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Original Studies |
IRCSS H. San Raffaele (P.M.P., L.D.M., G.V., C.V.P., S.C., M.C., E.P.S., A.E.P.), 20132 Milan, Italy; Cattedra di Clinica Medica Generale e Terapia Medica, Università Vita e Salute, (G.P.), 20132 Milan, Italy; University of Parma (I.Z.), 43100 Parma, Italy; University of Pavia (B.S.), 27100 Pavia, Italy; and Stanford University School of Medicine (G.R.), Stanford, California
Address correspondence and requests for reprints to: PierMarco Piatti, M.D., Metabolic Diseases Unit, Division of Medicine, IRCCS H. San Raffaele, Via Olgettina 60, 20132 Milano, Italy.
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Abstract |
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Introduction |
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Unfortunately, not all published data are consistent with the formulation outlined above. For example, there is evidence that NO production may be normal or actually is increased in patients and/or animal models of type 2 diabetes (9, 10, 11, 12). However, not all studies have confirmed this finding, and it is possible that elevated NO concentrations only occur in patients with type 2 early in the disease, at a time when vascular complications are absent (13, 14).
The present study was initiated in an effort to examine the relationship between NO and insulin resistance in a somewhat different fashion; namely, by focusing on healthy volunteers, without known vascular disease or manifest hyperglycemia, but likely to differ in their degree of insulin resistance. For this purpose, we enrolled healthy volunteers with or without a family history of type 2 diabetes. The participants with a positive family history had at least one sibling and one parent with a diagnosis of type 2 diabetes. In addition to quantifying their degree of insulin resistance and NO concentrations, we also determined concentrations of cyclic-GMP.
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Materials and Methods |
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. It can be seen that individuals with a
family history of type 2 diabetes, who also had IGT, had higher values
for both body mass index and ratio of waist to hip girth. There was no
other significant differences between the three groups.
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Insulin-mediated glucose disposal was determined by means of the low-dose insulin glucose infusion test (LDIGIT) (16). This approach is based on determining the steady-state plasma glucose (SSPG) and steady-state plasma insulin (SSPI) concentrations in response to a continuous low-dose infusion of insulin (25 mU/kg·h) and glucose (4 mg/kg·min) for 150 min. Samples for blood glucose and insulin determinations were taken at -5, 0, 10, 20, 30, 40, 50, 60, 90, 120, and every 5 min until 150 min. Because hepatic glucose production is completely suppressed, the insulin sensitivity index (ISI) was calculated by dividing the constant glucose infusion rate by the SSPI concentration during the last 30 min of each test and normalizing this ratio to the SSPG concentration in the same period (16).
All subjects kept a diary of food intake for 3 days before measurement of NO and cyclic-GMP to control for differences in food intake. Because nitrogen intake could influence the measurement of NO2-/NO3- levels (17), the amount of nitrogen consumed was calculated. This precaution was taken despite the fact that the half-life of nitrate, a major component of the end product of NO, is 3 h (18) and 12 h of overnight fast and should be sufficient to result in total clearance of nitrogen dietary intake. No differences were found in the daily energy intake of the three groups of subjects, for either total amount of calories consumed or the proportion of calories as carbohydrate, lipid, or protein. Furthermore, because animal and vegetable nitrogen daily intakes were identical in the three groups of subjects, it is unlikely that variations in any of these variables would influence the measurement of NO2-/NO3- levels. Finally, any systematic error should have affected all groups to a similar extent.
Plasma glucose was measured with a glucose-oxidase based method (Unimate 5 Glucose HK; Roche, Basel, Switzerland). Serum triglyceride (intra-assay, 1.7%; interassay 9.7%), total cholesterol, and HDL-cholesterol levels (intra-assay, 3.5%; interassay 8.8%) were measured using automated enzymatic spectrophotometric techniques adapted to Cobas Fara II (Roche). Quality control was assessed by examining systematic and random error on pooled sera at low and high levels. Serum insulin levels [intra-assay coefficient of variation (CV), 3.0%; interassay CV, 5.0%] were assayed with a Microparticle Enzyme Immunoassay (MEIA, IMX; Abbott Laboratories, Abbott Park, IL) in which the lowest insulin sensitivity is 6 pmol/L. Cross-reactivity with the measurement of proinsulin is less than 2%. Serum C-peptide levels (intra-assay CV, 3.0%; interassay CV, 3.0%) were assayed by RIA using commercial kits (Medical System, Genova, Italy). Serum creatinine levels were assayed by using a spectrophotometric automated method on Cobas Fara II (Crea Unimate 5; Roche). Cyclic-GMP was assayed with a RIA kit (Amersham International, Buckinghamshire, UK), and NO levels were assayed by measurement of the end products of their metabolism [i.e. nitrite and nitrate levels (NO2-/NO3-)], using enzymatic catalysis coupled with Griess reaction. Specifically, NO3- was reduced to NO2- by 0.1 U nitrate reductase, 5 x 10-6 M flavin adenine dinucleotide, and 250 x 10-6 M nicotinamide adenine dinucleotide phosphate (reduced form). Samples were incubated at 37C for 3 h, 8.8 U lactate dehydrogenase and 10-2 M pyruvate were added to each well, and the sample was incubated for 90 min at 37C. Finally, Griess reactives were added to each well, and the sample was read at 540 nm (19).
