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Original Studies |
Division of Endocrinology and Metabolic Diseases, University of Verona Medical School, I-37100 Verona, Italy
Address all correspondence and requests for reprints to: Enzo Bonora, M.D., Division of Endocrinology and Metabolic Diseases, Ospedale Civile Maggiore, Piazzale Stefani 1, I-37126 Verona, Italy.
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Abstract |
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Introduction |
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At present, no data are available regarding the effects of chronic smoking on insulin resistance in patients with NIDDM, a disease specifically characterized by a concomitant impairment of insulin secretion and insulin action (8, 9). The clarification of this aspect may be of clinical relevance for undertaking preventive and therapeutic strategies. In fact, cigarette smoking, by exacerbating the insulin resistance already present in NIDDM, might contribute to hyperglycemia, whose harmful effect in the development of diabetes chronic complications is well established (10, 11, 12). In addition, the smoking-related insulin resistance and/or the attendant compensatory hyperinsulinemia might accelerate atherosclerosis through direct or indirect mechanisms (13, 14, 15, 16, 17, 18, 19).
Thus, the aim of the present study was to examine whether chronic cigarette smoking has a deleterious impact on insulin sensitivity in patients with NIDDM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study population consisted of 40 consecutive, nonobese, noninsulin-treated patients with NIDDM, regularly attending the outpatient Diabetes Clinic of Verona and recruited over a period of 6 months to participate in a clinical trial on the effects of antihypertensive treatment on insulin sensitivity. Inclusion criteria were the following: age 30–70 yr; body mass index (BMI) <30 kg/m2; no treatment with insulin or with any other drug known to influence glucose metabolism; no history of recent acute illness or clinical evidence suggestive of kidney, liver, or endocrine diseases; no severe chronic diabetic complications (proliferative retinopathy, macroalbuminuria, symptomatic neuropathy, coronary, and other vascular diseases). Twenty-eight of them were cigarette smokers and 12 were nonsmokers. The former had been smoking more than 5 cigarettes per day for at least 20 yr, the latter either had never smoked (10 subjects) or had stopped smoking for at least 5 yr (2 subjects). The length of the period without smoking was chosen according to recent reports which estimated the period of the time necessary to eliminate most of the deleterious effect of smoking (20). The two groups were matched for sex, age, BMI, waist/hip ratio (WHR), diabetes duration, and treatment. This procedure resulted in the exclusion of 5 of the 45 subjects recruited for the above mentioned clinical trial. All participants underwent a medical history and physical examination. Nine subjects were treated with diet only; and the remaining 31 subjects, with diet and oral hypoglycemic agents (sulfonylurea plus metformin, n = 19; sulfonylurea alone, n = 12). In five subjects (3 smokers, 2 nonsmokers) treated with antihypertensive agents (calcium channel blockers, n = 2 and ACE-inhibitors, n = 3), the treatment was discontinued 4 weeks before entering the study. None of the subjects was taking other medications. BMI was calculated by dividing weight in kilograms by the square of height in meters. A tetrapolar bioimpedance analyzer (BIA-103, Akern, Florence, Italy) was used to measure body electrical resistance and to derive an estimate of total body fat content and fat-free mass (FFM)(21). The measure of body composition achieved with this technique is highly correlated with that generated by more sophisticated methods, including isotope dilution in the body (22). WHR was calculated by dividing waist circumference (measured midway between the lower rib margin and the iliac crest) by hip circumference (the widest circumference over the great trochanters). Blood pressure was measured in triplicate, with a standard mercury manometer, after the subjects had rested in the supine position for at least 10 min. Information on daily alcohol consumption (expressed as grams of alcohol consumed per day), coffee consumption (cups per day), and physical activity level (3 categories: light, moderate, intense) was collected from all participants by a questionnaire, as described elsewhere (23, 24). Trained subjects, i.e. those who exercised more than 4 h/week, were not included in the study. In all subjects, 24-h urine collections were obtained to assess albumin excretion rate (AER). Patients were classified as normoalbuminuric (AER < 20 µg/min), microalbuminuric (AER between 20 and 200 µg/min), and macroalbuminuric (AER > 200 µg/min). All subjects gave their written informed consent to participation in the study. The study protocol was approved by the Ethical Committee of the University of Verona Medical School.
