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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 242-249
Copyright © 2004 by The Endocrine Society

Metformin Therapy Increases Insulin-Stimulated Release of D-Chiro-Inositol-Containing Inositolphosphoglycan Mediator in Women with Polycystic Ovary Syndrome

Jean-Patrice Baillargeon, Maria J. Iuorno, Daniela J. Jakubowicz, Teimuraz Apridonidze, Na He and John E. Nestler

Department of Medicine (J.-P.B.), Université de Sherbrooke, Sherbrooke, J1H SN4 Canada; Departments of Medicine (M.J.I., T.A., N.H., J.E.N.) and Obstetrics and Gynecology (J.E.N.), Virginia Commonwealth University, Richmond, Virginia 23298-0111; and Hospital de Clinicas Caracas (D.J.J.), 1040 Caracas, Venezuela

Address all correspondence and requests for reprints to: John E. Nestler, M.D., Medical College of Virginia, P.O. Box 980111, Richmond, Virginia 23298-0111. E-mail: nestler{at}hsc.vcu.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Some actions of insulin are mediated by putative inositolphosphoglycan mediators, and a deficiency in D-chiro-inositol-containing inositolphosphoglycan (DCI-IPG) may contribute to insulin resistance in women with polycystic ovary syndrome (PCOS). Furthermore, similar effects of DCI and metformin, an insulin-sensitizing drug, have been demonstrated in PCOS women. To determine whether metformin improves insulin actions by increasing biologically active DCI-IPG in women with PCOS, we analyzed DCI-IPG during an oral glucose tolerance test in 19 obese women with PCOS before and after 4–8 wk of metformin or placebo. After treatment, the mean (±SE) area under the curve (AUC) during the oral glucose tolerance test of insulin (AUCinsulin) decreased significantly more in the metformin group, compared with the placebo group [-3574 ± 962 vs. +1367 ± 1021 µIU/min·ml (-26 ± 7 vs. +10 ± 7 nmol/min·liter), P = 0.003], but the AUC of DCI-IPG (AUCDCI-IPG) decreased similarly in both groups (-1452 ± 968 vs. -2207 ± 1021%/min, P = 0.60). However, the ratio of AUCDCI-IPG/AUCinsulin increased by 160% after metformin and decreased by 29% after placebo (P = 0.002 between groups). Moreover, metformin seemed to improve the positive correlation between AUCDCI-IPG and AUCinsulin but not placebo (r = 0.32, P = 0.68 at baseline; r = 0.52, P = 0.12 after metformin; and r = -0.39, P = 0.30 after placebo). We conclude that in obese women with PCOS, metformin may improve the action of insulin in part by improving insulin-mediated release of DCI-IPG mediators, as evidenced by increased bioactive DCI-IPG released per unit of insulin.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE POLYCYSTIC OVARY syndrome (PCOS) is a prevalent but poorly understood disorder associated with significant adverse short-term and long-term health consequences. It is defined by chronic anovulation and hyperandrogenism (1) and affects 6–10% of women of child-bearing age (2). PCOS is the most common cause of female anovulatory infertility in the United States and is associated with an increased risk of developing hypertension, dyslipidemia, impaired glucose tolerance or type 2 diabetes, and cardiovascular disease (2).

During the past decade, increasing evidence supports the central role of insulin resistance and compensatory hyperinsulinemia in the pathogenesis of the syndrome (3, 4). Obese and lean women with PCOS manifest insulin resistance independent of fat mass (3, 5) and administration of insulin-sensitizing drugs, such as metformin (3), troglitazone (3), and D-chiro-inositol (DCI) (6, 7) to both obese and lean women with the syndrome increases the frequency of ovulation and decreases circulating androgens.

Some actions of insulin may be effected by putative inositolphosphoglycan (IPG) mediators of insulin action (8, 9), and evidence suggests that a deficiency in a specific DCI-containing IPG (DCI-IPG) may contribute to insulin resistance in individuals with impaired glucose tolerance or type 2 diabetes (10, 11). In support of this idea, oral administration of DCI has been demonstrated to improve glucose tolerance while reducing insulin in both obese (6) and lean (7) women with PCOS and also to decrease serum androgens and improve ovulatory function.

