Mechanisms of insulin resistance in cystic fibrosis

Dana S. Hardin1,2,3, Adrian Leblanc3, Gailen Marshall2, and Dan K. Seilheimer3

1 University of Texas Southwestern Medical School, Dallas 75390; 2 University of Texas Health Science Center, and 3 Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cystic fibrosis (CF) is associated with a high incidence of diabetes. Studies evaluating causes of CF-related diabetes (CFRD) have consistently documented decreased insulin secretion. In patients with CFRD, insulin sensitivity has been documented to be decreased, but controversy exists in patients with normal or impaired glucose tolerance (IGT). We undertook this study 1) to reexplore insulin sensitivity in patients with IGT and 2) to evaluate potential mechanisms of insulin resistance in CF, including GLUT-4 translocation, elevation of serum cytokines, and free fatty acid (FFA) levels. We recruited nine CF subjects with impaired glucose tolerance (IGTCF) and nine age-, gender-, and body mass index-matched control volunteers. Each underwent a hyperinsulinemic euglycemic clamp (200 mU · m-2 · min-1) to measure insulin sensitivity. A muscle biopsy was obtained at maximal insulin stimulation for measure of GLUT-4 translocation with sucrose gradients. An oral glucose tolerance test and National Institutes of Health (NIH) clinical status scores were measured in all volunteers. We also measured tumor necrosis factor (TNF)-alpha levels and FFA in all subjects. Additionally, we report the results of TNF-alpha and FFA in 32 CF patients previously studied by our group. Results were that glucose disposal rate (GDR) was significantly lower in the CFIGT subjects than in controls, indicative of impaired insulin action. GLUT-4 translocation was impaired in CF and correlated with GDR. TNF-alpha levels were higher in all CF subjects than in controls and correlated with GDR. There was no difference in FFA between CF and control subjects. Modified NIH clinical status scores were inversely correlated with GDR and TNF-alpha levels. We conclude that IGTCF patients have decreased peripheral insulin sensitivity. Mechanisms include elevation of TNF-alpha and impaired translocation of GLUT-4.

insulin resistance; cytokines; tumor necrosis factor-alpha ; free fatty acids


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is a common inherited disease affecting one in 2,500 newborns. Diabetes and abnormal glucose tolerance are common in CF, affecting almost 75% of patients over the age of 25 (1, 16). The etiology of diabetes remains somewhat puzzling. Decreased insulin secretion has been well documented (19, 41) and occurs even in patients with normal glucose tolerance (NGT) (41, 42). The principal defects that cause decreased insulin secretion are fibrosis and fatty infiltration of beta -cells. However, autopsy studies (36, 53) do not correlate percentage of damaged islets with development of diabetes. Thus other defects must be present to account for the high incidence of abnormal glucose tolerance in this population. Previous studies by our group (24, 25) and others (38, 43) have documented decreased insulin sensitivity in CF patients with frank diabetes [CF-related diabetes (CFRD)]. However, controversy exists regarding insulin sensitivity in nondiabetic CF subjects, with reports of normal (12, 43), enhanced (3, 43), or decreased (4, 24, 25) insulin action. One purpose of our study was to reexamine peripheral insulin sensitivity in CF subjects with impaired glucose tolerance (IGT).

Many potential factors can cause decreased insulin sensitivity in CF, and exploration of these factors may help us better understand the discrepancies in various studies. The glucose transporter protein GLUT-4 is the major glucose transport protein involved in insulin-stimulated glucose disposal. Translocation of GLUT-4 from the intracellular compartment to the cell surface is necessary for the normal transport of glucose into the cell, and abnormalities in GLUT-4 trafficking have been reported in type 2 diabetics (17). Translocation of GLUT-4 relies on normal endocytosis (50, 51). CF is caused by genetic alteration of a transport-linked gene, the CF transmembrane conductance regulator (CFTR), and past studies (27, 45) have suggested that abnormal CFTR may alter normal endosome fusion and exocytosis. We measured GLUT-4 translocation with the use of sucrose gradients and differential centrifugation to test our hypothesis that abnormal translocation of GLUT-4 from the intracellular compartment to the plasma membrane occurs in CF patients.

Patients with CF experience chronic long-term infection, even when not acutely ill. In normal volunteers, Yki-Järvinen et al. (56) have demonstrated decreased insulin sensitivity during acute illness. Therefore, it is highly possible that insulin sensitivity is decreased in CF patients, secondary to chronic low-grade infection. Elevation of cytokines occurs during acute and chronic illness, and elevation of the cytokine tumor necrosis factor-alpha (TNF-alpha ) has been postulated to contribute to insulin resistance. Hotamisligil et al. (29) demonstrated increased TNF-alpha levels in insulin-resistant subjects, and Ofei et al. (47) demonstrated improved insulin sensitivity in type 2 diabetics by infusion of synthetic antibodies to TNF-alpha . Elevated TNF-alpha levels have been reported from bronchoalveolar lavage samples (8) and from plasma (39, 54) in CF patients. Given these previous reports, another purpose of our study was to correlate insulin sensitivity with plasma TNF-alpha levels in CF subjects and controls.

