Abstract
This study assessed the relationships of urinary D-chiro-inositol and myo-inositol excretions to indices of whole body glucose tolerance and total content and tyrosine phosphorylation of the insulin receptor (activation) in skeletal muscle of older, non-diabetic subjects. 15 adults (age 65 ± 8 y; BMI 27.9 ± 3.3 kg/m2; mean ± SD) completed duplicate assessments of oral (75-g OGTT) and intravenous (300 mg/kg body weight IVGTT) glucose tolerance challenges and 24-h urinary D-chiro-inositol and myo-inositol excretions. Skeletal muscle (vastus lateralis) biopsies were obtained at min 60 of the OGTTs. Subjects with higher urinary D-chiro-inositol excretion had higher insulin (Rho = 0.51, P ≤ 0.05) and C-peptide (Rho = 0.56, P ≤ 0.05) area under the curves, and lower insulin sensitivity index (Rho = -0.60, P ≤ 0.05) during the IVGTT. The urinary myo / D-chiro-inositol ratio was also inversely related to insulin area under the curve (Rho = -0.59, P ≤ 0.05). Urinary D-chiro-inositol (Rho = -0.60, P ≤ 0.05) and myo-inositol (Rho = -0.60, P ≤ 0.05) were inversely related to tyrosine phosphorylation of the insulin receptor (tyrosines 1162/1163), but not total content of the insulin receptor during the OGTT. The apparent relationships were modestly weakened when adjustments were made for sex. These findings support previous research linking higher urinary D-chiro-inositol excretion with a progressive decline in whole body glucose tolerance. This is the first report to link higher urinary D-chiro-inositol excretion to a blunted activation of skeletal muscle insulin receptor signaling in older, non-diabetic subjects.
Introduction
Peripheral insulin resistance is a component of the metabolic syndrome and the first step in the progression towards overt type 2 diabetes [1]. Researchers have related insulin resistance to a decrease in insulin-receptor binding due to a reduction in the number of insulin receptors, in addition to defects in the post-receptor insulin signaling cascade [2]. A defect in the post-receptor insulin signaling can potentially lead to impairment of glycosyl-phosphatidylinositol (GPI) / inositol phosphoglycan (IPG) pathway [3]. Insulin stimulates the hydrolysis of GPI to IPG, which contains various compounds such as myo-inositol, chiro-inositol (L and D-chiro-), glucosamine, galacosamine, and other residues [3]. Myo-inositol is the most abundant inositol that is synthesized from glucose [4]. Myo-inositol is converted to chiro-inositol (conversion determined by the ratio of myo-inositol to chiro-inositol) in normal rats [5]. There is evidence that in diabetic rats, there is minimal or no conversion of myo-inositol to chiro-inositol [6, 7]. This altered ratio may be due to a defect in the epimerase-type enzyme that catalyzes this reaction [6].
The IPG that contains the D-chiro-inositol has been suggested to exert biological effects as a potential second messenger or mediator of insulin action [8, 9] by dephosphorylating (activating) glycogen synthase and pyruvate dehydrogenase, which are critical rate-limiting steps of nonoxidative and oxidative glucose disposal [10, 11], respectively. In humans with impaired tolerance or type 2 diabetes, D-chiro-inositol content is low or undetectable in the muscle [12] and IPG-D-chiro-inositol is also less active or inactive [13]. This could possibly decrease the activation of glycogen synthase and pyruvate dehydrogenase and negatively impact the insulin stimulated GPI / IPG pathway.
Research findings document that subjects with type 2 diabetes also have altered urinary D-chiro-inositol excretion. In patients with uncontrolled type 1 and 2 diabetes, renal clearance and 24-h urinary excretion of D-chiro-inositol were greater than in the normal subjects [14]. The urinary loss of myo-inositol also increased with diabetes, but the urinary loss of D-chiro-inositol was disproportionately greater than the loss of urinary myo-inositol [14]. Similar results were found in patients with polycystic ovary syndrome (PCOS) that were insulin resistant when compared to normal, healthy women [15]. Urinary D-chiro-inositol excretion increases with poor glycemic control, hyperinsulinema, and diabetes [14, 15]. Obesity does not seem to influence urinary D-chiro-inositol excretion [12, 14, 15]. Researchers suggest that urinary D-chiro-inositol excretion was inversely associated with insulin sensitivity [15] with the possibility that urinary D-chiro-inositol excretion and myo / D-chiro-inositol ratio are predictors of insulin resistance [16-19].