Metformin, a well known insulin-sensitizing drug, has also demonstrated such benefits in women with PCOS in numerous randomized, placebo-controlled trials (3). Because the mechanism of action of metformin is complex and not completely understood and because it exerts effects similar to those of DCI in PCOS, we hypothesized that metformin improves insulin action in part by increasing insulin-mediated release of DCI-IPG in women with PCOS.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We analyzed the release of insulin and DCI-IPG during an oral glucose tolerance test (OGTT) in 19 obese [body mass index (BMI) >= 27.5 kg/m2] women with PCOS before and after 4–8 wk of therapy with metformin (Glafornil, North Medicamenta, Caracas, Venezuela) (n = 10) or placebo (n = 9). The subjects were part of a previously published randomized, placebo-controlled trial on PCOS (11 randomized to metformin and 13 to placebo) (12), and we used all the subjects with enough blood available for the assays. Women were 18–35 yr old and had PCOS, as defined by oligomenorrhea (fewer than six menstrual periods in the previous year) and hyperandrogenemia (elevated serum-free testosterone concentrations).

Hyperprolactinemia, thyroid dysfunction, and late-onset adrenal hyperplasia were excluded by the appropriate tests. All women had polycystic ovaries based on ultrasonographic findings, and none had taken any medication for at least 2 months before study or had diabetes mellitus. The initial clinical trial had been approved by the Institutional Review Boards of the Hospital de Clinicas Caracas and Virginia Commonwealth University, and each woman had given informed consent. The present retrospective study was approved by the Institutional Review Board of Virginia Commonwealth University.

The study protocol was detailed in the original report (12). On d 1 the women came to the hospital after a 12-h overnight fast, and blood samples were drawn at 0830, 0845, and 0900 h and pooled for the measurement of insulin, glucose, and steroids. At 0900 h, 75 g dextrose (Glycolab, Relab Laboratory, Caracas, Venezuela) was given orally. Blood samples were collected at 0, 60, and 120 min for determination of serum glucose and insulin concentrations and serum DCI-IPG bioactivity. The women were then randomized to treatment with either metformin (500 mg orally thrice daily) or placebo for 4–8 wk, and at the end of study the OGTT was repeated.

Insulin and glucose assays

Blood samples were centrifuged immediately, and sera were stored at -20 C until assayed. Serum-free testosterone was determined by RIA (Diagnostic Products, Los Angeles, CA). All other hormones were assayed as described previously (13, 14, 15). To avoid interassay variation, all samples were analyzed in duplicate in a single assay for each hormone. The intraassay coefficients of variation for insulin was 5.5%, and they were less than 10% for all the steroid hormone assays.

DCI-IPG insulin mediator bioactivity assay

Isolation of insulin mediator. Blood samples were centrifuged immediately, and sera were stored at -80 C until processed. DCI-IPG mediator was isolated from serum as described previously (10, 11, 16) with some modifications. Briefly, 1.0 ml serum samples (distilled water for blank control) were mixed with 3.0 ml extraction buffer consisting of formic acid (final concentration, 67 mmol/liter), 2-mercaptoethanol (1.34 mmol/liter), and EDTA (1.34 mmol/liter). Samples were boiled at 100 C for 5 min and cooled on ice for 45 min.

Subsequently, 20 mg activated charcoal (Sigma Chemical Co., St. Louis, MO) were added to each sample, which was then kept on ice for 10 min and centrifuged (60,000 x g, 60 min at 4 C). The supernatants were neutralized with NH4OH to pH 6.0 and centrifuged again (40,000 x g, 30 min at 4 C). The supernatants were then mixed with 1.2 mg anion exchange resin in formate form (AG 1-X8 Resin; Bio-Rad Laboratories, Hercules, CA). The slurries of resin and supernatants were agitated slowly overnight at 4 C. Then slurries were poured into polypropylene columns (BIO-RAD Poly-Prep, Bio-Rad Laboratories) and eluted stepwise with 4.8 ml distilled water and 8 ml (0.03 N) HCl (pH 1.75).

The pH 1.75 fractions were shell frozen and lyophilized twice. To remove inhibitors of pyruvate dehydrogenase (PDH) (17, 18) from the samples, lyophilized powder from each sample was suspended in 2.0 ml absolute ethanol, sonicated for 5 min, vortexed for 1 min, centrifuged for 20 min (500 x g), and the supernatant was discarded (16). Ethanol purification steps were repeated twice. Thereafter, samples were dried under stream of N2 and reconstituted with 200 µl distilled water.