Investigators (34, 49) have documented a close link between free fatty acid (FFA) levels and insulin resistance. Additionally, a relationship between FFA and TNF-alpha may exist independent of insulin sensitivity. One previous study (28) has described that elevated TNF-alpha levels result in decreased FFA levels. Levy et al. (39) reported a correlation between TNF-alpha levels and lipid and lipoprotein levels in CF. We therefore wished to evaluate total and individual FFA levels and their relationship to TNF-alpha and insulin sensitivity in CF subjects. Thus our study reexplores insulin sensitivity in CF patients with IGT and examines potential mechanisms of decreased insulin sensitivity.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

We recruited nine CF patients (2 female, 7 male), ages 22-32 yr, for participation in this study. Seven were recruited from the CF center clinics at Texas Children's Hospital and Methodist Hospital/Baylor College of Medicine. Two additional CF subjects were recruited from the University of Oklahoma, Tulsa, OK. All subjects were medically stable at the time of the study, with no hospital admissions for 6 wk and no home intravenous medications for >= 1 mo preceding the study. No subject had used oral or intravenous corticosteroids for >= 3 mo before the study. No subject was colonized with Burkholderia cepacia, and neither female subject was pregnant. All CF subjects had IGT, as determined by a 3-h oral glucose tolerance test (OGTT) and National Diabetes Data Group (NDDG) criterion (46) performed within 1 mo of the clamp. Clinical status of the CF subjects was measured using a modified National Institutes of Health (NIH) scoring system (52).

Nine control volunteers (3 female, 6 male) were recruited by advertisement. They were matched to CF subjects for body mass index, age, and sex. No one was an endurance-trained athlete, a physical state known to enhance insulin sensitivity (21), and none had an eating disorder. All control volunteers had normal physical examinations, complete blood counts, and serum chemistries. None had any history of chronic illness, and all were normally glucose tolerant by NDDG criteria. In all subjects, lean body mass (LBM) was measured by dual-energy X-ray absorptiometry.

All protocols were approved by the Committee for the Protection of Human Subjects at the University of Texas Health Science Center. CF and control subjects all gave written, informed consent.

In addition to subjects specifically recruited for this study, cytokine and FFA levels were measured in frozen plasma samples from 32 CF subjects and 19 controls who had been previously studied (24, 25) with the use of similar techniques but who had not had a muscle biopsy. The purpose of including this group was to enlarge the subject number for evaluation of the role of cytokines and FFA on insulin sensitivity. Inclusion of this group also allowed exploration in patients with NGT as well as frank diabetes.

In Vivo Measurements

Hyperinsulinemic euglycemic clamp. Each subject was admitted to the General Clinical Research Center the night before the clamp study and fasted for 12 h before the clamp study. Normal saline was administered overnight through either a 9-in. intravenous catheter placed in an antecubital vein or through an existing indwelling catheter. The next morning, a small intravenous catheter was placed retrograde in the subject's hand and kept warm with a heating pad. A primed continuous infusion of regular human insulin (Humulin, Eli Lilly, Indianapolis, IN) was administered at a rate of 200 mU · m-2 · min-1 for 3 h. Serum glucose was clamped (14) at euglycemia by infusion of 20% dextrose to maintain serum glucose at 88-95 mg/dl.

Plasma glucose was measured every 5-10 min from the retrograde intravenous catheter. Throughout the study, K2HPO4 was infused to prevent hypokalemia and hypophosphatemia. Maximum glucose disposal rate (GDR) was calculated on the basis of the glucose infusion rate needed to maintain euglycemia during the final 20 min of the clamp and before muscle biopsy.

Percutaneous muscle biopsy. A percutaneous muscle biopsy of the vastus lateralis was performed during the final 15 min of the clamp study on each subject. A 3.0-mm side-cutting Bergstrom needle (DePuy, Warsaw, IN) was inserted through a small incision in locally anesthetized skin (2% lidocaine) ~15 cm above the patella on the anterolateral thigh. Multiple passes were made with the biopsy needle to obtain >380 mg of muscle. After each pass with the biopsy needle, the muscle sample was immediately blotted on sterile cloth and then snap frozen in liquid nitrogen within 15 s. Frozen samples were stored at -70°C for future analysis of GLUT-4 translocation.