The primary purpose of this study was to assess the relationships of urinary D-chiro-inositol and myo-inositol to indices of glucose tolerance (glucose, insulin, C-peptide), insulin sensitivity, and skeletal muscle insulin receptor total content (independent of phosphorylation) and phosphorylation of specific tyrosine sites (1162/1163) necessary to activate the insulin receptor in older, non-diabetic individuals. We hypothesized that there would be a positive association between urinary inositol excretions and glucose, insulin, and C-peptide AUC. Furthermore, there would be a negative association between urinary inositol excretions and insulin sensitivity and total content and tyrosine phosphorylation of the insulin receptor.
Materials and Methods
Subjects
Sixteen subjects (6 men and 10 women) were recruited from the Little Rock and central Arkansas region to participate in this study. One woman discontinued participation in the study for reasons unrelated to the protocol. The inclusion criteria included: 1) aged ≥ 50 y, 2) body mass index 21-35 kg/m2, 3) no drug use known to alter carbohydrate metabolism, 4) no previous or current diagnosis of type 1 or type 2 diabetes mellitus or any other acute or chronic disease, 5) < 40 min of physical exercise 4 or more d/wk or the initiation of a structured exercise program in the past 3 mo, 6) body weight stability (< 2 kg change in the past 3 mo), 6) no impaired renal function (serum creatinine concentration < 150 μmol/L), and 7) women postmenopausal for at least 2 y. A pre-study evaluation included a physical examination, resting electrocardiogram, 75g OGTT, routine blood and urine chemistries, and subjects signing a consent form. This information was not used for baseline testing during the study. Monetary compensation was given to each subject for participating. The study protocol, consent form, and recruitment flyers were approved by the Human Research Advisory Committee, University of Arkansas for Medical Sciences, Little Rock, AR.
Study Design
The data presented in this report were obtained from subjects who participated in a randomized, placebo-controlled, double-blinded intervention study designed to assess the effects of pinitol (INZITOL™, CAS #10284-63-6, 3-O-methyl-1,2,4 cis-3,5,6 trans hexahydroxycyclohexane, 96% purity based on high performance liquid chromatography analysis, Humanetics Corporation, Chanhassen, MN) supplementation on whole-body glucose tolerance, insulin sensitivity, and insulin action in skeletal muscle. Testing and evaluations were conducted within 3-d periods before (baseline) and at the end of a 6-wk intervention (post) during which time each subject was provided a controlled energy and macronutrient-defined diet. As described previously [20], there were no differences at baseline or post between the Placebo (n=8) and Pinitol (n=7) groups and no significant changes over time occurred for any of the primary outcomes, except for an ∼12% decrease in whole body insulin sensitivity during the oral glucose tolerance test (OGTT). Also there were no group-dependent differences in responses from baseline to post for any outcomes except fasting plasma C-peptide concentration during the OGTT. Since the treatment (Placebo vs Pinitol) and time (baseline vs. post-study) did not statistically influence the results, for the current investigation, the data from the Placebo and Pinitol groups were combined (n=15) and the baseline and post results for each subject were averaged (pooled).
Testing Procedures
At baseline and post, OGTTs and IVGTTs were performed on consecutive days, with the subject residing at a General Clinical Research Center overnight between tests. Subjects were in a 12-h fasting state before starting the OGTT and IVGTT. A catheter was inserted into an antecubital vein, a blood sample was drawn, and the subject consumed a sugar-free beverage with nothing (placebo) or 1000 mg pinitol (only for Pinitol group at post testing) dissolved into it. The subject then consumed a 75g glucose solution (OGTT) within 5 min or was administered 300 mg glucose/kg body weight by intravenous injection of precisely portioned quantities of a 50% (wt:v) glucose solution over 60-s period (IVGTT). During the OGTT, blood was drawn at 30, 60, 90, 120, 150, and 180 min. During the IVGTT, blood was taken at 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 22, 25, 30, 40, 50, 60, 70, 90, 100, 120, 140, 160, and 180 min. All blood samples were collected into heparinized tubes, centrifuged at 3000 rpm, and aliquots of plasma were stored at -20°C until analyzed for plasma glucose, insulin, and C-peptide concentrations.
