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
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ABSTRACT |
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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 · m2 · 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)-
levels and FFA in all
subjects. Additionally, we report the results of TNF-
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-
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-
levels. We conclude that IGTCF patients have
decreased peripheral insulin sensitivity. Mechanisms include elevation
of TNF-
and impaired translocation of GLUT-4.
insulin resistance; cytokines; tumor necrosis factor-; free
fatty acids
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INTRODUCTION |
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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 -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-
(TNF-
) has been postulated to contribute to insulin resistance.
Hotamisligil et al. (29) demonstrated increased TNF-
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-
. Elevated TNF-
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-
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- may exist
independent of insulin sensitivity. One previous study
(28) has described that elevated TNF-
levels result in
decreased FFA levels. Levy et al. (39) reported a
correlation between TNF-
levels and lipid and lipoprotein levels in
CF. We therefore wished to evaluate total and individual FFA levels and
their relationship to TNF-
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.
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METHODS |
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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 forNine 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 · m2 · 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.
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
-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.
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-.
The cytokine TNF-
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 |
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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.
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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
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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.
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TNF-
TNF- 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-
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-
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.
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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-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- ![]() |
DISCUSSION |
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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- levels in CF
subjects than in controls. Similar findings have been reported by
others (39, 54). TNF-
levels increase during acute
illness, and previous studies indicate that TNF-
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-
.
Hotamisligil et al. (28) have demonstrated that TNF-
affects the phosphorylation cascade of insulin signaling at the
receptor level, and Ofei et al. (47) demonstrated
improvement in insulin sensitivity by giving TNF-
receptor
antibodies. Although we could not find a correlation between TNF-
levels and GDR or GLUT-4 levels, we did find a correlation between
clinical status scores and TNF-
levels. Thus it is likely that
TNF-
levels either reflect or contribute to worsened clinical
status. Without a direct link to glucose disposal, we are reluctant to
state that TNF-
causes insulin resistance in CF; however,
measurement of insulin resistance after use of TNF-
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- levels. These studies suggest a considerable need for future research in the area of
CF-related diabetes and insulin sensitivity.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge critical review of the manuscript by Drs. Patrick Brosnan and Robert Moore, and subject recruitment by Dr. Piers Blackett.
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FOOTNOTES |
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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.
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