In Vitro Measurements

Analytical analysis used during the clamp. Plasma glucose concentrations were measured during the clamp study by means of the glucose oxidase method with a YSI glucose analyzer (Yellow Springs Instrument, Yellow Springs, OH). Serum-free insulin levels were measured using radioimmunoassay and a double-antibody technique (Coat-A-Count; Diagnostic Products, Los Angeles, CA) (44).

Skeletal muscle fractionation and marker enzyme analyses. Muscle samples were removed from the freezer, weighed, and immediately placed in buffer (pH 7.4) containing 20 mmol/l Tris base, 1 mmol/l EDTA, 0.25 mmol/l sucrose, and protease inhibitors, including leupeptin, aprotinin, pepstatin, and phenylmethylsulfonyl fluoride. The tissue was homogenized on ice by use of a Polytron (Brinkmann, Westbury, NY) for four 5-s bursts at a setting of four. Further homogenization was obtained using a Caframo stirrer-type homogenizer (Arthur H. Thomas, Philadelphia, PA). The homogenate was then centrifuged at 2,000 rpm at 4°C for 15 min. The resulting postnuclear supernatant represented the total membrane fraction.

Membrane subfractionation was carried out according to Holloszy et al. (26), as modified by Garvey et al. (17). Briefly, the total membrane fraction was placed on a discontinuous sucrose gradient (25, 30, and 35%, wt/vol) and centrifuged at 150,000 g at 4°C for 16 h in a swinging bucket rotor. Membranes were collected from each sucrose layer and washed by 10-fold dilution in the Tris-EDTA-sucrose buffer. Finally, the membrane proteins were recovered by high-speed centrifugation (45,000 rpm) at 4°C for 120 min. The final pellet from each sucrose layer was collected and resuspended in buffer. These samples were frozen for later analysis of GLUT-4 content.

Immunoblot analyses. Each homogenate and membrane fraction was analyzed for protein content according to the method of Bradford (9), with crystalline bovine serum albumin serving as the standard. Membrane proteins (50 µg) were solubilized in Laemmli sample buffer (37) and resolved by SDS-PAGE on 1.5-mm slab gels containing 10% polyacrylamide. Proteins were then electrophoretically transferred to nitrocellulose filters (55). The filters were incubated with specific antiserum against the carboxy terminus of rat GLUT-4 (Eastacre Biologicals, Southbridge, MA), followed by incubation with 125I-labeled protein A, and then quantified using gamma -counting. To quantitatively compare different gels, 50 µg of protein of internal standard (both a rat standard and a human standard) were loaded onto every gel.

To standardize recovery of GLUT-4, we measured total protein in the homogenate and in each of the membrane fractions in all individuals of both subgroups. To determine the purity and recovery of plasma membranes, we measured the plasma membrane marker enzyme 5'-nucleotidase in the homogenate and in each sucrose fraction according to the methods of Avruch and Wallach (5).

Measurement of FFA. Serum levels of oleic, palmitic, and stearic FFA were measured by radioisotope dilution in the subjects specifically recruited for this study, as well as in frozen samples (never thawed before assay) collected during our previous studies. Briefly, 250 µl of internal standard solution (400 µmol/l heptadecanoic acid) were added to 150 µl of plasma. FFA were extracted by adding hydrochloric acid-hexane-1-propanol (1:25:25 vol/vol/vol). Derivatization was carried out with addition of N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide-acetonitril (1:1 vol/vol) to produce t-butyldimethylsilyl derivatives. Quantitation of the fatty acid derivatives was carried out by gas chromatography-mass spectrometry (Hewlett-Packard 5989A system) in the electron impact mode. The ratio of internal standard to each fatty acid was calculated using standard containing the internal standard and fatty acids between 0 and 600 µl. Plasma levels were determined using calibration graphs and measured ion ratios. Samples were processed by Metabolic Solutions Laboratory, Boston, MA.

TNF-alpha . The cytokine TNF-alpha was measured as previously described (2) by means of ELISA (Millennia, Diagnostic Product, Los Angeles, CA).

Statistical Analysis

All results are given as means ± SD. Statistical significance between mean data in CF and controls was determined using Student's t-test. The cytokine and FFA measures were analyzed by ANOVA. Linear regression analysis was used for correlations according to the method of Pearson. Significance was assessed at the P < 0.05 level. All analyses were performed by a statistician using an SAS software package.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

All CF subjects specifically recruited for this study had IGT, and control volunteers were normally glucose tolerant. Table 1 reviews the subject characteristics, including OGTT results. The data for serum cytokines and FFA include the previously collected samples (24, 25). These subjects were also categorized by OGTT. Eight had IGT, 11 had NGT, and 10 were diabetic. All previous control volunteers were normally glucose tolerant.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Subject characteristics