Muscle biopsy
During the OGTT at min 50, a small portion of each subject’s dominant leg was anesthetized with 4 mL of a 1% lidocaine solution. After the 60 min blood sample was drawn a sample of muscle from the vastus lateralis was obtained by the percutaneous needle biopsy technique (a 6-mm Bergstrom biopsy needle; Microsurgical Instruments) with applied suction [21]. The extracted muscle tissue was quickly blotted to remove blood, fat, or connective tissue. Then the muscle tissue was frozen and stored in liquid nitrogen until analyzed for total content and tyrosine phosphorylation of the insulin receptor. The muscle samples were obtained in an oral-glucose stimulated hyperglycemic and hyperinsulinemic state that corresponded with maximum plasma insulin concentration [22].
Body composition and 24-h urine collections
At baseline and post, in the morning after a 12-h fast and urine void, percentage of body fat and fat-free mass were estimated from body density using a whole body plethysmographer (BOD POD, Life Measurement) and the two-compartment model equation of Siri [23]. Timed 24-h urine collections were made during day 2 of dietary control. The urine was collected into a disposable 4-L polyethylene container and 10 mL of 1.0 normal acetic acid added as a preservative. Specific gravity was measured and an aliquot was placed in polypropylene tubes and stored at -20°C until analyzed for inositols.
Analytical methods and calculations
During the OGTT and IVGTT, plasma glucose concentrations were analyzed using an oxidase method standard for a Beckman glucose analyzer (model 6517, Beckman Instruments). Plasma insulin and C-peptide concentrations were analyzed using double antibody radioimmunologic procedures, as described by Engdahl et al. [24]. The integrated area under the curve (AUC) for plasma glucose, insulin, and C-peptide were calculated using the trapezoidal method [25]. For the OGTT, hepatic and whole body (hepatic plus peripheral) insulin sensitivities were estimated using the formulas described by Matsuda and Defronzo [26] and 24-h urinary myo-inositol and D-chiro-inositol were prepared and analyzed using mass spectrometry as previously described [20]. The myo / D-chiro-inositol ratio was calculated from the measured amounts of urinary myo-inositol and D-chiro-inositol excretions. The muscle samples taken during the OGTT were homogenized in a buffer containing protease and phosphatase inhibitors, measured for protein with a BCA kit and then analyzed for total insulin receptor content and insulin receptor phosphotryosine pYpY1162/1163 activation using ELISA kits from Biosource International. For the IVGTT, insulin sensitivity, acute insulin response to glucose, and glucose effectiveness were estimated using the MINMOD Millennium computer program (version 5.0, 2001) [27].
Statistical Analyses
The data are reported as mean ± SD. The correlations between variables were established using the Spearman Rho non-parametric ranked correlation test. Spearman’s partial rank-order correlation was used to control for potential confounders such as BMI and sex. A two-sample t-test was used to assess the differences between men and women and various parameters, such as subject characteristics, indices of glucose tolerance and insulin sensitivity during OGTT and IVGTT, urinary inositol excretion, and total content and tyrosine phosphorylation of the insulin receptor. Statistical significance was assigned if P ≤ 0.05. All data processing and calculations were performed using Statistical Analyses Systems Software (SAS Institute Inc., Cary, NC, version 9.1).