To confirm that DCI was present in the insulin IPG mediator fraction purified from the serum, a sample of this material was subjected to size exclusion chromatography, and the resulting eluent fractions were assayed for DCI.

Serum (800 µl) collected at the 90-min time point of an OGTT was treated as described above, lyophilized, dissolved in 1.2 ml aqueous formic acid (pH 4.0), and injected onto a 1.6 x 90-cm column containing P2 gel (Bio-Rad Laboratories). The column was eluted with dilute aqueous formic acid (pH 4) at a flow rate of 0.2 ml/min, and fractions were collected at 2.5 ml intervals. The fractions were concentrated, and the residues were hydrolyzed (6N HCl, 48 h, 100 C) so that DCI present would be in the free, nonglycosylated form. The hydrolyzed fractions were lyophilized and analyzed for DCI using a Dionex HPLC system with PAD detector and CarboPac MA1 column (4 x 250 mm), eluting with 200 mM NaOH at 0.4 ml/min.

To confirm the DCI content of the peak from the serum sample, the extract from H4IIE rat hepatoma cells incubated with [3H]DCI and treated with 10 nmol/liter insulin was placed onto a P2 sizing column and eluted in conditions identical to those of the serum sample. This P2 peak of DCI content from the plasma sample corresponded to the one of the two P2 peaks of radioactivity obtained from extract of [3H]DCI-treated H4IIE rat hepatoma cells. The other peak of H4IIE cell extract matched the retention volume of free DCI.

The DCI content (2 nmol) from the processed OGTT serum sample eluted as a single peak from the P2 sizing column in fractions 49–54. The elution volume of the peak was lower (implying higher molecular weight) than free DCI (fractions 58–62) under these conditions. The P2 peak of DCI content from the serum sample corresponded to the more rapidly eluting one of two peaks of radioactivity obtained from a 1-butanol extract of [3H] DCI-treated H4IIE rat hepatoma cells. The other peak of radioactivity from the H4IIE cell extract matched the retention volume of free chiro-inositol. Interestingly, in three experiments, pretreatment of the H4IIE cells with 10 nM insulin increased the counts by 26.4 ± 2.5% relative to cells not stimulated with insulin in the more rapidly eluting peak of H4IIE cell extract corresponding with the DCI peak from the serum sample.

PDH phosphatase bioactivity assay. To date, it has not been possible to measure the content of extracted DCI-IPG because its structure and exact mass are still unknown, and no specific antibody suitable for an immunoassay has yet been developed. However, its has been well established that the DCI-IPG insulin mediator fraction has pyruvate dehydrogenase phosphatase (PDP) stimulating activity (Fig. 1Go) (19, 20). PDP is necessary for activation of PDH complex by dephosphorylation (21, 22, 23), which increases the rate of production of the reduced form of ß-nicotinamide adenine dinucleotide (NADH) from ß-nicotinamide adenine dinucleotide (NAD). The assay with this sequence of reaction was used to determine DCI-IPG mediator bioactivity as described previously (24), with some modifications (Fig. 1Go).



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  		FIG. 1. Outline of the two stages of the PDH bioactivity assay. PDHi, Inactive phosphorylated form of PDH; CoA, coenzyme A.

 
Before each DCI-IPG bioactivity assay was run, the basal activity of PDH was standardized using a PDH inhibition curve with different concentrations of ATP because ATP converts active PDH to its inactive, phosphorylated form (25).

First, PDH was activated by mixing 10 µl of assay buffer [1 mmol/liter imidazole (pH 7.0), 2 mmol/liter dithioretitol, 10 mmol/liter MgCl2, 0.1 mmol/liter CaCl2, and 0.5 mg/ml BSA], 10 µl PDH (Sigma Chemical Co.), 10 µl PDP, and 10 µl of different concentrations of ATP (range, 0.5–4 mM) into the wells of a 96-well microtiter plate and by preincubating the plate for 30 min at 37 C. This reaction was terminated with 5 µl NaF (210 mmol/liter). The PDP used in this assay was prepared from beef hearts, and its concentration was adjusted so that 10 µl PDP activated 0.045 µU/ml PDH complex to half its maximal activity in presence of 10 µmol of Mg2+.