Statistical analysis
Results are expressed as mean ± SEM. Comparisons between groups were performed using one-way ANOVAs, followed by Scheffe-F test when appropriate. Relationships between different variables were analyzed by Pearson correlations. Statistical significance was accepted at the P = 0.05 level.
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Results |
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. By definition, the plasma
glucose concentration was higher 120 min after 75 g oral glucose
in those with IGT as compared with the other two groups. In addition,
this group had significantly higher insulin concentrations, both
fasting and postglucose load, as well as higher triglyceride
concentrations.
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and Fig. 1. The SSPG concentrations were
significantly higher in those with IGT as compared with other two
groups. In addition, the SSPG concentrations were higher in the NGT
group with a family history of type 2 diabetes than in those without a
family history. It should be emphasized that the higher SSPG
concentrations were achieved despite the fact that the SSPI
concentrations were also higher in those with a positive family
history. This phenomenon is most likely the result of a failure to
inhibit endogenous insulin secretion in these individuals, as attested
to by their much higher C-peptide concentrations during the infusion.
To compensate for the differences in SSPI, the ISI was calculated, and
Table 3 shows that the ISI was lower in both groups with a positive
family history of type 2 diabetes (i.e. they were more
insulin resistant than those with a negative family history).
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. It can be seen that
concentrations of NO2-/NO3- were
higher and cyclic-GMP lower in both groups with a family history of
type 2 diabetes. Furthermore, these values did not seem to differ as a
function of glucose tolerance in those with a family history.
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illustrates the Pearson correlation
coefficients between the ISI and the metabolic variables listed in
Table 2
. These results demonstrate that the more insulin resistant
(lower ISI), the higher triglyceride and
NO2-/NO3- concentration and the
lower the cyclic-GMP concentration. The relationships between the ISI
and glucose and HDL-cholesterol concentrations were not statistically
significant.
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. The correlation coefficients between
the 120-min insulin response and both plasma triglyceride (r =
0.47, P < 0.001) and
NO2-/NO3- (r = 0.39,
P < 0.001) concentration remained highly significant
(data not shown). However, the relationship between insulin response
and cyclic-GMP was somewhat lower (r = -0.22, P =
0.08), whereas that between insulin response and both triglyceride
(r = 0.47, P < 0.001) and HDL-cholesterol (r
= -0.31, P < 0.02) increased. |
Discussion |
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The study most similar to our present study is that of Petrie et al. (1), who measured insulin-mediated glucose disposal in 19 healthy volunteers with the hyperinsulinemic, euglycemic clamp technique and defined the relationship between this value and an estimate of NO production based on the forearm vasoconstrictor responses to Ng-monomethyl-L-arginine. On the basis of these measurements, they concluded that endothelial NO synthesis and insulin sensitivity are positively related in healthy volunteers (i.e. the more insulin sensitive, the greater the NO synthesis). One obvious difference between the results of the two studies is the method of assessing insulin sensitivity: the clamp vs. the LDIGIT method. However, this difference seems unlikely to account for the disparity for at least two reasons. First, there is evidence that values of insulin resistance obtained with the two methods are significantly correlated (16). Second, the ISI and insulin response to oral glucose were also highly correlated (r = -0.69, P < 0.001), a relationship very similar to that described in a nondiabetic population between specific estimates of insulin resistance and insulin response to glucose (20, 21).
