclonal PKC or I[kappa]K-[alpha] antibodies (Santa Cruz, CA) at a dilution of 1:1,000 were added to the membranes. Membranes were subjected to enhanced chemiluminescence reagent (Western blot chemiluminescence reagent; Dupont). Multiple autoradiographs (to establish linearity) were quantitated using the National Institutes of Health (NIH) image analysis software (free distribution by NIH, Bethesda, MD). PKC-[beta]I and -[beta]II both ran as doublets when blotted with their respective antibodies. For purposes of comparison and because the two bands sometimes merged, both bands were included in the densitometric analysis.

PKC enzyme assay, PKC activity was determined as described elsewhere (18) using the PKC enzyme assay system (Amersham Life Science). Skeletal muscle was homogenized and the cytosolic and particulate fractions separated as described above. Samples were incubated at 37[degrees]C for 15 min. The phosphorylated peptide was separated using binding paper disks, and disks were counted for 10 min using Beckman LS 6500 (Beckman Instruments, Fullerton, CA).

DAG and ceramide content. The measurement of DAG content was determined as described (28). In brief, lipids were extracted from muscle biopsies using chloroform:methanol:PBS + 0.2% SDS (1:2:0.8). Diacylglycerol kinase and [[gamma][sup.-32]P]ATP (15[micro]Ci/[micro]mol cold ATP) were added to extracts, and the reaction was stopped using chloroform:methanol (2:1). Samples were run on thin-layer chromatography plates in chloroform:acetone:methanol:acetic acid:water (100:40:20:20:10). The DAG and ceramide bands were counted in a Beckman LS 6500 (Beckman Instruments).

Statistical analysis. All data are expressed as means [+ or -] SE. Statistical analysis was performed using the SAS program (SAS Institute, Cary, NC). ANOVA with repeated measures was used to determine the differences in [G.sub.Rd], PKC activity and distribution, I[kappa]B-[alpha] abundance, and DAG and ceramide content across time points. A Student’s t test and paired nonparametric test (Wilcoxon’s signed-rank test) for each time point was then performed if the overall comparison was statistically significant. Because of our finding that PKC measurements in human muscle tend to be more variable than in rodents (S.I. and N.R., unpublished data), all measurements, including a few that were >2 SD from the group mean, were included in the statistical analysis.


Glucose, insulin, FFA, and [G.sub.Rd]. Basal glucose concentrations were 5.1 [+ or -] 0.1 and 5.3 [+ or -] 0.3 mmol/l in the insulin (study 1) and the insulin plus lipid (study 2) groups, respectively (Fig. 1). Mean clamp glucose concentrations were 5.3 [+ or -] 0.2 and 5.1 [+ or -] 0.2 mmol/l in the two groups. Basal insulin levels were 49 [+ or -] 20 and 66 [+ or -] 17 pmol/l, and mean insulin levels during the clamps were 428 [+ or -] 19 and 403 [+ or -] 11 pmol/l in the insulin and insulin plus lipid groups, respectively.


Plasma FFA concentrations decreased from 470 [+ or -] 117 [micro]mol/l (at 0 min) to 66 [+ or -] 15 [micro]mol/l (at 360 min, P < 0.001) in the insulin group. In the insulin plus lipid group, plasma FFA increased from 308 [+ or -] 77 [micro]mol/l (at 0 min) to 1,259 [+ or -] 274 [micro]mol/l (at 360 min, P < 0.001).

[G.sub.Rd] rose from 10.9 [+ or -] 0.9 [micro]mol * [kg.sup.-1] * [min .sup.-1](at 0 min) to 52.7 [+ or -] 8.2 [micro]mol * [kg.sup.-1] * [min.sup.-1] (at 360 min) in the insulin group and from 11.8 [+ or -] 0.9 to 30.0 [+ or -] 5.3 [micro]mol * [kg.sup.-1] * [min.sup.-1] in the insulin plus lipid group. The difference between the two groups of 43% (P < 0.05) at 360 min was similar to that in previous reports (3,5,8). Likewise, in agreement with earlier studies (3-5,8), no significant difference was observed between the groups at ~2 h.