Experimental design
The protocol consisted of two studies carried out on separate, nonconsecutive days. The studies were performed at 0800 h, after an overnight fast. Patients were asked to refrain from smoking for at least 8 h before study, to eliminate the acute effects of smoking. Medications were withheld on the morning of the metabolic tests to avoid observing the acute effects of the drugs.
Study 1 consisted of a 75-g oral glucose tolerance test. Plasma glucose, insulin, and C-peptide concentrations were measured at baseline and 30, 60, 90, 120, 180, and 240 min after the glucose load. Baseline blood sample was used to measure also total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and glycosylated hemoglobin A1c.
Study 2 consisted of a 4-h euglycemic hyperinsulinemic clamp, as
originally described by De Fronzo et al. (25), associated
with 3-[3H]-D-glucose infusion and
indirect calorimetry, as previously reported in detail (26, 27). A
20-gauge Teflon catheter was inserted into an antecubital vein for the
infusion of all test substances. A second catheter was inserted
retrogradely into a wrist vein for blood sampling and kept patent with
the infusion of normal saline. The hand was inserted into a hot (
60
C) box to achieve the arterialization of the venous blood. At 0830
h, a prime-continuous infusion of
3-[3H]-D-glucose was started and maintained
for the following 150 min. The infusion rate of tritiated glucose was
0.15 µCi/min. The prime for tracer glucose was calculated by dividing
the glucose pool (plasma glucose concentration times glucose
distribution volume, assumed to be 25% of body weight) by the
estimated basal glucose turnover (11 mmol/min·kg FFM when fasting
plasma glucose was below 11.1 mmol/L, and 14 mmol/min·kg FFM when it
was 
11.1 mmol/L) and then multiplying the result by the tracer
infusion rate. During the last 50 min of tracer equilibration, samples
were drawn every 10 min, for the determination of plasma tritiated
glucose specific activity and insulin and free fatty acid (FFA)
concentrations. At the end of the 150-min tracer equilibration period,
a 4-h euglycemic insulin clamp was performed. Insulin was given as a
prime-continuous infusion (20 mU/min·m2 of body surface
area), and a 20% glucose solution was infused at a rate periodically
adjusted to achieve and maintain an arterialized plasma glucose
concentration of 5 mmol/L. Tritiated glucose infusion was
discontinued at the beginning of the insulin clamp, resumed 120 min
later at the rate of 0.30 µCi/min, and thereafter continued until the
end of the study. Also, this infusion of tritiated glucose was preceded
by a priming bolus. The prime dose of labeled glucose was calculated by
dividing the glucose pool by the product of 1.1 times
GIR100–120 and then multiplying the result by the tracer
infusion rate. GIR100–120 was the glucose infusion rate
during the time interval of 100–120 min of the glucose clamp. It was
multiplied by 1.1 to take into account the expected 10% of average
increase in glucose infusion from 100–120 min to 180–240 min (26, 27). The rationale for interrupting the tracer administration during
the first 2 h of the insulin clamp was that, by resuming the
tracer infusion in a near-steady state, the time of equilibration for
labeled precursor and products would be significantly shortened (28).
Indeed, with such a methodological approach, we have previously shown
that a steady-state of plasma glucose specific activity is achieved in
the fourth hour of insulin clamp at both low and high insulin infusion
rates (28, 29). Blood samples for the determination of plasma glucose
specific activity and insulin and FFA concentrations were collected at
time 180, 190, 200, 210, 220, 230, and 240 min during the insulin
clamp. Blood was collected in heparinized tubes and promptly
centrifuged (T = 4 C, 4000 x g) and the plasma
decanted and stored at -20 C until analyzed. Between time -50 and 0
min and between 180 and 240 min, oxygen consumption and carbon dioxide
production rates were measured by continuous indirect calorimetry
(Deltatrac, Sensormedics, Anaheim, CA), and the data were used to
compute the rates of glucose and lipid oxidation (30). Protein
oxidation rate was estimated from urinary nitrogen excretion during the
baseline and insulin-stimulated periods (30).
