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P a t h o p h y s i o l o g y / C o m p l i c a t i o n s
O R I G I N A L
Altered D-Chiro-Inositol Urinary Clearance
in Women With Polycystic Ovary Syndrome
EAN-PATRICE BAILLARGEON, MD, MSC
TEIMURAZ APRIDONIDZE, MD
VANTHIA DIAMANTI-KANDARAKIS, MD
MARIA J. IUORNO, MD
ICHARD E. OSTLUND, JR., MD
JOHN E. NESTLER, MD
zone (3), and D-chiro-inositol (DCI)(10 –12), to both obese and lean womenwith the syndrome increases the fre- OBJECTIVE — Evidence suggests that some actions of insulin are effected by inositolphos-
phoglycan (IPG) mediators. We hypothesize that a deficiency in D-chiro-inositol (DCI) and/or a DCI-containing IPG (DCI-IPG) may contribute to insulin resistance in humans.
RESEARCH DESIGN AND METHODS — To assess this possibility in polycystic ovary
(IPG) mediators of insulin action (13,14), syndrome (PCOS), we determined insulin sensitivity (S by frequently sampled intravenous and evidence suggests that a deficiency in glucose tolerance test), plasma and urinary DCI and myo-inositol (MYO) levels (by gas chroma-tography/mass spectrometry), and the release of insulin and DCI-IPG during the oral glucose tolerance test (area under the curve [AUC]) in 23 women with PCOS and 26 normal women.
phoglycan (DCI-IPG) may contribute toinsulin resistance in individuals with im- RESULTS — Women with PCOS were heavier than control subjects (P ϭ 0.002 for BMI), but
paired glucose tolerance or type 2 diabe- also had decreased S (P Ͻ 0.001) and increased AUC women, even when corrected for BMI. The urinary clearance of DCI (uCl suggest that a deficiency in DCI-IPG con- almost sixfold in PCOS compared with normal women (P ϭ 0.001), but not MYO clearance (P ϭ tributes to insulin resistance in PCOS as correlated inversely with S when all women were analyzed together (n ϭ 49, r ϭ 0.50, P Ͻ 0.001) and was one of the three best independent parameters predicting S . Finally, was decreased threefold in women with PCOS (P Ͻ 0.001).
CONCLUSIONS — uCl
is inversely correlated with insulin sensitivity in women and is a strong independent predictor of insulin resistance in multivariate models. PCOS, which is characterized by insulin resistance, is associated with a selective increase in uCl DCI-IPG release in response to insulin. These findings are consistent with a defect in tissue availability or utilization of DCI in PCOS that may contribute to the insulin resistance of the tion. The idea that a deficiency in DCI-IPG, related perhaps to an actual or Diabetes Care 29:300 –305, 2006
functional deficiency of the precursorDCI, contributes to the insulin resistance Polycysticovarysyndrome(PCOS)is oping cancer, hypertension, dyslipide- ofPCOSisfurthersupportedbyevidence
mia, impaired glucose tolerance or type 2 P C O S w o m e n e n h a n c e s i n s u l i n - evidence supports the central role of in- sized that a defect in an alternative insu- 6 –10% of women of childbearing age (3– acts as a mediator of insulin action, con- tributes to the pathophysiology of the in- anovulatory infertility in the U.S. and is associated with an increased risk of devel- ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● From the 1Department of Medicine, Universite´ de Sherbrooke, Sherbrooke, Canada; the 2First Department of Medicine–Endocrine Section, University of Athens Medical School, Athens, Greece; the 3Department of Medicine, Washington University School of Medicine, St. Louis, Missouri; the 4Department of Medicine, release of the putative DCI-IPG mediator.
Virginia Commonwealth University, Richmond, Virginia; and the 5Department of Obstetrics and Gynecol- ogy, Virginia Commonwealth University, Richmond, Virginia.
Address correspondence and reprint requests to Jean-Patrice Baillargeon, MD, MSc, Universite´ de Sher- brooke, Sherbrooke, QC J1H 5N4, Canada. E-mail: jp.baillargeon@usherbrooke.ca.
Received for publication 10 June 2005 and accepted in revised form 23 October 2005.