To investigate whether the lipid-induced insulin resistance was associated with alterations in DAG, ceramide, and PKC, we have assessed DAG and ceramide content, total PKC activity, and the distribution of specific PKC isoforms in muscle biopsies taken at 0, ~2, and ~6 h.

Total PKC activity. No change in total PKC activity was observed in either the insulin plus lipid or the insulin alone groups after ~2 h (Fig. 2a and b). However, after ~6 h, significant increases in PKC activity in the membrane (~4-fold) and cytosolic (~3-fold) fractions were observed in the insulin plus lipid group, suggesting that increases in total PKC protein and translocation to the cell membrane had occurred.


PKC distribution of specific isoforms. To determine which PKC isoforms were responsible for the increased PKC activity in the membrane and cytosolic fractions of the subjects infused with lipid, Western analysis was performed using isoform-specific antibodies. As shown in Figs. 2c and d and Fig. 3, a significant (~6-fold) increase in membrane PKC-[delta] protein compared with the value at 0 h was observed in subjects infused with insulin plus lipid at ~6 h, whereas no increase was observed in the group infused with insulin alone. As with PKC activity, no change in abundance in either fraction was observed at ~2 h.


A similar increase (~8-fold) of membrane PKC-[beta]II was observed in the insulin plus lipid group at ~6 h (Fig. 2e and f and Fig. 3). However, in contrast to PKC-[delta], PKC-[beta]II content in the cytosol was increased ~4-fold. Thus, the increase in membrane PKC-[beta]II protein at ~6 h was attributable to an increase in its synthesis as well as translocation. Interestingly, membrane-associated PKC-[beta]II abundance was increased ~3-fold at ~2 h compared with 0 time in the lipid-infused group, although the difference did not achieve statistical significance (P = 0.058). The doublets seen in PKC-[beta]II and [beta]I (Fig. 3) may reflect different phosphorylation states of the proteins.

No significant alterations in PKC-[epsilon], -[theta], or -[zeta] protein content occurred in the cytosolic or membrane fractions of either group (Table 2 and Fig. 3). PKC-[beta]I in the membrane appeared to show the same trend as PKC-[beta]II; however, due to the small number of muscles biopsy samples available (n = 3), the observed increase in its content was not statistically significant (Table 2).

DAG and ceramide content. Infusion of insulin plus lipids (but not insulin) resulted in a >3-fold increase in DAG mass in muscle biopsied at ~6 h but not at 2 h (Fig. 2g). In contrast, no significant increase in ceramide was observed at any time (not shown).

I[kappa]B-[alpha] degradation. The abundance of I[kappa]B-[alpha] was significantly decreased (~3-fold) in muscle biopsied from subjects infused with insulin plus lipids for ~6 h compared with its preclamp value and compared with the value in insulin alone group (Fig. 2h and Fig. 3). No change was seen at 2 h.


Raising plasma FFA levels during a euglycemic-hyperinsulinemic clamp causes insulin resistance (diminished rate of [G.sub.Rd]) in human muscle within 3-6 h (6). The results of the present study demonstrate that the development of insulin resistance in this setting was associated temporally with 1) a fourfold increase in total membrane-associated PKC activity, 2) translocation of the PKC-[beta] and -[delta] isoforms from the cytosol to the cell membrane, 3) a threefold increase in DAG mass, and 4) a 70% decrease in the abundance of I[kappa]B-[alpha], an inhibitor of NF[kappa]B. In addition, no change in the content of ceramide, a molecule implicated in the pathogenesis of fatty acid-induced insulin resistance in cultured muscle cells (24), was found.