Analytical determinations
Plasma glucose was measured by the glucose-oxidase method (31) on a Beckman Glucose Analyzer (Beckman Instruments, Fullerton, CA). Plasma 3-[3H]-D-glucose specific activity was determined as described in detail elsewhere (26, 27). Insulin and C-peptide were measured by double-antibody RIAs (32). Insulin concentration was measured by using a commercial double-antibody method (human insulin-specific RIA method; Linco Research, Inc., St. Louis, MO), in which cross-reactivity to proinsulin and Des 31, 32 proinsulin was less than 0.2%. HbA1c was measured by high performance liquid chromatography (normal range = 3.5–5.5%) (33). Total cholesterol, HDL cholesterol, and triglycerides were assessed by the methods of Seidel et al. (34), Warnick et al. (35), and Wahlefeld (36), respectively. Serum low-density lipoprotein cholesterol was calculated with the equation of Friedewald et al. (37). Plasma FFA concentration was determined by a spectrophotometric method (38). Urinary nitrogen concentration was measured on the samples collected during the basal and insulin-stimulated periods, according to Kjeldhal (39). Urinary AER was determined by using an RIA method on 24-h urine collections, after excluding proteinuria caused by urinary tract infection.
Calculations
A steady-state plateau of plasma-tritiated glucose specific activity was achieved both during the last 50 min of the basal period and during the last hour of insulin clamp. Therefore, in both periods, the rate of body glucose appearance equals the rate of glucose disposal (mg/min) and was computed according to the equation: tracer infusion rate (dpm/min) divided by steady-state tracer specific activity (dpm/mg). Because, in the postabsorptive state, the inputs of glucose into the circulation are endogenous (liver and, perhaps, kidney), the basal rate of the endogenous glucose production equals the rate of total glucose appearance. During the insulin/glucose infusion, the rate of endogenous glucose production was computed by subtracting the exogenous glucose infusion rate from the isotopically determined rate of glucose appearance. The rates of carbohydrate and lipid oxidation were calculated from the nonprotein respiratory quotient, as previously described (30). Nonoxidative glucose disposal, which primarily reflects muscle glycogen formation (40), was calculated by subtracting the rate of glucose oxidation from the rate of whole-body glucose disposal.
Statistical analysis
All data are presented as mean ± SE throughout
the paper. The following statistical tests were carried out: unpaired
Student’s t test, one-way ANOVA, analysis of covariance,
and 
-square test (for categorical variables). A two-way ANOVA was
used to compare plasma glucose, C-peptide, and insulin responses to
oral glucose load in smokers and nonsmokers. Because nonparametric
statistical tests (i.e. Mann-Whitney U-test, Kruskal Wallis
rank test) yielded very similar results to parametric tests, the latter
are presented. To improve skewness and kurtosis of the distributions,
triglycerides, insulin, and FFA concentrations were logarithmically
transformed for statistical analyses and then back-transformed to their
natural units, for presentation in tables. Distributions of all other
variables were normal. P-values less than 0.05 were
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. They were comparable for sex,
age, BMI, body composition, WHR, daily alcohol consumption, degree of
physical activity, diabetes duration, and treatment. Furthermore, the
prevalence of microalbuminuria did not differ significantly between the
two groups. Smokers had significantly higher plasma triglyceride and
lower HDL cholesterol concentrations. Systolic blood pressure and
plasma levels of glycosylated HbA1c tended to parallel plasma
triglycerides, but no statistically significant differences were
detected between smokers and nonsmokers.
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shows that plasma glucose
response after oral glucose load was similar in the two groups, whereas
plasma insulin and C-peptide responses were significantly higher in
smokers than nonsmokers (P < 0.01, by two way
ANOVA).
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, rates of glucose
metabolism were not significantly different between the two groups in
the basal state. During insulin infusion, the rates of total,
oxidative, and nonoxidative glucose disposal were significantly lower
in smokers than nonsmokers. Endogenous glucose production was
suppressed to the same extent during insulin infusion in smokers and
nonsmokers. Lipid oxidation and plasma FFA concentrations did not
differ significantly between the two groups in the basal state but were
suppressed to a lesser extent during insulin infusion in smokers than
nonsmokers.