Hyperinsulinemic Euglycemic Clamp

Glucose levels were clamped at 5.0 ± 0.7 mM in all subjects. At an insulin infusion rate of 200 mU · m-2 · min-1, peripheral insulin levels were similar in controls (C) and CF subjects (C, 298 ± 16; CF, 303 ± 12 mU/ml; P = 0.1). Figure 1 compares the mean GDR, as normalized for LBM, in the CF subjects with IGT (IGTCF) and controls specifically recruited for this study. Even when not normalized for LBM, GDR was significantly less in IGTCF than in controls (CF, 11.4 ± 2.5; C, 16.2 ± 0.9; P = 0.002).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Insulin sensitivity, depicted as glucose disposal rate (GDR), was measured using a 200 mU · m-2 · min-1 hyperinsulinemic euglycemic clamp [peripheral insulin levels >300 µU/ml in all subjects, with no differences between cyctic fibrosis (CF) and control subjects]. Data were normalized for lean body mass (LBM). This demonstrates that insulin sensitivity is decreased in CF patients with impaired glucose tolerance (IGT).

GLUT-4 Subfractionation

Sucrose gradient subfractionation of membranes results in three distinct layers, each corresponding to different subcellular locations. Although rodent studies (26, 35) suggest distinct compartments for plasma membrane-associated vesicles, human studies (17) report plasma membrane-associated proteins throughout each subfraction. Therefore, in humans, sucrose subfraction layers are best described by density (17). The 35% sucrose layer is consistent with dense membrane vesicles that are more closely located in the intracellular (plasma membrane-associated) compartment. With the assumption of normal translocation after insulin stimulation, less GLUT-4 should be found in this compartment. The 25 and 30% layers contain lighter vesicles, which are sarcolemma enriched. After insulin stimulation, more GLUT-4 should be in the lighter vesicle compartments. Our results demonstrate that, despite insulin stimulation, the GLUT-4 content of intracellular-associated layer was significantly higher in CF subjects compared with controls (Figs. 2A, 3), corresponding to significantly lower GLUT-4 content in cell surface-associated subfraction (Figs. 2B, 3). These findings are consistent with abnormal subcellular localization of GLUT-4. The 30% layer results are as follows: GLUT-4 content dpm/min: CF, 100 ± 41; C, 210 ± 50; P = 0.04. Additionally, the content of GLUT-4 in the 25% sucrose gradient positively correlated with GDR in all subjects (r = 0.61, P = 0.04). Protein recovery was similar between controls and CF for each subfraction. 5'-Nucleotidase activity was similar in CF and controls.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   GLUT-4 translocation was measured using sucrose gradients (35, 30, and 25% wt/vol) and differential centrifugation. GLUT-4 in each sucrose subfraction was isolated and quantitated using SDS-PAGE immunoblot analysis, anti-GLUT-4 antibody, and 125I label. Mean GLUT-4 content (cpm) is depicted for the CF and control subgroups. A: GLUT-4 content is depicted in the 35% sucrose fraction (dense fraction associated with intracellular components) and demonstrates that more GLUT-4 is found in this subfraction in CF subjects than in controls. B: GLUT-4 content in the 25% sucrose fraction (lighter-density fraction associated with cell surface components) is depicted and demonstrates less GLUT-4 in CF subjects than in controls.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   Representative autoradiograph demonstrating differences in GLUT-4 content in the 35, 30, and 25% sucrose fractions from 2 individual CF patients and 1 control. GLUT-4 migrated as a 47-kDa protein.

TNF-alpha

TNF-alpha levels were measured in plasma from subjects recruited for this study as well as from previous study subjects. Although our current study evaluated only CF subjects with IGT, our previous studies had also measured insulin sensitivity in CF subjects with NGT or diabetes. There was no significant difference between the interleukin (IL)-6 or IL-1beta levels between controls and any CF subgroup.

TNF-alpha levels were significantly higher in all CF subgroups than in controls. Results averaged for all CF subjects and controls are demonstrated by Fig. 4. TNF-alpha levels were not significantly different in CF subgroups; however, CF subjects with IGT (IGTCF) and diabetes (DMCF) tended to have higher levels than CF subjects with NGT (NGTCF) (TNF-alpha pg/ml: IGTCF, 120 ± 5; DMCF, 130 ± 4; NGTCF, 90 ± 5; P = nonsignificant). There was no correlation between GDR and any of the cytokines measured.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Tumor necrosis factor-alpha (TNF-alpha ) levels from 38 CF subjects and 28 control volunteers. TNF-alpha levels are clearly elevated in the CF subjects.

FFA

The FFA palmitic, oleic, and stearic acid were measured in subjects recruited specifically for this study, as well as in previous study subjects (24, 25). Total FFA were similar between CF subjects and controls (Total FFA mmol/ml: CF, 405 ± 247; C, 374 ± 178; P = 0.6). Similarly, there were no differences in CF and control subjects in levels of individual FFA levels. There was no correlation between levels of either total FFA or individual FFA levels and GDR or TNF-alpha .