The increase in PKC activity was presumably related to the increase in the concentration of DAG, a potent allosteric activator of both conventional and novel PKC isoforms (12-14). It was associated with translocation of the PKC isoforms-[beta]II and -[delta] (but not PKC-[epsilon] or -[theta]) to the membrane fraction, which is generally considered a sign of PKC activation (12). In contrast, in a study in rats, in which lipid was infused during euglycemic-hyperinsulinemic clamping, Griffin et al. (17) reported translocation of PKC-[theta]. PKC activity and DAG mass were not measured in their study nor was the timing of PKC-[theta] translocation relative to the development of insulin resistance investigated. Whether the reported differences in PKC isoform responses to lipid infusions, i.e., PKC-[beta] and -[delta] in humans (this study) and -[theta] in rats (17), reflect species differences remains to be established. This question aside, these studies together clearly suggest that the insulin resistance caused by fat infusion during a euglycemic-hyperinsulinemic clamp is associated with activation of one or more PKC isoforms. The data do not allow us to state with certainty that changes in PKC antedate the insulin resistance, although a distinct trend for PKC-[beta]II translocation to occur before the other events (Fig. 2e and f and Fig. 3) is at least suggestive. DAG content was not altered at 2 h; however, an early increase in de novo synthesized DAG could have been missed. Future studies with a larger number of subjects and at times between 2 and 6 h could resolve this question.

A linkage between DAG-associated PKC activation and insulin resistance in skeletal muscle had been suggested by early studies in denervated rat muscle (16). In keeping with this, increases in DAG content and altered PKC activity and distribution have been observed in a wide variety of insulin-resistant states in rodents including fat-feeding, obesity, glucose infusion, inactivity, and type 2 diabetes (11,15,29,30). Furthermore, improvement in insulin sensitivity after exercise in the fat-fed rat is associated with a restoration of PKC distribution toward a control pattern (30). More recently, alterations in PKC activity and distribution have been found in obese, insulin-resistant humans, both with (31) and without (19) diabetes. In general, alterations in PKC-[theta] and -[epsilon] have been described in the rat, whereas in humans alterations in both PKC-[beta] (19 and this study) and PKC-[theta] (31) have been reported. The significance of these differences in the isoform(s) affected remains to be established.

Increases in intramuscular triglycerides and DAG (10) presumably occur during a lipid infusion because the increased FFA uptake exceeds its oxidation. Presumably, observed increases in DAG (and PKC) would have been less pronounced without the glucose and insulin infusions (15), because the high rate of glucose uptake during the clamp increases the intramuscular concentration of malonyl-CoA (15), an inhibitor of carnitine palmitoyl transferase-1. This would decrease the oxidation of FFA by inhibiting their transfer from the cytosol into mitochondria and secondarily increase their esterification. In addition, a high rate of glucose uptake would increase the concentration of [alpha]-glycerophosphate.

Ceramide is another FFA metabolite linked to insulin resistance. Increased levels of ceramide were first described in insulin-resistant muscle of the Zucker rat by Turinsky et al. (32). Since then, several studies have shown that C2-ceramide can inhibit insulin-stimulated glucose transport, glycogen synthesis, and Akt activation in adipocytes. In addition, exogenous palmitate has been shown to inhibit insulin-stimulated glycogen synthesis and Akt activation in C2C12 myotubes by a mechanism dependent on increased de novo synthesis of ceramide (24). In the present study, we found no increase in ceramide mass in skeletal muscle of lipid-infused men, suggesting that ceramide did not contribute to their insulin resistance. On the other hand, because ceramide synthesis was not assessed, the possibility that a small de novo synthesized ceramide pool might have played a role cannot be ruled out.

Activation of PKC could lead to insulin resistance by several mechanisms. PKC has been shown to serine/ threonine phosphorylate both the insulin receptor (19,33-35) and IRS-1 (36,37), leading to impaired insulin signaling. In keeping with this possibility, inhibition of insulin-stimulated PI3-kinase activation and decreased IRS tyrosyl phosphorylation have been shown to accompany the increase in membrane-associated PKC-[theta] in rats infused with lipid during euglycemic-hyperinsulinemic clamping (17).