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). This
relationship remained statistically significant, even after adjustment
for potential confounders such as BMI, WHR, systolic blood pressure,
plasma triglyceride, and HbA1c concentrations, as well as plasma levels
of FFA during insulin infusion (F = 5.1; P =
0.006).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the degree of insulin sensitivity in patients with NIDDM was worsened by concomitant cigarette smoking, we failed to observe significant differences in the degree of metabolic control of diabetes. However, fasting glucose levels and glycated hemoglobin concentrations tended to be higher in smokers than nonsmokers. Over a long-term period, these mildly higher glucose levels might result in a greater propensity to develop chronic diabetic complications, as recently demonstrated by results from the Diabetes Control and Complications Trial (41).
Smoking patients also were characterized by higher plasma insulin levels, higher plasma triglycerides, lower HDL cholesterol, and tendentially higher systolic blood pressure than their nonsmoking counterparts. All these features are typical hallmarks of the so-called insulin resistance syndrome (18, 19). Thus, cigarette smoking seems to be an environmental factor capable of making the insulin resistance of patients with NIDDM fully blossom with all insulin resistance syndrome phenotypic attributes. The consequence of this phenomenon would be an increased cumulative cardiovascular risk. On the other hand, insulin resistance itself also might have a direct effect on atherosclerosis, as suggested by recent cross-sectional studies (16, 17, 42).
In the present study the smoking group was selected to match the nonsmoking group in several clinical aspects, including chronic diabetic complications. This selection process might have eliminated the possibility of finding more striking metabolic differences between the two groups.
In our study, the two groups of patients were of comparable sex, age, BMI, WHR, alcohol consumption, physical activity level, glycometabolic control, and diabetes duration and treatment, i.e. variables known to affect insulin action and glucose tolerance (8, 9, 18, 19, 24). Additionally, no significant difference was found in the prevalence of microalbuminuria, which has been reported to be associated with a more severe degree of insulin resistance in patients with NIDDM (43). Thus, it seems reasonable to speculate that the difference in insulin sensitivity we observed between the two groups was secondary to the cigarette smoking and, theoretically, could be caused by the direct effects of nicotine, carbon monoxide, or other agents in tobacco smoke. In this regard, Eliasson et al. (44) have recently reported that, in healthy men, the long-term use of nicotine gum is associated with hyperinsulinemia and insulin resistance, thus suggesting that nicotine is the major constituent in cigarette smoke that leads to insulin resistance and to various manifestations of the insulin resistance syndrome.
The two groups of patients significantly differed in plasma triglyceride levels. Because hypertriglyceridemia in patients with NIDDM seems to be associated with a more severe degree of insulin resistance (45), one might postulate that the difference in insulin sensitivity we observed between smokers and nonsmokers is partly dependent on hypertriglyceridemia. Indeed, it is possible that higher plasma triglycerides of smokers could independently contribute to the more severe degree of insulin resistance of these subjects or, alternatively, that they could mediate the deleterious effect of smoking on insulin sensitivity. However, a role of smoking, independent of plasma triglycerides, is supported by the dose-response relationship found between the number of cigarettes smoked per day and insulin sensitivity, as well as by the results of the multivariate analysis (which included also plasma triglycerides).
In conclusion, although this study is cross-sectional and cannot, by its same nature, prove a cause-effect relationship, the present results indicate that chronic cigarette smoking can exert a deleterious impact on insulin sensitivity in patients with NIDDM and may bring a more complete expression of the insulin resistance syndrome. Because insulin resistance has been involved, by direct or indirect mechanisms, in the pathogenesis of hyperglycemia and macroangiopathy, smoking cessation in NIDDM patients might favorably affect not only cardiovascular risk, but also long-term metabolic control. Prospective studies, comparing NIDDM subjects who stopped smoking or continued to smoke, are needed to substantiate this hypothesis, especially because smoking cessation often results in weight gain, and this might adversely affect insulin sensitivity. An ancillary conclusion from the present results is that the smoking status should be carefully considered in case-control studies, including subjects with NIDDM.
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Acknowledgments |
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Footnotes |
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Received May 6, 1997.
Revised July 16, 1997.
Accepted July 21, 1997.
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