Clinical Status Scores

The mean NIH clinical status score for the IGTCF subjects specifically recruited for this study was 65 ± 8. Insulin sensitivity (GDR) correlated with NIH clinical status scores (r = 0.75, P = 0.042). There was no correlation between GLUT-4 content in any subfraction and NIH clinical status score. There was an inverse correlation between NIH clinical status score and TNF-alpha levels (r = 0.71, P = 0.03). There was no correlation between free FFA and clinical status scores.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CF patients have a very high incidence of diabetes (1), and insulin resistance plays a role in the high frequency of CFRD (24, 25). However, controversy remains regarding insulin sensitivity in CF patients with abnormal glucose tolerance who do not have CFRD. Our group (23-25) has reported decreased insulin sensitivity in CF subjects with NGT and IGT as well as in patients with CFRD. Similarly, Austin et al. (4) described decreased insulin sensitivity in CF subjects with IGT. However, Moran et al. (43) have reported normal peripheral insulin sensitivity in these patients. Other groups have not clearly distinguished results by glucose tolerance but have reported normal or enhanced insulin sensitivity in CF (3, 12, 38). It is with this controversy in mind that we recruited CF subjects with IGT for participation in these studies. Our current findings confirm previous reports (4, 24, 25) of decreased peripheral insulin sensitivity in CF subjects with IGT. The CF subjects participating in our previous studies, and those in this study, tend to have worse clinical status than CF subjects studied by others (43). We (24) have previously described the relationship between worsened clinical status and insulin resistance. Therefore, it seems likely that patients who have clinically worse disease, as described by worse pulmonary function or other parameters, are more insulin resistant than patients with less severe disease. To date, we have not found enhanced insulin sensitivity in any CF study subject, regardless of clinical status. Perhaps findings by others represent an early metabolic compensation in patients with mild disease. Treatment differences among the CF centers could also explain the discrepancy.

Skeletal muscle is the principal tissue utilized for glucose disposal (13), and GLUT-4 is the principal glucose transporter protein (6) found in skeletal muscle. Studies in diabetic rodents (22) report decreased GLUT-4 quantity in skeletal muscle; however, studies in humans with type 2 diabetes have described normal GLUT-4 quantity in plasma membrane samples from skeletal muscle (48). Investigators have now focused on decreased GLUT-4 function, rather than quantity, as the principal disorder associated with human insulin resistance (30, 31). Recently, Garvey et al. (17) have described abnormal subcellular location of GLUT-4 in type 2 diabetics and insulin-resistant volunteers without diabetes. They report that this abnormal distribution is associated with decreased translocation of GLUT-4.

We measured GLUT-4 translocation by use of methods similar to those of Garvey et al. (17), with muscle samples collected at maximum glucose disposal. Circulating insulin levels were higher than 300 µU/ml, thus allowing us to measure insulin-stimulated GLUT-4 translocation. Our results demonstrate preferential targeting of GLUT-4 to intracellular-associated sucrose gradient fractions in our IGTCF subjects and correspond to lower amounts of GLUT-4 in the lighter vesicle (cell surface-associated) layers. We also found a correlation between GDR and GLUT-4 levels in the cell surface layer (25% sucrose fraction). The differences in GLUT-4 levels between subfractions cannot be explained by differences in protein recovery or by recovery of plasma membranes. Thus our study suggests that one cause of insulin resistance in CF is abnormal translocation of GLUT-4.

One study (43) previously reported no difference between GLUT-4 quantity in CF subjects and controls studied under basal (non-insulin-stimulated) conditions; thus differences in translocation could not be discerned. Furthermore, biopsies were obtained from the exocrine-insufficient subgroup, which was composed of three NGT patients and four IGT subjects. All of these subjects had enhanced insulin sensitivity; thus GLUT-4 levels in the plasma membrane would not be expected to be low. The lack of basal difference in GLUT-4 quantity between CF subjects and controls suggests that our findings under insulin-stimulated conditions result from translocation differences rather than from inherent differences in GLUT-4 quantity.

CF is caused by mutation of a transport-related protein, the CFTR (33). Mutations of this protein have been associated with defects in translocation of ions in the gut (18, 32), and several groups (7, 45) have described abnormal endosomal packaging as a result of CFTR. GLUT-4 resides in a discrete cellular compartment (20, 40) and requires normal endosomal recycling for translocation to the cell surface (27, 51). Thus it is possible that the CFTR mutation, by altering endosomal function, could decrease GLUT-4 trafficking. However, other reasons for IGT, such as chronic elevation of cytokines, could also be responsible for this finding. Further research in this area will be helpful.