Another possibility is that activation of PKC caused insulin resistance by Increasing oxidative stress and by activating IKK and/or the NF[kappa]B pathway. It has been demonstrated that FFA and hyperglycemia can increase oxidative stress and activate NF[kappa]B in endothelium and that these effects are mediated by PKC (38-41). Activation of NF[kappa]B can be initiated by the phosphorylation of the inhibitor I[kappa]B and its subsequent release from NF[kappa]B (42). Activation of PKC can set these events in motion by directly phosphorylating I[kappa]B (42) or by causing the generation of reactive oxygen species that can secondarily activate I[kappa]B-kinase (IKK-[beta]) (Fig. 4). In fact, phosphorylation by IKK-[beta] is considered the main pathway by which I[kappa]B-[alpha] is released from NF[kappa]B and subsequently subjected to ubiquitination and proteosomal degradation. The result is a decrease in I[kappa]B-[alpha] mass and movement of NF[kappa]B from the cytosol to the nucleus. That such a series of events occurred in the present study is strongly suggested by the marked decrease in I[kappa]B-[alpha] mass in the subjects infused with lipid.


Two recent studies have implicated IKK and, by inference, I[kappa]B and NF[kappa]B in file pathogenesis of insulin resistance in rodent skeletal muscle. Kim et al. (22) have shown in rats that the inhibition of insulin-stimulated PI3-kinase activation caused by infusing lipid can be prevented by a high dose of salicylate (an inhibitor of IKK-[beta]). Inhibiting IKK-[beta] would stabilize I[kappa]B and prevent activation of NF[kappa]B. The same group has found that lipid infusion during a euglycemic-hyperinsulinemic clamp does not cause insulin resistance in IKK-[beta] knockout mice, whereas overexpressing IKK-[beta] increases insulin resistance (22,23). The results of the present study offer additional support for a role of the IKK-[beta]/I[kappa]B-([alpha]/NF[kappa]B pathway in the pathogenesis of fatty acid-induced insulin resistance in human muscle, although as already noted, activation of PKC could cause insulin resistance by direct effects on IRS and other molecules in the insulin-signaling cascade. Whether IKK-[beta] is altered in this situation, and if so, whether it too has direct effects on insulin signaling remains to be determined. A hypothetical scheme that depicts how the metabolic events initiated by infusing lipid/heparin during euglycemic-hyperinsulinemia could lead to changes in I[kappa]B and NF[kappa]B and insulin resistance is presented in Fig. 4.

Lastly, the IKK-[beta]/I[kappa]B-[alpha]NF[kappa]B pathway is a major pro-inflammatory pathway (20), and inflammatory processes are now recognized to play a pivotal role in the pathogenesis of coronary artery disease (43). Hence, activation of the IKK-[beta]/[kappa]B-([alpha]/NF[kappa]B pathway by FFA may explain at least some of the increased prevalence of coronary artery disease in obese patients with type 2 diabetes, because almost all of these patients have increased plasma FFA levels and are insulin resistant.


Study subjects

Insulin +

Insulin lipid P

Sex 6 men 6 men NS

Age (years) 30.3 [+ or -] 3.8 40.7 [+ or -] 3.4 NS

Height (cm) 178.3 [+ or -] 2.9 183.8 [+ or -] 3.4 NS

Weight (kg) 74.0 [+ or -] 3.5 85.6 [+ or -] 5.7 NS

Lean body mass (kg) 61.7 [+ or -] 1.4 66.0 [+ or -] 2.3 NS

BMI (kg/[m.sup.2]) 23.1 [+ or -] 13 25.3 [+ or -] 1.4 NS

Data are means [+ or -] SE.