Abbreviations: AUC
, area under the bioactivity curve for DCI-IPG during OGTT; AUC under the insulin curve during OGTT; DCI, D-chiro-inositol; DCI-IPG, DCI-containing inositolphosphogly- can; IPG, inositolphosphoglycan; MYO, myo-inositol; OGTT, oral glucose tolerance test; PCOS, polycysticovary syndrome; uCl , urinary clearance of D-chiro-inositol; WHR, waist-to-hip ratio.
A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion technique. As a control, we also assessed the urinary clearance of myo-inositol 2006 by the American Diabetes Association.
(MYO), an inositol not believed to influ- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ence insulin sensitivity. The findings indi- DIABETES CARE, VOLUME 29, NUMBER 2, FEBRUARY 2006 Baillargeon and Associates
the specific activation of phosphate dehy- clearance and deficient insulin-stimulated intravenous glucose tolerance test as de- drogenase phosphatase, as previously val- scribed by Bergman and colleagues (18 – 20). At time zero, 300 mg/kg dextrose was RESEARCH DESIGN AND
venously 20 min later. A total of 27 blood METHODS — A total of 23 women
glucose were collected over the 3-h dura- 8.5%, respectively, for the absolute values jects were evaluated at the General Clini- of basal and peak DCI-IPG bioactivity.
Commonwealth University Health System.
assay to the other, and therefore from sub- nation of tissue insulin sensitivity (Si).
ject to subject, the water-blank activity (elevated serum total or free testosterone ately, and sera were stored at Ϫ70°C until percentage of its bioactivity at baseline (0 previously described (22–24). Serum free known to affect insulin sensitivity for at lin concentrations and relative bioactivi- (40 g/l). To avoid interassay variation, all ties of DCI-IPG to the oral administration of glucose by calculating the areas under strual cycles, normal androgen levels, and single assay for each hormone. The intra- assay coefficient of variation (CV) for the trapezoidal rule. Results not normally dis- have any history of gestational diabetes or family history of a first-degree relative with diabetes. They were free of disorders tance, such as hypertension or dyslipide- results are reported as means, or geomet- with 95% CIs. P values Ͻ0.05 were con- [2H6]racemic chiro-inositol and [2H6]myo- sidered significant. All analyses were per- woman gave written informed consent.
inositol were added to plasma or urine as then purified, derivatized with pentaflu- groups were made with the Student’s two- diet high in legumes or fruits, the women tailed t test, and equalities of variances State College, PA), and analyzed in a neg- were tested with the Brown-Forsythe test.
anced mixed diet for at least 3 days before P values of Welch ANOVA tests were re- studied during the equivalent of the fol- ported, as indicated. Correlation analysis ane as the reagent gas, as previously re- tion test. Correction for BMI and/or Si was performed with multiple linear regression lieu of anovulatory women with PCOS.
ately, and sera were stored at Ϫ70°C until scribed (17). To date, it has not been pos- both. BMI status was categorized as obese sible to measure the content of extracted to collect all their urine for the next 24 h.
first one, the dependant variable was DCI urinary clearance, the exposition variable DIABETES CARE, VOLUME 29, NUMBER 2, FEBRUARY 2006 Altered DCI urinary clearance in PCOS
Table 1 —Clinical and biochemical characteristics of women with PCOS and normal control women
Data are means (95% CIs) unless otherwise indicated. To convert values for total testosterone to nanomoles per liter, multiply by 0.0347; to convert values for freetestosterone to picomoles per liter, multiply by 34.7; and to convert values for insulin to picomoles per liter, multiply by 6.9. Note that only significant partial P values(‡, §, or ʈ) after correction for BMI and/or S are reported (multiple linear regression analyses). FSIVGGT, frequently sampled intravenous glucose tolerance test.