Thereafter, the production of the reduced form of NADH was started by adding into each well 5 µl sodium pyruvate mixture (3 mmol/liter ADP and 3 mmol/liter Na pyruvate) and 45 µl reaction mixture [82.5 mmol/liter imidazole, 82.5 mmol/liter NAD, 1.65 mmol/liter thiamine pyrophosphate chloride, and 3.3 mmol/liter coenzyme A]. The resultant mixture was then incubated at room temperature for 2 min, and NADH production rate was determined spectrophotometrically by measuring the rate of absorbance change, at 340 nm, during four time points over 45 sec with a microplate reader (VersaMAX; Molecular Devices, Sunnyvale, CA). The measurements of reaction rate, reported in milliOD/min, were plotted for each ATP concentration, and the appropriate concentration of ATP, which inhibited PDH activity to 10% of its maximal bioactivity, was determined.

The sample bioassay used the same steps as described above, except 40 µl purified serum sample were added to the incubation mixture with 10 µl ATP at the predetermined appropriate concentration. Patient samples were measured in duplicates on the same microplate.

DCI-IPG insulin mediator extracted from a control pooled serum was assayed with samples on the same plate each time the PDH assay was run. Based on this control serum, which was assayed at seven different times in duplicates, the interassay coefficient of variation of the bioassay was 17.4%. The PDH activity intraassay coefficient of variation was 6.7%, based on 22 assays of the control serum done on the same microplate. To determine the variability of the entire assay method, the basal (0 min) and peak (60 min) OGTT samples from a subject were separated into three aliquots and put through the DCI-IPG mediator extraction and PDH assay steps. Coefficients of variation were 10.7 and 8.5%, respectively, for the absolute values of samples basal and peak DCI-IPG bioactivity. Finally, we determined that the mediator bioactivity for the same control serum kept at -80 C and assayed at an 18-month interval was not changed (data not shown), which means DCI-IPG insulin mediator bioactivity is stable after a prolonged freezing.

To adjust for variation in basal PDH activity from one assay to the other, and therefore from subject to subject, the water-blank activity was subtracted from the bioactivity of DCI-IPG released into serum during OGTT, which was then expressed as the percentage of its bioactivity at baseline (0 min).

PDH bioactivity dilution curve. The dependency of PDH activity on content of DCI-IPG mediator is represented in Fig. 2AGo. For this experiment, DCI-IPG was extracted from 1.0 ml of pooled serum samples, obtained 60 min after oral administration of 75 g glucose, and reconstituted with 100 µl water, i.e. twice as concentrated as the study samples. Different volumes of this solution of extracted DCI-IPG were used in the PDH assay. However, because we did not have a measurement of the actual content of mediator, we expressed the different contents of DCI-IPG as volume instead of concentration. The results are the average of five replicates and expressed as percentage relative to water-blank.



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  		FIG. 2. A, Dependency of PDH bioactivity on content of DCI-IPG. DCI-IPG was extracted from 1.0 ml of pooled blood samples, obtained 60 min after oral administration of 75 g glucose and reconstituted with 100 µl water. Different volumes of this solution were used in the PDH assay. We expressed the different contents of DCI-IPG as volume instead of concentration because it is not possible actually to measure the true content of DCI-IPG. Values are the averages of five replicates expressed as percentage relative to water-blank ± SD. B, Results of DCI-IPG bioactivity in nine subjects included in the present study, expressed as percentage relative to water-blank, for all time points of the OGTTs before and after treatment. The short dashed line represents the mean, and the long dashed lines indicate the range.

 
We were also able to determine the percentage DCI-IPG bioactivity relative to water-blank for nine subjects of the present study. Figure 2BGo illustrates their results at all time points of the OGTTs before and after treatment. As shown, all these results fell within the linear part of the dose-response curve of our PDH assay.

Statistical analysis

We analyzed the response of serum insulin concentrations and the relative bioactivity of DCI-IPG to the oral administration of glucose by calculating the areas under the respective response curves (AUCs) by the trapezoidal rule. Serum AUCDCI-IPG/AUCinsulin ratios were not normally distributed and were therefore log transformed for statistical analyses and then reported backtransformed in their original units.

The relative DCI-IPG bioactivities during OGTT were compared with 100% at 0 min using one sample mean two-tailed z-test, and a Bonferroni corrected {alpha}-level of 0.025 was used to account for multiple comparisons (two comparisons). Insulin levels during OGTT were compared for overall differences using ANOVA and for difference with levels at 0 min using Dunnett’s test.