Distribution of PKC isoforms in the membrane and cytosolic fractions


0h -2h


[beta]I 0.03 [+ o r -] 0.03 0.05 [+ o r -] 0.02

[epsilon] 0.27 [+ o r -] 0.13 0.84 [+ o r -] 0.75

[theta] 0.44 [+ o r -] 0.18 0.98 [+ o r -] 0.86

[zeta] 0.06 [+ o r -] 0.61 0.07 [+ o r -] 0.01


[beta]I 0.02 [+ o r -] 0.02 0.04 [+ o r -] 0.02

[epsilon] 0.34 [+ o r -] 0.19 0.21 [+ o r -] 0.08

[theta] 0.29 [+ o r -] 0.11 0.14 [+ o r -] 0.05

[zeta] 0.03 [+ o r -] 0.01 0.05 [+ o r -] 0.01

Glycerol Lipid

-6h 0h


[beta]I 0.086 [+ o r -] 0.05 0.05 [+ o r -] 0.02

[epsilon] 0.16 [+ o r -] 0.06 0.21 [+ o r -] 0.11

[theta] 0.14 [+ o r -] 0.04 0.23 [+ o r -] 0.11

[zeta] 0.07 [+ o r -] 0.01 0.10 [+ o r -] 0.001


[beta]I 0.03 [+ o r -] 0.01 0.06 [+ o r -] 0.02

[epsilon] 0.22 [+ o r -] 0.09 0.25 [+ o r -] 0.11

[theta] 0.14 [+ o r -] 0.06 0.15 [+ o r -] 0.06

[zeta] 0.05 [+ o r -] 0.01 0.06 [+ o r -] 0.003


-2h -6h


[beta]I 0.17 [+ o r -] 0.03 0.52 [+ o r -] 0.3

[epsilon] 0.55 [+ o r -] 0.43 0.23 [+ o r -] 0.05

[theta] 0.45 [+ o r -] 0.29 0.20 [+ o r -] 0.05

[zeta] 0.10 [+ o r -] 0.01 0.11 [+ o r -] 0.01


[beta]I 0.08 [+ o r -] 0.02 0.07 [+ o r -] 0.04

[epsilon] 0.18 [+ o r -] 0.08 0.02 [+ o r -] 0.01

[theta] 0.18 [+ o r -] 0.06 0.13 [+ o r -] 0.01

[zeta] 0.04 [+ o r -] 0.003 0.05 [+ o r -] 0.001

Data are means [+ or -] SE. n = 6 for PKC-[theta] and [epsilon],

and n = 3 of densitometric units for PKC-[beta]I and -[epsilon].

See legend to Fig. 2C and D for details


This work was supported by National Institutes of Health Grants R01-AG-07988 (to G.B.) and P01-HL-55854/JDF F 99 6004 (to N.R.), the General Clinical Research Center branch of the National Center for Research Resources (Grant RR-349), and a grant from the Juvenile Diabetes Foundation (JDF 1-20004-319) (to N.R.). In addition, S.I.I. was the recipient of a mentor-based training award from the American Diabetes Association and a traineeship from the NHLBI (HL 07224-25) during the course of these studies.

The authors thank the nurses of the General Clinical Research Center for help with the studies and for excellent patient care, and Constance Harris Crews for typing the manuscript. They also gratefully acknowledge the advice of Dr. John Keaney in carrying out the I[kappa]B-[alpha] studies.


(1.) Gorden E: Non-esterified fatty acids in blood of obese and lean subjects Am J Clin Nutr 8:740-747, 1960

(2.) Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD: Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020-1024, 1988

(3.) Boden G, Jadali F, White J, Liang Y, Mozzoli M, Chen X, Coleman E, Smith C: Effects of fat on insulin-stimulated carbohydrate metabolism in normal men. J Clin Invest 88:960-966, 1991

(4.) Boden G, Chen X: Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest 96:1261-1268, 1995

(5.) Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW, Shulman GI: Mechanism of free fatty acid-induced insulin resistance in humans. J Clin Invest 97:2859-2865, 1996

(6.) Boden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46:3-10, 1997

(7.) Santomauro AT, Boden G, Silva ME, Rocha DM, Santos RF, Ursich MJ, Strassmann PG, Wajchenberg BL: Overnight lowering of free fatty acids with Acipimox improves insulin resistance and glucose tolerance in obese diabetic and nondiabetic subjects. Diabetes 48:1836-1841, 1999