In our present study, we have found higher TNF-alpha levels in CF subjects than in controls. Similar findings have been reported by others (39, 54). TNF-alpha levels increase during acute illness, and previous studies indicate that TNF-alpha from sources such as fat (29) and serum (28) affects insulin sensitivity at the level of skeletal muscle. One group (11) has demonstrated downregulation of GLUT-4 in cultured muscle cells and adipocytes by administration of TNF-alpha .

Hotamisligil et al. (28) have demonstrated that TNF-alpha affects the phosphorylation cascade of insulin signaling at the receptor level, and Ofei et al. (47) demonstrated improvement in insulin sensitivity by giving TNF-alpha receptor antibodies. Although we could not find a correlation between TNF-alpha levels and GDR or GLUT-4 levels, we did find a correlation between clinical status scores and TNF-alpha levels. Thus it is likely that TNF-alpha levels either reflect or contribute to worsened clinical status. Without a direct link to glucose disposal, we are reluctant to state that TNF-alpha causes insulin resistance in CF; however, measurement of insulin resistance after use of TNF-alpha neutralizing antibodies would be interesting.

Studies in type 2 diabetes have described decreased insulin sensitivity when circulating FFA levels are elevated (34, 49). Elevation of FFA has been demonstrated in CF (10, 39); thus we wanted to determine a possible relationship between elevated FFA levels and insulin resistance in our subjects. We did not find any difference between FFA levels in control and those in CF subjects, nor did we find differences in individual FFA levels between these two groups. Therefore, it is not surprising that we did not find a relationship between FFA levels and insulin sensitivity. FFA levels were measured at baseline only; thus we cannot rule out specific interactions between fatty acid metabolism and insulin resistance in the fed state. Future studies may elucidate such a relationship.

In conclusion, our current study has supported previous findings by our group (24, 25) and others (4) of decreased peripheral insulin sensitivity in CF patients with impaired glucose tolerance. Additionally, this study has elucidated potential mechanisms of decreased insulin sensitivity, specifically abnormal subcellular localization of GLUT-4 and higher-than-normal TNF-alpha levels. These studies suggest a considerable need for future research in the area of CF-related diabetes and insulin sensitivity.


    ACKNOWLEDGEMENTS

We gratefully acknowledge critical review of the manuscript by Drs. Patrick Brosnan and Robert Moore, and subject recruitment by Dr. Piers Blackett.


    FOOTNOTES

This work was supported by National Institutes of Health Grants 1K08-DK-02365-01 (D. S. Hardin) and M01-RR-02558 (Clinical Research Center, University of Texas) and a grant from the National Cystic Fibrosis Foundation (D. S. Hardin).

Address for reprint requests and other correspondence: D. S. Hardin, Univ. of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas TX 75390-9063 (E-mail: dana.hardin{at}utsouthwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 November 2000; accepted in final form 22 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aarsland, A, Chinkes D, and Wolfe R. Hepatic and whole-body fat synthesis in humans during carbohydrate overfeeding. Am J Clin Nutr 65: 1774-1782, 1997[Abstract].

2.   Agarwal, SK, and Marshall GD. In vivo alteration in type-1 and type-2 cytokine balance: a possible mechanism for elevated total IgE in HIV-infected patients. Hum Immunol 59: 99-105, 1998[ISI][Medline].

3.   Ahmad, T, Nelson R, and Taylor R. Insulin sensitivity and metabolic clearance rate of insulin in cystic fibrosis. Metabolism 43: 163-167, 1994[ISI][Medline].

4.   Austin, A, Kalhan SC, Orenstein D, Nixon P, and Arslanian S. Roles of insulin resistance and beta -cell dysfunction in the pathogenesis of glucose intolerance in cystic fibrosis. J Clin Endocrinol Metab 79: 80-85, 1994[Abstract].

5.   Avruch, J, and Wallach DFH Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta 233: 334-347, 1971[ISI][Medline].

6.   Bell, GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukomoto H, and Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care 13: 198-208, 1990[Abstract].

7.   Biwersi, J, Emans N, and Verkman AS. Cystic fibrosis transmembrane conductance regulator activation stimulates endosome fusion in vivo. Proc Natl Acad Sci USA 93: 12484-12489, 1996[Abstract/Free Full Text].

8.   Bonefield, TL, Panuska JR, and Konstan NM. Inflammatory cytokines in cystic fibrosis lungs. Am J Respir Crit Care Med 152: 2111-2116, 1995[Abstract].

9.   Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle protein-dye bonding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

10.   Clandinin, MT, Zuberbuhler P, Brown NE, Kielo ES, and Goh YK. Fatty acid pool size in plasma lipoprotein fractions of cystic fibrosis patients. Am J Clin Nutr 62: 1268-1275, 1995[Abstract].