The two reports also differ in the methods used to quantify NO production; a chemical estimate in the current study, as compared with a functional assay based on the vasodilatory response to inhibition of NO synthesis. The chemical estimate we used as an index of NO production involved measurement of NO2-/NO3-, stable degradation products of NO in biological solutions, and there is evidence that NO2-/NO3- serves as an accurate indicator of NO production in vivo (18, 22). The method used by Petrie et al. (1) to assess NO production is a functional assay, dependent on inhibition of enzyme responsible for the generation of NO and the ability of the endothelium to respond to the change in NO production. As such, it is possible that the insulin-resistant patients studied by Petrie et al. (1) did not have a defect on NO production, but rather an abnormal endothelial response to NO. In other words, it is the biological effect of NO, not its production, that is abnormal in insulin-resistant subjects.
It is currently believed that NO interacts with soluble guanylate cyclase, leading to elevation of cyclic-GMP concentrations (22, 23), and Albert et al. (24) have recently shown that inhaled NO produced a 3-fold elevation in plasma cyclic-GMP concentrations. Given evidence that cyclic-GMP concentrations are increased in response to NO and the view that cyclic-GMP is the effector messenger of NO, it becomes possible to reconcile apparently conflicting data. The results of this study demonstrated that cyclic-GMP concentrations were lower in both groups of subjects with a family history of type 2 diabetes. A simple approach to the relationship between the ISI, NO, and cyclic-GMP is to propose that insulin resistance is associated with an impairment in the ability of NO to generate its messenger (cycling-GMP), leading to a decrease in cyclic-GMP generation and a relative decline in the ability of insulin to produce vasodilation. The increase in NO2-/NO3- associated with insulin resistance could then represent an effort to compensate for the defect in cyclic-GMP production. This hypothesis is consistent with the results of an earlier study from our laboratory, showing that insulin-resistant subjects with metabolic Syndrome X had higher plasma concentrations of NO, both in the basal state and in response to insulin stimulation as compared with an insulin-sensitive control group (25). However, despite the higher levels of NO, insulin-stimulated vasodilation was impaired in those with the metabolic Syndrome X. Thus, in this instance, insulin-induced vasodilation was blunted in insulin-resistant subjects, not because of an inability to produce NO, but rather due to an inhibition of NO action presumably secondary to the generation of cyclic-GMP. Although quite speculative, this formulation is consistent with the current results, the conclusion by Petrie et al. (1) of a relationship between insulin resistance and the endothelial response to inhibition of NO synthesis, as well as the evidence that the vasodilatory response to insulin is decreased in insulin-resistant individuals (4, 5).
In conclusion, the results presented are consistent with the hypothesis that plasma NO concentrations are higher than normal in insulin-resistant individuals and the defect in insulin-mediated vasodilation in these individuals is secondary to a defect in the generation of the effector molecule cyclic-GMP. On the other hand, it is important to emphasize that the situation is certainly more complex. For example, our laboratory (25) has also shown that although individuals with the cardiac Syndrome X are relatively insulin resistant compared with the control group, their basal NO concentrations were similar to control values and did not increase in response to insulin. However, these subjects also had a blunted vasodilatory response to insulin. Obviously, the cause of the blunted vasodilatory response in those with cardiac Syndrome X cannot be the same as individuals with the metabolic Syndrome X. One explanation for this disparity is the suggestion by Egashira et al. (26) that impaired vasodilation in patients with cardiac Syndrome X is due to a defect in NO synthesis. The fact that NO levels were not increased in these subjects may be because the degree of insulin resistance was not severe as that in those with the metabolic Syndrome X. In support of this hypothesis is that individuals with cardiac Syndrome X also had a normal value for insulin-stimulated glucose storage.
Finally, it must be emphasized that our conclusion that NO production is increased in insulin-resistant subjects is not the only interpretation of our results. For example, higher NO concentrations in insulin-resistant subjects could be due to decreased excretion, rather than increased production of NO. Similarly, even if it is assumed that increased production is the cause of higher NO2-/NO3- levels, it is possible that this was due to stimulation of NO production in noninsulin-sensitive tissues (e.g. liver or kidney). Finally, there may be an increase in the catabolic rate of cyclic-GMP in insulin-resistant subjects, and the lower cyclic-GMP concentrations need not be due to an impairment in the ability of NO to generate the messenger. Obviously, these issues must be clarified to substantiate our suggestion that NO production is increased in insulin-resistant individuals and that the impaired endothelial function described by others (1, 4, 5, 27, 28) in these individuals is due to a defect in the generation of cyclic-GMP by NO.
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Footnotes |
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Received November 15, 1999.
Revised February 25, 2000.
Accepted March 21, 2000.
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References |
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