(8.) Boden G, Chen X, Ruiz J, White JV, Rossetti L: Mechanisms of fatty acid-induced inhibition of glucose uptake. J Clin Invest 93:2438-2446, 1994

(9.) Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI: Effects of free fatty acids on glucose transport mid IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 103:253-259, 1999

(10.) Boden G, Lebed B, Schatz M, Homko C, Lemieux S: Effects of acute changes of plasma free fatty acids on intramyocellular fat content and insulin resistance in healthy subjects. Diabetes 50:1612-1617, 2001

(11.) Schmitz-Peiffer C, Browne CL, Oakes ND, Watkinson A, Chisholm DJ, Kraegen EW, Biden TJ: Alterations in the expression and cellular localization of protein kinase C isozymes epsilon and theta are associated with insulin resistance in skeletal muscle of the high fat-fed rat. Diabetes 46:169-178, 1997

(12.) Farese R: 2000. Protein kinase C. In Diabetes Mellitus: A Fundamental and Clinical Text. Philadelphia, Lippincott, p. 239-251

(13.) Bronfman M, Morales MN, Orellana A: Diacylglycerol activation of protein kinase C is modulated by long-chain acyl-CoA. Biochem Biophys Res Commun 152:987-992, 1988

(14.) Nishizuka Y: Protein kinase C and lipid signaling for sustained, cellular responses. Faseb J 9:484-496, 1995

(15.) Ruderman NB, Saha AK, Vavvas D, Witters LA: Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol 276: E1-E18, 1999

(16.) Heydrick SJ, Ruderman NB, Kurowski TG, Adams HB, Chen KS: Enhanced stimulation of diacylglycerol and lipid synthesis by insulin in denervated muscle: altered protein kinase C activity and possible link to insulin resistance. Diabetes 40:1707-1711, 1991

(17.) Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI: Free fatty acid-induced insulin resistance is associated with activation of protein kinase C-[tau] and alterations in the insulin signaling cascade. Diabetes 48:1270 1274, 1999

(18.) Cortright RN, Azevedo JL Jr, Zhou Q, Sinha M, Pories WJ, Itani SI, Dohm GL: Protein kinase C modulates insulin action in human skeletal muscle. Am J Physiol Endocrinol Metab 278:E553-E562, 2000

(19.) Itani SI, Zhou Q, Pories W J, MacDonald KG, Dohm GL: Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity. Diabetes 49:1353-1358, 2000

(20.) Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066-1071, 1997

(21.) Luberto C, Yoo DS, Suidan HS, Bartoli GM, Hanun YA: Differential effects of sphingomyelin hydrolis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts. J Biol Chem 275: 14760-14766, 2000

(22.) Kim JK, Rim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI: Prevention of fat-induced insulin resistance by salicylate. J Clin Invest 198:437-446, 2001

(23.) Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE: Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkebeta. Science 293:1673-1677, 2001

(24.) Schmitz-Peiffer C, Craig DL, Biden TJ: Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. J Biol Chem 274:24202-24210, 1999

(25.) Shimoyama R, Ray TK, Savage, CR Jr, Owen OE, Boden G: In vivo and in vitro effects of antiinsulin receptor antibodies. J Clin Endocrinol & Metab 59:916-23, 1984

(26.) Steele R, Wall JS, DeBodo RC, Altszuler N: Measurement of size and turnover rate of body glucose pool by the isotope dilution method. Am J Physiol 187:15-24, 1956

(27.) Lukaski HC: Methods for the assessment of human body composition: traditional and new. Am J Clin Nutr 46:537-556, 1987

(28.) Preiss J, Loomis CR, Bishop WR, Stein R, Niedel JE, Bell RM: Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem 261:8697-8600, 1986

(29.) Turinsky J, Bayly BP, O’Sullivan DM: 1,2-Diacylglycerol and ceramide levels in rat skeletal muscle and liver in vivo: studies with insulin, exercise, muscle denervation, and vasopressin. J Biol Chem 265:7933-7938, 1990