Comparisons between groups at baseline were made with a two-tailed t test. To assess the treatment effects between groups, the changes in each variable (after treatment minus baseline) were compared also using a two-tailed t test. Correlation analyses were performed using Pearson’s correlation (JMP 4.0; SAS Institute, Cary, NC). All results are reported as means ± SE, except for graphics in which means and 95% confidence intervals (CIs) are presented. P < 0.05 were considered significant, except for Bonferroni correction, when indicated.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Baseline characteristics

The women in the metformin and placebo groups did not differ significantly with respect to age, BMI, waist to hip ratio, blood pressure, or serum total or free testosterone concentrations (Table 1Go). They also did not differ at baseline with respect to AUCinsulin and the relative bioactivity of DCI-IPG (AUCDCI-IPG) curves during OGTT as well as the ratio of AUCDCI-IPG/AUCinsulin. However, the baseline fasting serum insulin was lower in the metformin group than in the placebo group [14.7 ± 3.4 vs. 26.7 ± 3.5 µIU/ml (106 ± 24 vs. 193 ± 25 pmol/liter), P = 0.02].


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  		TABLE 1. Clinical characteristics and serum hormone concentrations of women with the PCOS at baseline

 
Anthropometric measurements and serum sex steroid concentrations

Weight and BMI did not change after treatment, but the waist to hip circumference ratio decreased only in the metformin group, which was significantly different from the placebo group (P = 0.003) (Table 2Go). Systolic and diastolic blood pressures also decreased significantly more in the metformin group after treatment (P = 0.04 and P = 0.02, respectively).


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  		TABLE 2. Changes in clinical characteristics and serum hormone concentrations after the administration of metformin or placebo for 4–8 wk in women with the PCOS

 
Changes in serum total testosterone concentrations did not differ between groups (P = 0.16), but free testosterone concentrations decreased significantly only in the metformin group, which were significantly different, compared with the placebo group (P = 0.008).

Serum insulin levels and the bioactivity of DCI-IPG after a glucose load

At baseline, we observed a significant increase in the bioactivity of DCI-IPG (P = 0.008, using a Bonferroni corrected {alpha}-level of 0.025) and in serum insulin levels (ANOVAs P < 0.001) 60 and 120 min after 75 g glucose, compared with 0 min (Fig. 3Go).



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  		FIG. 3. Changes in DCI-IPG bioactivity and insulin levels during OGTTs administered to women with PCOS in both groups (A) at baseline; the metformin group (B) after 4–8 wk of treatment; and the placebo group (C) after 4–8 wk of treatment. Results are the mean and 95% CI. To convert values for insulin to picomoles per liter, multiply by 7.2. *, P <= 0.008 for comparison with 0 min using one-sample mean z-test with a Bonferroni corrected {alpha}-level of 0.025. {dagger}, P < 0.05 for comparison with 0 min using ANOVA test for overall differences and Dunett’s test for difference with 0 min.

 
After treatment, the bioactivity of DCI-IPG during OGTT increased significantly in the metformin group at 60 min, compared with 0 min (P < 0.001) but not at 120 min (P = 0.04, using a Bonferroni corrected {alpha}-level of 0.025). After placebo, the bioactivity of DCI-IPG did not change significantly during the OGTT (P = 0.60 at 60 min and P = 0.50 at 120 min, compared with 0 min). Insulin levels increased significantly both in the metformin and placebo groups at 60 and 120 min during OGTT, compared with 0 min (ANOVAs P = 0.003 and P < 0.001, respectively).

Serum insulin and the bioactivity profiles of DCI-IPG

After treatment, the changes in serum fasting insulin concentrations were comparable in the metformin and placebo groups (P = 0.14) (Table 2Go). However, the mean AUCinsulin decreased significantly only in the metformin group, and this decrease was significantly different from the increase in the placebo group [-3574 ± 962 vs. +1367 ± 1021 µIU/min·ml (-26 ± 7 vs. +10 ± 7 nmol/min·L), respectively, P = 0.003] (Fig. 4Go).



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  		FIG. 4. Changes in mean AUCinsulin during OGTTs after the administration of metformin or placebo for 4–8 wk in women with the PCOS. Values are the mean change and 95% CI. To convert values for AUCinsulin to nanomoles per liter, multiply by 0.0072.