Results
Subject Characteristics
The subject characteristics, indices of glucose tolerance and insulin sensitivity of the men and women who participated in this study are presented in Table 1. The men and women did not differ in their BMI, HbAlc, indices of glucose tolerance, and insulin sensitivity. Age, height, body density, and fat-free mass (FFM) were higher and percent body fat and fat mass were lower in men compared to women (P ≤ 0.05). This older population was studied because they were at risk for developing type 2 diabetes. The majority of participants studied were classified as having impaired fasting glucose and / or impaired glucose tolerance. The 15 subjects were classified as follows: fasting glucose, 5 men and 6 women normal (< 5.6 mmol/L), 1 man and 3 women impaired ( 5.6 – 6.9 mmol /L); 2-hr glucose during OGTT, 3 men and 2 women normal (< 7.8 mmol/L), 3 men and 7 women impaired ( 7.8 – 11.0 mmol/L), and 1 man diabetic (> 11.1 mmol/L). The subjects’ urinary inositol excretions and skeletal muscle insulin receptor content and phosphorylation are listed in Table 2.
Table 1.
Subject characteristics and OGTT and IVGTT parameters
| Parameters | All (n=15) | Men (n=6) | Women (n=9) |
|---|---|---|---|
| Age, y | 65 ± 8 | 67 ± 10* | 64 ± 8 |
| Height, cm | 170.5 ± 10.9 | 178.2 ± 10.5* | 165.4 ± 8.1 |
| Body weight, kg | 81.2 ± 10.5 | 83.4 ± 15.4 | 79.7 ± 6.3 |
| Body mass index, kg/m2 | 27.9 ± 3.3 | 26.1 ± 2.5 | 29.2 ± 3.4 |
| Body density, g/mL | 1.01 ± 0.02 | 1.03 ± 0.01* | 0.99 ± 0.01 |
| Body fat, % | 39.1 ± 9.8 | 29.3 ± 5.2* | 45.6 ± 5.6 |
| Fat mass, kg | 31.8 ± 8.8 | 24.8 ± 7.5* | 36.5 ± 6.3 |
| Fat-free mass, kg | 49.3 ± 10.1 | 58.5 ± 9.5* | 43.2 ± 4.0 |
| Hemoglobin A1c, % | 5.4 ± 0.4 | 5.2 ± 0.4 | 5.5 ± 0.4 |
| Oral Glucose Tolerance Test | |||
| Glucose, fasting, mmol/L | 5.4 ± 0.5 | 5.6 ± 0.5 | 5.3 ± 0.5 |
| Insulin, fasting, pmol/L | 73 ± 30 | 84 ± 36 | 65 ± 24 |
| C-peptide, fasting, pmol/L | 591 ± 263 | 643 ± 208 | 556 ± 301 |
| Glucose, AUC, mmol/L • 180 min | 494 ± 172 | 498 ± 227 | 492 ± 140 |
| Insulin, AUC, nmol/L • 180 min | 46 ± 19 | 52 ± 25 | 42 ± 13 |
| C-peptide, AUC, nmol/L • 180 min | 259 ± 79 | 273 ± 103 | 250 ± 64 |
| Hepatic insulin sensitivity index | 0.48 ± 0.17 | 0.39 ± 0.11 | 0.53 ± 0.18 |
| Whole body insulin sensitivity index | 3.97 ± 1.30 | 3.41 ± 1.15 | 4.34 ± 1.32 |
| Intravenous glucose tolerance test | |||
| Glucose, fasting, mmol/L | 5.3 ± 0.5 | 5.4 ± 0.6 | 5.2 ± 0.5 |
| Insulin, fasting, pmol/L | 62 ± 24 | 72 ± 30 | 55 ± 17 |
| C-peptide, fasting, pmol/L • 180 min | 596 ± 223 | 583 ± 241 | 583 ± 241 |
| Glucose, AUC, mmol/L • 180 min | 361 ± 63 | 358 ± 93 | 363 ± 38 |
| Insulin, AUC, nmol/L • 180 min | 10 ± 5 | 13 ± 6 | 9 ± 3 |
| C-peptide, AUC, nmol/L • 180 min | 76 ± 30 | 92 ± 31 | 65 ± 26 |
| Insulin sensitivity, pmol/L • min | 7.48 ± 4.42 | 8.64 ± 4.17 | 8.64 ± 4.17 |
| Acute insulin response to glucose, nmol/L |
13.66 ± 8.37 | 11.83 ± 8.54 | 11.83 ± 8.54 |
| Glucose effectiveness, min-1 | 0.013 ± 0.006 | 0.013 ± 0.006 | 0.013 ± 0.006 |
Mean ± SD
P ≤ 0.05 (Men vs. Women)
AUC = Area under the curve
Table 2.