11.   Cornelius, P, Lee M, Marlowe M, and Pekala PH. Monokine regulation of glucose transporter mRNA in L6 myotubes. Biochem Biophys Res Commun 165: 429-436, 1998.

12.   Cucinotta, D, Nibali SC, Arrigo T, Di Benedetto A, Magazzu G, Di Cesare E, and Costantino A. Beta cell function, peripheral sensitivity to insulin and islet cell autoimmunity in cystic fibrosis patients with normal glucose tolerance. Horm Res 34: 33-38, 1990[ISI][Medline].

13.   DeFronzo, RA, Jacot E, Jequier E, Maeder E, and Feller JP. The effect of insulin on the disposal of intravenous glucose: results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30: 1000-1007, 1981[ISI][Medline].

14.   DeFronzo, RA, Tobin JD, and Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E214-E223, 1979[Abstract/Free Full Text].

15.   Finegood, DT, Bergman RN, and Vranic M. Estimation of endogenous glucose production during hyperinsulinemic-euglycemic glucose clamps. Diabetes 36: 914-924, 1987[Abstract].

16.   Finkelstein, SM, Wielinski CL, Elliott GR, Warwick WJ, Barbosa J, Wu S, and Klein DJ. Diabetes mellitus associated with cystic fibrosis. J Pediatr 112: 373-377, 1988[ISI][Medline].

17.   Garvey, WT, Maianu L, Zhu J, Brechtel-Hook G, Wallace P, and Baron AD. Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. J Clin Invest 101: 2377-2386, 1998[Abstract/Free Full Text].

18.   Grubb, BR. Ion transport across the murine intestine in the absence and presence of CFTR. Comp Biochem Physiol 118A: 277-282, 1997[ISI].

19.   Handwerger, S, Roth J, Gorden P, Di Sant'Agnese P, Carpenter DF, and Peter G. Glucose intolerance in cystic fibrosis. N Engl J Med 281: 451-461, 1969[ISI][Medline].

20.   Haney, JM, Slot JW, Piper RC, James DE, and Mueckler M. Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type. J Cell Biol 114: 689-699, 1991[Abstract].

21.   Hardin, DS, Azzarelli B, Edwards J, Wigglesworth J, Maianu L, Brechtel G, Johnson A, Baron A, and Garvey WT. Mechanisms of enhanced insulin sensitivity in endurance-trained athletes. J Clin Endocrinol Metab 80: 2437-2446, 1995[Abstract].

22.   Hardin, DS, Dominguez J, and Garvey WT. Muscle group-specific regulation of GLUT-4 glucose transporters in control, diabetic, and insulin-treated diabetic rats. Metabolism 42: 1310-1315, 1993[ISI][Medline].

23.   Hardin, DS, LeBlanc A, Lukenbaugh S, Para L, and Seilheimer DK. Increased rates of proteolysis associated with insulin resistance in cystic fibrosis. J Pediatr 10: 948-956, 1998.

24.   Hardin, DS, LeBlanc A, Lukenbaugh S, and Seilheimer DK. Insulin resistance is associated with decreased clinical status in cystic fibrosis. J Pediatr 6: 948-956, 1997.

25.   Hardin, DS, LeBlanc A, Para L, and Seilheimer DK. Hepatic insulin resistance and defects in substrate utilization in cystic fibrosis. Diabetes 48: 1082-1087, 1999[Abstract].

26.   Holloszy, JO, Klip A, Ramal T, and Young DA. Insulin-induced translocation of glucose transporters in rat hindlimb muscles. FEBS Lett 224: 224-232, 1987[ISI][Medline].

27.   Holman, GD, Lo Leggio L, and Cushman SW. Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools. J Biol Chem 269: 17516-17525, 1994[Abstract/Free Full Text].

28.   Hotamisligil, GS, Budavari A, Murray D, and Spiegelman BM. Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. J Clin Invest 94: 1543-1549, 1994[ISI][Medline].

29.   Hotamisligil, GS, Shargill NS, and Spiegelman BM. Adipose expression of tumor necrosis factor-alpha : direct role in obesity-linked insulin resistance. Science 259: 87-91, 1993[ISI][Medline].

30.   Hunter, SJ, and Garvey WT. Insulin action and insulin resistance: diseases involving defects in insulin receptors, signal transduction, and the glucose transport effector system. Am J Med 105: 331-345, 1998[ISI][Medline].

31.  James DE. Targeting of the insulin-regulatable glucose transporter (GLUT-4). Biochem Soc Trans 22: 101-105.

32.   Kartner, N, Augustinas O, Jensen TJ, Naismith AL, and Riordan JR. Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland. Nat Genet 1: 321-327, 1992[ISI][Medline].

33.   Kerem, B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, and Tsui L. Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080, 1989[ISI][Medline].