 
The decrease in mean AUCDCI-IPG was slightly significant only in the placebo group (-2207 ± 1021%/min), but the observed decreases in both groups were not statistically different (-1452 ± 968%/min, P = 0.60) (Fig. 5Go). However, the ratio of AUCDCI-IPG/AUCinsulin after treatment increased by 160% in the metformin group and decreased by 29% in the placebo group and was 2.3 times higher after metformin than after placebo (P = 0.002) (Fig. 6Go).



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  		FIG. 5. Changes in mean relative bioactivity AUCDCI-IPG during OGTTs after the administration of metformin or placebo for 4–8 wk in women with PCOS. Values are the mean change and 95% CI.

 


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  		FIG. 6. Changes in the ratio of the AUCinsulin/AUCDCI-IPG curve during OGTTs after the administration of metformin or placebo for 4–8 wk in women with PCOS. Values are the geometric mean change and 95% CI. The ratios were log transformed for statistical analysis and are reported backtransformed in their original units, so the change represents the ratio of the result after treatment compared with baseline (no change is represented by a ratio of 1.0).

 
Correlations between AUCinsulin and AUCDCI-IPG

At baseline, there was no correlation between the AUCDCI-IPG and the AUCinsulin for the two groups combined (r = 0.32, P = 0.68) (Fig. 7AGo), but the positive correlation improved in the metformin group after treatment (r = 0.52), although it was not statistically significant (P = 0.12) (Fig. 7BGo). After treatment, the correlation in the placebo group was not positive and remained poor (r = -0.39), and it was not statistically significant (P = 0.30) (Fig. 7CGo).



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  		FIG. 7. Correlation between AUCDCI-IPG and AUCinsulin before (A) and after the administration of metformin (B) or placebo (C) for 4–8 wk in women with PCOS. Closed circles represent women with PCOS and open circles, normal controls. To convert values for AUCinsulin to micro-international units per minute per milliliter, divide by 0.0072.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study assessed the effect of metformin treatment on the insulin-stimulated release of a putative DCI-IPG during an OGTT in women with PCOS. Metformin, or a corresponding placebo, was administered to obese women with PCOS for 4–8 wk. After treatment, areas under the relative bioactivity curve of DCI-IPG during OGTT decreased slightly and similarly in both groups. In contrast, AUCinsulin during OGTT decreased significantly more in the metformin group compared with placebo. Consequently, the ratio of AUCDCI-IPG/AUCinsulin increased significantly more after metformin therapy, i.e. the percent release of bioactive DCI-IPG per unit of insulin increased 2.3-fold more after metformin than after placebo.

If DCI-IPG insulin mediator has a significant role in glucose metabolism, it would not be surprising that its release after a glucose load is relatively constant after metformin because glucose levels are also stable in women with PCOS with normal glucose tolerance. Therefore, the AUCDCI-IPG/AUCinsulin ratio is probably a better way to assess insulin-mediated DCI-IPG release than AUCDCI-IPG alone.

It is assumed that the release of the DCI-IPG mediator is stimulated by insulin during an acute oral glucose load in humans and not by glucose or other factors. Indeed, insulin is greatly increased during OGTT and has been shown to stimulate directly the release of DCI-IPG in vitro (26). There is no evidence that glucose can stimulate directly the release of DCI-IPG, and the increase of other growth factors during OGTT are unlikely to be responsible for the increase in bioactive DCI-IPG, as discussed by Shashkin et al. (16).

An increase in insulin mediators activating PDH has been demonstrated in healthy young subjects after glucose ingestion (27), and Shashkin et al. (16) reported that the relative increase in the bioactivity of DCI-IPG during an OGTT is abolished in obese men with type 2 diabetes, compared with healthy men. These findings are consistent with the observation that the concentrations of DCI in IPG preparations from muscle biopsies obtained during euglycemic-hyperinsulinemic clamp studies were detectable in normal subjects at baseline and increased 6-fold during the clamp, as opposed to undetectable levels of DCI throughout the clamp in patients with type 2 diabetes (11).