Urinary inositol excretions and skeletal muscle insulin receptor total content and phosphorylation
| All (n=15) | Men (n=6) | Women (n=9) | |
|---|---|---|---|
| Urine | |||
| Myo-inositol, μmol/d | 181 ± 101 | 206 ± 102 | 164 ± 103 |
| (21, 164, 394) | (104, 175, 394) | (21, 149, 317) | |
| Myo-inositol, μmol/kg FFM • d | 3.75 ± 2.23 | 3.49 ± 1.58 | 3.93 ± 2.65 |
| (0.46, 3.11, 7.53) | (1.97, 3.10, 6.57) | (0.46, 3.13, 7.53) | |
| D-chiro-inositol, μmol/d | 45 ± 26 | 60 ± 24 | 35 ± 22 |
| (1, 45, 95) | (36, 53, 95) | (1, 44, 59) | |
| D-chiro-inositol, μmol/kg FFM • d | 0.90 ± 0.46 | 1.01 ± 0.28 | 0.83 ± 0.55 |
| (0.03, 0.88, 1.45) | (0.69, 0.98, 1.40) | (0.03, 0.88, 1.49) | |
| Myo/D-chiro ratio | 5.43 ± 4.18 | 3.45 ± 0.99 | 6.74 ± 5.00 |
| (2.50, 4.24, 17.50) | (2.50, 3.21, 4.68) | (2.76, 4.42, 17.52) | |
|
#Muscle insulin receptor content, ng/μg protein |
|||
| Total | 0.42 ± 0.12 | 0.36 ± 0.07 | 0.47 ± 0.12 |
| Phosphotyrosine pY1162/1163 | 0.20 ± 0.07 | 0.16 ± 0.04 | 0.24 ± 0.08 |
Mean ± SD (low, median, high); n=15
n=14
Urinary D-chiro-inositol excretion
The 24-h urinary D-chiro-inositol excretion (μmol/d) was inversely related to insulin sensitivity and positively related to insulin and C-peptide AUC (Table 3), but not related to glucose AUC during the IVGTT. These observations were not observed during the OGTT. When adjusted for BMI, the apparent relationship with urinary D-chiro-inositol excretion (μmol/d) remained significant for C-peptide AUC (Rho = 0.59, P = 0.03) and insulin sensitivity (Rho = -0.58, P = 0.04), but not for insulin AUC (Rho = 0.45, P = 0.12). The relationships between urinary D-chiro-inositol excretion (μmol/d) and insulin AUC (Rho = 0.42, P = 0.15), C-peptide AUC (Rho = 0.53, P = 0.06), and insulin sensitivity (Rho = -0.51, P = 0.08) were not significant after adjusting for sex. However, when urinary D-chiro-inositol excretion was expressed as μmol/kg FFM • d, urinary D-chiro-inositol excretion was positivity correlated with C-peptide AUC (Table 3) and remained significant after adjusting for BMI (Rho = 0.62, P = 0.02) and sex (Rho = 0.61, P = 0.03).
Table 3.
Correlation between urinary inositol excretions and indices of glucose tolerance, insulin sensitivity, and tyrosine phosphorylation of the insulin receptor
| Urinary inositols | Insulin AUC mmol/L • 180 min |
C-peptide AUC mmol/L • 180 min |
Insulin Sensitivity Index mmoL/L • 180 min |
#Phosphotyrosine T1162/1163 Insulin Receptor |
|---|---|---|---|---|
| Myo, μmol/d | 0.22 | 0.32 | −0.38 | −0.60* |
| Myo, μmol/ kg FFM • d | 0.15 | 0.22 | −0.29 | −0.42 |
| D-chiro, μmol/d | 0.51* | 0.56* | −0.60* | −0.60* |
| D-chiro, μmol/ kg FFM • d | 0.34 | 0.52* | −0.42 | −0.51 |
| Myo/D-chiro ratio | −0.59* | −0.36 | 0.41 | 0.03 |
Data were assessed using Spearman Rho non-parametric ranked correlation
Rho, P < 0.05; n = 15
n=14.