(30.) Bell KS, Schmitz Peiffer C, Lim-Fraser M, Biden TJ, Cooney GJ, Kraegen EW: Acute reversal of lipid induced muscle insulin resistance is associated with rapid alteration in PKC-theta localization. Am J Physiol Endocrinol Metab 279:E1196-E1201, 2000

(31.) Itani SI, Pories WJ, Macdonald KG, Dohm GL: Increased protein kinase C theta in skeletal muscle of diabetic patients. Metabolism 50:553-557, 2001

(32.) Turinsky J, O’Sullivan DM, Bayly BP: 1,2 Diacylglycerol and ceramide levels in insulin resistant tissues of the rat in vivo. J Biol Chem 265:16880-16885, 1990

(33.) Takayama S, White MF, Kahn CR: Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem 263:3440-3447, 1988

(34.) Bollag GE, Roth RA, Beaudoin J, Mochly-Rosen D, Koshland DE Jr: Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity. Proc Natl Acad Sci U S A 83:5822-5824, 1986

(35.) Chin JE, Dickens M, Tavare JM, Roth RA: Overexpression of protein kinase C isoenzymes alpha, beta I, gamma, and epsilon in cells overexpressing the insulin receptor: effects on receptor phosphorylation and signaling. J Biol Chem 268:6338-6347, 1993

(36.) De Fea K, Roth RA: Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612. Biochemistry 36:12939-12947, 1997

(37.) Ravichandran LV, Esposito DL, Chen J, Quon MJ: Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin. J Biol Chem 276:3,543-3549, 2001

(38.) Pieper GM, Riaz ul H: Activation of nuclear factor-kappaB in cultured endothelial cells by increased glucose concentration: prevention by calphostin C. J Cardiovasc Pharmacol 30:528-532, 1997

(39.) Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H: High glucose level and flee fatty acid stimulate reactive oxygen species production through proteIn kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 49:1939-1945, 2000

(40.) Dichtl W, Nilsson L, Goncalves I, Ares MP, Banfi C, Calara F, Hamsten A, Eriksson P, Nilsson J: Very low-density lipoprotein activates nuclear factor-kappaB in endothelial cells. Circ Res 84:1085-1094, 1999

(41.) Coudronniere N, Villalba M, Englund N, Altman A: NF-kappa B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-theta. Proc Natl Acad Sci U S A 97:3394-399, 2000

(42.) Ghosh S, Baltimore D: Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature 344:678-682, 1990

(43.) Ross R: Atherosclerosis–an inflammatory disease. N Engl J Med 340:115-126, 1999

Samar I. Itani, (1) Neil B. Ruderman, (1) Frank Schmieder, (2) and Guenther Boden (3)

From the (1) Diabetes Unit, Section of Endocrinology and Departments of Medicine and Physiology, Boston University Medical Center, Boston, Massachusetts; the (2) Department of Surgery and the General Clinical Research Center, Temple University Hospital, Philadelphia, Pennsylvania; and the (3) Division of Endocrinology, Diabetes, mid Metabolism and the General Clinical Research Center, Temple University Hospital, Philadelphia, Pennsylvania.

Address correspondence and reprint requests to Dr. Guenther Boden, Temple University Hospital, 3401 N. Broad St., Philadelphia, PA 19140. E-mail:

Received for publication 6 March 2002 and accepted in revised form 6 May 2002. Posted on the World Wide Web at rapidpubs.shtml on 7 June 2002.

DAG, diacylglycerol; FFA, free fatty acid; [G.sub.Ra], total body glucose appearance; [G.sub.Rd], total body glucose disappearance; IKK, I[kappa]B kinase; IMCL-TG, intramyocellular triglyceride; NF, nuclear factor; NIH, National Institutes of Health; PI, phosphatidylinositol; PKC, protein kinase C.

COPYRIGHT 2002 American Diabetes Association

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