34.   Kim, JK, Wi JK, and Youn JH. Plasma free fatty acids decrease insulin-stimulated skeletal muscle gluscose uptake by suppressing glycolysis in conscious rats. Diabetes 45: 446-453, 1996[Abstract].

35.   Klip, A, Ramlal T, Young DA, and Holloszy JO. Insulin-induced translocation of glucose transporters in rat hindlimb muscles. FEBS Lett 21: 224-230, 1987.

36.   Kopito, LE, and Shwachman H. The pancreas in cystic fibrosis: chemical composition and comparative morphology. Pediatr Res 10: 742-749, 1976[Abstract].

37.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

38.   Lanng, S, Thorsteinsson B, Roder ME, Nerup J, and Koch C. Insulin sensitivity and insulin clearance in cystic fibrosis patients with normal and diabetic glucose tolerance. Clin Endocrinol (Oxf) 41: 217-223, 1994[ISI][Medline].

39.   Levy, E, Gurbindo C, Lacaille F, Paradis K, Thibault L, and Seidman E. Circulating tumor necrosis factor-alpha levels and lipid abnormalities in patients with cystic fibrosis. Pediatr Res 34: 162-166, 1993[Abstract].

40.   Marette, A, Burdett E, Douen A, Vranic M, and Klip A. Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle. Diabetes 41: 1562-1569, 1993[Abstract].

41.   Mohan, V, Alagappan V, Snehalatha C, Ramachandran A, Varadachari T, and Viswanathan M. Insulin and C-peptide responses to glucose load in cystic fibrosis. Diabete Metab 11: 376-379, 1985[ISI][Medline].

42.   Moran, A, Diem P, Klein D, Levitt MD, and Robertson RP. Pancreatic endocrine function in cystic fibrosis. J Pediatr 118: 715-723, 1991[ISI][Medline].

43.   Moran, A, Pyzdrowski KL, Weinreb J, Kahn BB, Smith SA, Adams KS, and Seaquist ER. Insulin sensitivity in cystic fibrosis. Diabetes 43: 1020-1026, 1994[Abstract].

44.   Morgan, CR, and Lazaro A. Immunoassay of insulin: two antibody system. Plasma insulin levels in normal subdiabetic and diabetic rats. Diabetes 12: 115-118, 1963[ISI].

45.   Morris, AP, and Frizzell RA. Vesicle targeting and ion secretion in epithelial cells: implications for cystic fibrosis. Annu Rev Physiol 56: 371-397, 1994[ISI][Medline].

46.   National Diabetes Data Group. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28: 1039-1057, 1979[ISI][Medline].

47.   Ofei, F, Hurel S, Newkirk J, Sopwith M, and Taylor R. Effects of an engineered human anti-TNF-alpha antibody (CDP571) on insulin sensitivity and glycemic control in patients with NIDDM. Diabetes 45: 881-885, 1996[Abstract].

48.   Pedersen, O, Bak JF, Andersen PH, Lund S, Moller DE, Flier JE, and Kahn BB. Evidence against altered expression of GLUT 1 or GLUT 4 in skeletal muscle of patients with obesity or NIDDM. Diabetes 39: 865-870, 1990[Abstract].

49.   Roden, M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW, and Shulman GI. Mechanism of free fatty acid-induced insulin resistance in humans. J Clin Invest 97: 2859-2865, 1996[Abstract/Free Full Text].

50.   Slot, JW, Geuze HJ, Gigengack S, James DE, and Lienhard GE. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci USA 88: 7815-7819, 1991[Abstract].

51.   Slot, JW, Geuze HJ, Gigengack S, Lienhard GE, and James DE. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 113: 123-135, 1991[Abstract].

52.   Sockrider, MM, Swank PR, Seilheimer DK, and Schidlow DV. Measuring clinical status in cystic fibrosis: internal validity and reliability of a modified NIH score. Pediatr Pulmonol 17: 86-96, 1994[ISI][Medline].

53.   Soejima, K, and Landing BH. Pancreatic islets in older patients with cystic fibrosis with and without diabetes mellitus: morphometric and immunocytologic studies. Pediatr Pathol 6: 25-46, 1986[Medline].

54.   Suter, S, Schaad B, Roux-Lombard P, Girardin E, Grau G, and Dayer JM. Relation between tumor necrosis factor-alpha and granulocyte elastase-alpha 1-proteinase inhibitor complexes in the plasma of patients with cystic fibrosis. A Rev Respir Dis 140: 1640-1644, 1989[ISI].

55.   Towbin, H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979[Abstract].

56.   Yki-Järvinen, H, Sammalkorpi K, and Koivisto VA. Severity, duration and mechanisms of insulin resistance during acute infections. J Clin Endocrinol Metab 69: 317-323, 1989[Abstract].


Am J Physiol Endocrinol Metab 281(5):E1022-E1028
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society