*Unequal variance t test. †Geometric means. ‡P Ͻ 0.05 when corrected for BMI and S . §P Ͻ 0.05 when corrected for BMI. ʈP Ͻ 0.05 when corrected for S .
significantly to the model (partial P Ͻ with normal control subjects (P ϭ 0.035), significantly higher (P ϭ 0.043) (Table 1).
testosterone, and Si. These confounders were selected based on scientific literature and because they were significantly asso- was highly significant (P Ͻ 0.001). This ciated with the dependant variable in uni- statistical significance persisted even after variate analyses. To assess the effect of correction for BMI (P ϭ 0.015). In con- on next highest partial F test (forward tested with each new variables added into concentrations, 24-h urinary excretion of MYO, and urinary clearance of MYO (P Ն levels were significantly higher in women analysis, the dependant variable was S with PCOS than in control women (P Ͻ 0.001), even after correction for BMI and Si (P ϭ 0.006 and P Ͻ 0.001, respec- tively). Furthermore, fasting insulin levels independently of obesity (P ϭ 0.006), a and total testosterone. These confounders (P ϭ 0.047) and areas under the insulin were also significantly associated with the curves during OGTT (AUCinsulin) (P Ͻ dependant variable in univariate analyses.
PCOS status (P ϭ 0.02), and a significant in normal control subjects, and Si values were significantly lower (P Ͻ 0.001).
obesity (P ϭ 0.005). To illustrate this in- AUCinsulin and Si remained significantly different between groups after correction analysis, the best model to predict S for BMI (P ϭ 0.001 and P ϭ 0.002, re- significantly increased by 14-fold in obese assessed using all variables of Table 1.
found with the first two stepwise regres- [13.1–77.2] vs. 2.2 [0.7–7.3], P Ͻ 0.001), even after correction for Si and Plasma concentrations of DCI were signif- total testosterone (partial P ϭ 0.003). No DIABETES CARE, VOLUME 29, NUMBER 2, FEBRUARY 2006 Baillargeon and Associates
(partial P ϭ 0.001). This interaction hasalready been explained above.
CONCLUSIONS — The aim of this
study was to test the hypothesis that
women with PCOS would exhibit abnor-
mal metabolism of DCI and deficient in-
sulin-stimulated release of DCI-IPG,
which would correlate with their de-
creased sensitivity to insulin. Indeed, we
found that women with PCOS, when
compared with normal control women,
had a greater than fivefold increase in
uClDCI and a circulating concentration of
DCI that was reduced by half. These ab-
normalities persisted even when cor-
rected for differences in BMI between the
Figure 1—Correlation between urinary clearance of DCI and S when women with PCOS and normal women were analyzed together. Results are represented back-transformed in their original firmed with two-way ANOVA. Further-more, insulin sensitivity correlatedinversely and robustly with uClDCI, and women (2.9 [1.1–7.3] vs. 2.9 [1.6 –5.3], levels (partial P ϭ 0.02). This first inter- action has already been detailed above.
insulin-stimulated release of the putative tween uClDCI and Si identified in the en- serve as an internal control, the parame- tire group by univariate analysis (Fig. 1) ters of a different inositol, MYO, were also than in normal control subjects (P ϭ assessed. The levels and urinary clearance 0.025) (Table 1). Because insulin released the effect of uClDCI (partial P ϭ 0.002) was adjusted successively for total testos- tus (adjusted R2 ϭ 0.55 and P Ͻ 0.001 for [37.8 nmol ⅐ minϪ1 ⅐ lϪ1] vs. 2,312 ␮IU ⅐ minϪ1 ⅐ mlϪ1 [16.0 nmol ⅐ minϪ1 ⅐ lϪ1], the relationship between uClDCI and Si significant interaction was found between total testosterone levels and uClDCI (par- testosterone and Si. Furthermore, uClDCI tial P ϭ 0.04), which means that total tes- remained strongly associated with insulin resistance after correction for potential was an inverse correlation between S DCI) (r ϭ Ϫ0.60, P ϭ 0.002, n ϭ 24).
DCI, which was relatively strong (r ϭ This association in subgroup analysis was tional predictors of insulin sensitivity and 0.50) and highly significant (P Ͻ stronger after correction for BMI (partial 0.001) (Fig. 1). Correlation between S P Ͻ 0.001). No association between total women (n ϭ 23, r ϭ Ϫ0.39, P ϭ 0.07), Ͻ3.9 ml/min (P ϭ 0.51, predictors of insulin resistance, along but not in control subjects (P ϭ 0.63).
n ϭ 24), even after correction for BMI.