Urinary D-chiro-inositol excretion (μmol/d) was negatively correlated with the insulin receptor phosphotyrosine 1162/1163 (Table 3 and Figure 1) during OGTT, but not correlated to the total insulin receptor content. When adjusted for BMI, the relationships between urinary D-chiro-inositol excretion and the insulin receptor phosphotyrosine 1162/1163 remained significant (Rho = -0.62, P = 0.03) and a trend when adjusted for sex (Rho = -0.50, P = 0.08).
Figure 1.
Correlations between urinary D-chiro-inositol and myo-inositol excretions and tyrosine phosphorylation of the insulin receptor, n=14. IGT = impaired glucose tolerance.
Urinary myo-inositol excretion
Urinary myo-inositol excretion (μmol/d) was not correlated to indices of glucose tolerance and insulin sensitivity (Table 3). Urinary myo-inositol excretion was negatively correlated to the insulin receptor phosphotyrosine 1162/1163 (Table 3 and Figure 1) during OGTT, but not correlated to total insulin receptor content. When corrected for BMI, the relationship with the insulin receptor phosphotyrosine 1162/1163 remained significant for urinary myo-inositol excretion (Rho = -0.61, P = 0.03), but was not maintained when adjusted for sex (Rho = -0.52, P = 0.08). When myo-inositol was expressed as μmol/kg FFM • d, the correlations were not significant after adjusting for BMI (Rho = -0.43, P = 0.14) and sex (Rho = -0.42, P = 0.17).
Urinary myo / D-chiro-inositol ratio
Urinary myo / D-chiro-inositol ratio was negatively correlated to insulin AUC during the IVGTT (Table 3) and was not maintained when adjusted for BMI (Rho = -0.52, P = 0.07) and sex (Rho = -0.21, P = 0.51). There were no statistically significant associations between urinary myo / D-chiro-inositol ratio and glucose AUC, C-peptide AUC, and insulin sensitivity. Urinary myo / D-chiro-inositol ratio was not correlated to the total content and tyrosine phosphorylation of the insulin receptor.
Discussion
The current study documents that higher urinary D-chiro-inositol is related to indices of glucose tolerance and insulin sensitivity. Campbell et al. [22] reported that 24-h urinary excretions of myo-inositol and D-chiro-inositol were positively related to plasma C-peptide AUC (indirect indicator of insulin secretion) during an OGTT in a group of older, overweight subjects. Also, there was an inverse correlation between myo / D-chiro-inositol ratio and C-peptide AUC [22]. Ostlund et al. [14] reported that 24-h urinary D-chiro-inositol excretion was positively correlated with fasting plasma glucose, glycated hemoglobin, and urinary glucose in patients with diabetes [14]. Baillargeon et al. [15] showed women that secreted greater amounts of urinary D-chiro-inositol also had decreased insulin sensitivity (inverse relationship). The present and previous human studies [14, 15, 22] show that subjects with higher urinary D-chiro-inositol excretion had greater insulin secretion (indicated by insulin and C-peptide AUC) and lower insulin sensitivity. The current study findings were in a selection of older, non-diabetic subjects and as a whole (group mean) had normal insulin sensitivity. However, when individuals were observed separately, there were various degrees of insulin sensitivity.
The relationships between urinary D-chiro-inositol excretion and insulin sensitivity, insulin AUC, C-peptide AUC, and the insulin receptor phosphotyrosine 1162/1163 were apparent, just not maintained when adjusted for sex. In addition, the relationship between urinary myo-inositol excretion and the insulin receptor phosphotyrosine 1162/1163 was not maintained when adjusted for sex. Campbell et al. [22] also found that the correlation between urinary inositol excretions and C-peptide AUC were strongly influenced by sex. When compared to women, men had a greater excretion of urinary D-chiro-inositol and myo-inositol and higher C-peptide AUC measured during an OGTT [22]. However, a sex effect was not evaluated. This could likely reflect the small sample size. It is evident that the men and women in the current study had differences in FFM. When D-chiro-inositol was expressed in μmol/kg FFM • d, the relationships between urinary D-chiro-inositol and C-peptide AUC remained even after adjusted for sex. Limited data have shown that men and women have differences in peripheral insulin sensitivity and insulin receptor binding [28]. Older women had higher peripheral insulin sensitivity and higher insulin receptor binding than men [28]. In opposition to the study findings, Ostlund et al. [14] found no sex-related differences in urinary D-chiro-inositol and myo-inositol excretions. However, the study was conducted in younger individuals [14].