Similarly, adipocytes isolated from nonobese, insulin- resistant, and diabetic rats did not release IPGs in response to insulin as opposed to adipocytes of control rats (28). Moreover, four different states of insulin resistance, i.e. glucocorticoid treatment (29, 30), streptozotocin-induced diabetes (31), aging (32), and obesity (33), have been associated with the inability of insulin to release IPGs from isolated rat hepatocytes. These results were observed with insulin concentrations as low as 14–139 µIU/ml (100–1000 pmol/liter), which are comparable with the mean insulin concentrations during OGTT in our women with PCOS at baseline [i.e. 20–88 µIU/ml (144–634 pmol/liter)]. This suggests that insulin resistance is the main factor impeding the release of bioactive DCI-IPG by insulin, rather than obesity or diabetes, and supports the possibility of such a defect in women with PCOS because they have significant insulin resistance (5).

Our results demonstrate that bioactive DCI-IPG insulin mediator is released in serum after a glucose load in obese women with PCOS. The relative bioactivity of DCI-IPG increased significantly and paralleled the increase in insulin levels (Fig. 3AGo).

The release of the bioactivity of DCI-IPG during OGTT was significantly less pronounced and delayed after placebo, compared with metformin (Fig. 3Go, B and C). At that time, the women in the placebo group probably were more insulin resistant because their insulin levels and AUCinsulin were much higher. This is concordant with the observations in type 2 diabetic patients and insulin-resistant rat models.

Because insulin is thought to mediate the release of DCI-IPG after a glucose load (16), one would expect to observe a correlation between insulin levels and the increase in DCI-IPG bioactivity during an OGTT in normal subjects. In insulin-resistant states, however, this correlation should be abolished. We found that in obese women with PCOS, there was an absence of a correlation between AUCDCI-IPG and AUCinsulin (Fig. 7AGo).

After metformin treatment, but not after placebo administration, a positive correlation emerged, which missed attaining statistical significance. It would have been advisable to increase the power of this analysis by increasing the number of subjects, but unfortunately it was not possible. However, this observation corroborates the suggestion that insulin-resistant women with PCOS have a blunted release of DCI-IPG in response to insulin that might be improved by the insulin-sensitizing drug metformin.

The primary mechanism of action of metformin is to reduce hepatic glucose production (34, 35), and it has been shown to inhibit glycogenolysis (35) and increase lipogenesis (36) in the liver or hepatocytes. Because the main actions of DCI-IPGs are to stimulate PDP activity (9, 10, 19, 20, 37), the limiting enzyme of insulin-induced lipogenesis, and glycogen synthase phosphatase activity (9, 37), the limiting enzyme of insulin-induced glycogenesis, they are expected to inhibit hepatic neoglucogenesis as well.

DCI-IPGs have also been shown to lower blood glucose when injected to streptozotocin-diabetic, but not to normal, rats (38). In another study with the same model, DCI-IPGs decreased blood glucose without inducing hypoglycemia (39). These clinical effects are similar to the normoglycemic property of metformin. Therefore, it is plausible that metformin improves hepatic glucose production in insulin- resistant states by restoring insulin-stimulated release or activity of IPG mediators containing DCI.

We conclude that in obese women with PCOS, metformin therapy significantly decreased serum insulin concentrations without changing the release of DCI-IPG during OGTT. Therefore, the release of bioactive DCI-IPG per unit of insulin was much higher after metformin than after placebo, and the correlation between the release of DCI-IPG and insulin also seemed to improve after metformin. Collectively, these findings suggest that metformin may enhance the action of insulin in PCOS in part by improving insulin-mediated release of the DCI-IPG mediator, as evidenced by increased serum DCI-IPG bioactivity released per unit insulin after a glucose load.


   Acknowledgments
 
We thank Dr. Mark Sleevi (INSMED Inc.) for his contribution to the characterization of DCI content in isolated IPG mediator and for helpful review of this manuscript.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants R01HD35629 and K24HD40237 (both to J.E.N.) and grants from the Fond de Recherche en Santé du Québec (to J.-P.B.).

Abbreviations: AUC, Area under the curve; BMI, body mass index; CI, confidence interval; DCI, D-chiro-inositol; DCI-IPG, DCI-containing IPG; IPG, inositolphosphoglycan; NAD, ß-nicotinamide adenine dinucleotide; NADH, reduced form of NAD; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; PDH, pyruvate dehydrogenase; PDP, pyruvate dehydrogenase phosphatase.

Received March 13, 2003.

Accepted September 19, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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