These results might be related to the lim- ited numbers of women in each group.
above were considered was S ϭ ␣ ϩ strongly suggest a contribution of abnor- mal handling of DCI to the insulin resis- tivariate analysis when the effect of group R2 ϭ 0.73, P Ͻ 0.001). The three pa- status (partial P ϭ 0.14) was adjusted suc- cessively for BMI, Si, total testosterone dently predicting Si were therefore PCOS or not) was not a predictor of insu- levels, and WHR (adjusted R2 ϭ 0.49 and AUCinsulin (negative association, partial P Ͻ 0.001 for model). However, signifi- P Ͻ 0.001), uClDCI (negative associa- tion, partial P ϭ 0.01), and the interac- BMI and group status (partial P Ͻ 0.001) tion between uClDCI and total testosterone DIABETES CARE, VOLUME 29, NUMBER 2, FEBRUARY 2006 Altered DCI urinary clearance in PCOS
tissues. The consequence is an intracellu- lar deficiency of DCI and, ultimately, of the DCI-IPG mediator of insulin action.
which suggests a role of insulin resistance sponse to stimulation by insulin results in a further decrease in insulin sensitivity ported that the relative increase in the bio- (i.e., aggravation of insulin resistance).
Hence, a “vicious cycle” is initiated whereby insulin resistance is amplified in and diabetic subjects. Urinary DCI excre- PCOS through the induction of a defect in ulations, ranging from 2.1 to 96.0 ␮mol/ suffers from the lack of direct evidence for mean 24-h urinary excretion of DCI of 3.7 ␮mol/day (Table 1 reports geometric opposed to undetectable levels through- tion in our study. This is because it is dif-means), which was comparable to the 2.1 out the clamp in patients with type 2 di- ␮mol/day reported by Ostlund et al. (26) abetes (16). Finally, a recent study pathway in vivo.
using the identical gas chromatography/ the action of insulin in obese women with icant results, even in multivariate analy- multiple linear regression models built for several purification steps and did not use sumed in the diet, and it is unlikely that a stability. Such validation in another pop- dietary deficiency could substantially al- ulation would be of great interest. Second, ute in part to these differences (29).
are also possible, such as an abnormality ditions associated with insulin resistance, in tissue/cellular uptake of DCI and/or in- such as diabetes, impaired glucose toler- ance, and familial history of diabetes.
these latter mechanisms should be associ- uClDCI is inversely correlated with insulin Ostlund et al. (26), however, reported an ated with a normal or increased circulat- sensitivity in women, and this correlation observe. Finally, it is possible that more than one metabolic abnormality is present also one of only three variables that best predicted insulin sensitivity in our study.
sistent with a defect in tissue availability PCOS, which correlates inversely with in- findings and the findings of Ostlund et al.
sulin sensitivity, is consistent with two contribute to the insulin resistance of the possible scenarios. One is that the defect causes insulin resistance. The other is that of insulin action. These findings offer a chiro-inositol confounds interpretation of insulin resistance, or more likely the hy- the studies of diabetic individuals, espe- (10 –12), which reported that oral admin- cially if diabetes was not well controlled.
clearance of DCI. It is also possible that other salutary effects consistent with an lease of bioactive DCI-IPG per unit of in- environmental “insult” causing insulin re- relevant to other disorders characterized sistance leads to a compensatory hyperin- lease is coupled with the release of insulin induces a defect that increases renal clear- ance of DCI, and this leads to a reduction Acknowledgments — This study was sup-
AUCDCI-IPG-to-AUCinsulin is likely a more in circulating DCI and its availability to ported in part by National Institutes of Health DIABETES CARE, VOLUME 29, NUMBER 2, FEBRUARY 2006 Baillargeon and Associates
Grants R01HD35629 (to J.E.N.), K24HD40237 (to J.E.N.), and R01DK58698 (to R.E.O.) and the Fond de Recherche en Sante´ du Que´bec (to puter program to calculate insulin sensi- polycystic ovary syndrome. N Engl J Med 11. Iuorno MJ, Jakubowicz DJ, Baillargeon JP, cose tolerance test. Comput Methods Pro- References
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