Due to the lack of previous research on the relationship between urinary D-chiro-inositol excretion and insulin signaling, we started our evaluation at the initial steps of the insulin-signaling pathway with measurements of total insulin receptor content and activation of the insulin receptor at specific sites (1162/1163). Previous research has documented that depending on an individual’s insulin-resistant state, urinary inositol excretion and tyrosine phosphorylation of the insulin receptor can be affected. In people with type 2 diabetes and PCOS, urinary D-chiro-inositol excretions were elevated [14, 15] and tyrosine phosphorylation of the insulin receptor were reduced [29]. The present study found an inverse correlation between urinary inositol excretions and the insulin receptor phosphotyrosine 1162/1163 before adjustments were made for BMI and sex. The autophosphorylation of the insulin receptor is obligatory for insulin sensitivity as it initiates propagation of the insulin signaling cascade finally culminating in the translocation of glucose transporters (GLUT4) and activation of glucose transport. It has been proposed that activation of the insulin receptor can lead to activation of large G-proteins [30]. Then the large G-protein can activate phospholipase C or D, which hydrolyzes GPI at the cell surface and release of IPG extracellularly [30]. The IPG containing D-chiro-inositol must reenter the cell through the myo / D-chiro-inositol transporter [31] in order to exert biological activities as an insulin second messenger [8, 9]. One of the biological effects is activation of glycogen synthase. In skeletal muscle samples from people with type 2 diabetes, IPG-chiro-inositol was reduced or undetectable, whereas IPG-myo-inositol remained the same [12, 13]. After administration of insulin, the chiro-inositol concentrations remained undetected and myo-inositol concentrations increased then decreased after 15 min [12]. We would speculate that if people with type 2 diabetes have reduced phosphorylation of the tyrosine insulin receptor then the GPI / IPG pathway would be altered, subsequently reducing IPG-D-chiro-inositol activation of glycogen synthase.
It is generally recognized that urinary D-chiro-inositol excretion is altered or abnormal with worsening glucose control [14, 15, 22]. The proposed mechanisms for elevated urinary D-chiroinositol excretion include reduced renal threshold for D-chiro-inositol [15], abnormal tissue/cellular uptake of D-chiro-inositol [31], and / or abnormal intracellular processing of D-chiro-inositol (myo-inositol conversion to D-chiro-inositol) [7]. The current study supports previous studies [14, 15, 22] that found elevated urinary D-chiro-inositol excretion associated with increased insulin and C-peptide AUC and decreased insulin sensitivity. Caution is warranted not to over-interpret the present findings because this was a small observational study with results apparently influenced by the sex of the subjects and mechanisms were not evaluated.
In conclusion, the present study in non-diabetic older subjects support previous research linking higher urinary D-chiro-inositol excretion with a decline in whole body glucose tolerance, and extend them to include a blunted activation of tyrosine phosphorylation of the insulin receptor in skeletal muscle. The current study was not designed to evaluate sex differences, but the potential that sex influenced these relationships underscores that this issue needs to be further explored in larger groups of men and women who have a wide range of insulin sensitivities.
Acknowledgements
Sincere thanks go to the volunteers who participated in this study, the University of Arkansas for Medical Sciences General Clinical Research Center dietary and nursing staffs, and Andreana Robertson for help with statistical analysis. Hannah Morse-Carrithers, MS, RD, James Fluckey, Ph.D., Matthew Hulver, Ph.D., and Zonda Birge, MS, are thanked for their technical and analytical contributions to this research. The Humanetics Corporation is thanked for providing the pinitol powder.
Support: NIH P50 AT00477, NIH R29 AG13409, NIH R01 AG021911, and NIH M01 RR14288
Footnotes
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