- Email: [email protected]
The effect of combined inositol supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity
Accepted Manuscript The effect of combined Inositols supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity Francesca Ferrari, MD, Fabio Facchinetti, MD, Alejandra E. Ontiveros, MD, Robyn P. Roberts, MD, Mia M. Saade, Sean C. Blackwell, MD, Baha M. Sibai, MD, Jerrie S. Refuerzo, MD, Monica Longo, MD, PhD PII:
S0002-9378(16)30254-X
DOI:
10.1016/j.ajog.2016.05.038
Reference:
YMOB 11127
To appear in:
American Journal of Obstetrics and Gynecology
Received Date: 5 March 2016 Revised Date:
18 May 2016
Accepted Date: 24 May 2016
Please cite this article as: Ferrari F, Facchinetti F, Ontiveros AE, Roberts RP, Saade MM, Blackwell SC, Sibai BM, Refuerzo JS, Longo M, The effect of combined Inositols supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity, American Journal of Obstetrics and Gynecology (2016), doi: 10.1016/j.ajog.2016.05.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The effect of combined Inositols supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity
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Francesca Ferrari MD1,2, Fabio Facchinetti MD2, Alejandra E. Ontiveros MD1, Robyn P. Roberts MD1, Mia M. Saade1, Sean C. Blackwell MD1, Baha M. Sibai MD1, Jerrie S. Refuerzo MD1, Monica Longo MD, PhD1 1
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Division of Maternal Fetal Medicine, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Texas Health Science Center at Houston, Houston, Texas; 2 Department of Obstetrics & Gynecology, University of Modena and Reggio Emilia, Modena, Italy Corresponding Author:
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Francesca Ferrari, M.D. Division of Maternal Fetal Medicine Department of Obstetrics & Gynecology, University of Modena and Reggio Emilia, Via del Pozzo 71 41124 Modena, Italy Office: +39 059 4225334 E-Mail: [email protected]
The authors report no conflict of interest
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Financial support: Partially supported by the Larry Gilstrap Center
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Presented as oral presentation # 69 at the 36th Annual Meeting of the Society of Maternal Fetal Medicine, held in Atlanta, February 1-6, 2016.
Word count: Manuscript: 3090/3000; abstract: 442/500
Figure 3 can be included in the print issue of the Journal.
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ACCEPTED MANUSCRIPT Condensation Inositols supplementation improves metabolic profile and blood pressure only in pregnancies
Short Title
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Inositols effect in pregnancies with metabolic syndrome and obesity
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complicated by metabolic syndrome.
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ACCEPTED MANUSCRIPT ABSTRACT Background: Myoinositol (MI) and D-chiro-inositol (DCI) improve insulin resistance in women with obesity, gestational diabetes and in post-menopausal women with metabolic syndrome (MS). We previously reported that offspring born to hypertensive dams lacking endothelial nitric oxide
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synthase (eNOS) and fed high fat diet (HFD) develop metabolic like syndrome (MLS) phenotype. Objective: To investigate the effect of a mixture of MI/DCI supplementation during pregnancy on the maternal metabolic profile in pregnancies complicated by MLS and obesity using a pregnant model.
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mouse
Study Design: Female heterozygous eNOS-/+ mice with moderate hypertension were placed on
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HFD for 4 weeks to induce a MLS phenotype. Similarly, wild type (WT) C57BL/6 mice were placed on high fat diet (HFD) for 4 weeks to induce a murine obesity model. Mice were then bred with WT males. On gestational day 1 (GD1), dams were randomly allocated to receive either a mixture of MI/DCI in water (7.2/0.18 mg/ml respectively) or water as control (placebo). At term,
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(GD18), maternal weights, systolic blood pressure (SBP) and glucose tolerance test (GTT) were obtained. Dams were then sacrificed; pups and placentas were weighed and maternal blood collected. Serum levels of metabolic biomarkers relevant to diabetes and obesity (ghrelin, GIP,
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GLP-1, glucagon, insulin, leptin, resistin) were measured by multiplex elisa assay (Bio-Rad). Analysis was done comparing MLS-MI/DCI-treated versus MLS-non-treated mice and obese-
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MI/DCI-treated
versus
obese-non-treated
mice.
Results: Mean SBP was lower in MLS pregnant mice treated with MI/DCI compared to placebo (p=0.04), whereas there was no difference in SBP between treated and placebo obese pregnant mice. Pregnant MLS mice treated with MI/DCI showed lower glucose values during the GTT and in area under the curve (AUC, MI/DCI: 17512.5 ± 3984.4 vs. placebo: 29687.14 ± 8258.7; p=0.003), but no differences were seen in the obese pregnant mice. Leptin serum levels were lower in the MLS-MI/DCI-treated mice compared with the placebo group (MI/DCI: 16985 ± 976.4 pg/dl vs. placebo: 24181.9 ± 3128.2 pg/dl, p= 0.045). No other differences were seen in any of the 3
ACCEPTED MANUSCRIPT remaining serum metabolic biomarkers studied in MLS and in obese pregnant mice. Maternal weight gain was not different in the pregnant MLS dams, while it was lower in the obese MI/DCItreated dams compared to the placebo group (MI/DCI: 10.9 ± 0.5 g vs 12.6 ± 0.6 g, p=0.04). Fetal and placental weights did not differ between MI/DCI-treated and non-treated pregnant dams with
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MLS and obesity.
Conclusion: Combined Inositols treatment during pregnancy improves blood pressure, glucose levels at GTT and leptin levels in pregnant dams with MLS phenotype, but not in obese pregnant
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dams. In addition, Inositols treatment was associated with lower gestational weight gain in the
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obese, but not in the MLS pregnant dams.
Key words: Inositol, Metabolic syndrome, Obesity, Insulin Resistance, Blood Pressure, eNOS
KO
mice
and
C57BL/6J
mice,
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Pregnancy,
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ACCEPTED MANUSCRIPT INTRODUCTION Metabolic syndrome (MS) and obesity are growing causes of morbidity and mortality worldwide. MS is a major risk factor for cardiovascular disease (CVD) and is defined by the National Institutes of Health (NIH) by having at least three of the following conditions:
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hypertension, elevated fasting plasma glucose, central obesity, elevated triglycerides or low highdensity lipoprotein cholesterol1,2. MS etio-pathogenesis and long-term consequences are still largely unknown and preventive strategies have not yet been identified3. The impact of MS and obesity
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during pregnancy is substantial: indeed, all metabolic changes that develop during pregnancy have well-known effects not only on maternal and fetal health during pregnancy but they also act as a
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catalyst for future health throughout later life4,5. Epidemiological and animal studies have shown that pregnancies complicated by MS and obesity are at risk for premature CVD, gestational diabetes and preeclampsia and predispose the offspring to an increased risk of cardiovascular and metabolic disease later in life6-8.
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Inositols are a family of simple carbohydrates naturally found in several foods. Inositols exist in 9 possible stereoisomers, 2 of which are predominantly found in eukaryotic cells: Myoinositol (MI) and D-chiro inositol (DCI) 9. The exact molecular mechanisms of action of MI and DCI have not
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yet been fully elucidated, however it is well established that MI and DCI have different roles as mediators of insulin, which lead to different functions within the cells. MI is the precursor of
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inositol triphosphate, a second messenger, responsible for the regulation of many hormones such as insulin, thyroid-stimulating hormone, and follicle-stimulating hormone. The activation of phospholipids-containing MI by insulin, activates glucose-transporters, such as GLUT-4, and increases the cell membrane permeability to glucose, which gets into the cell and is immediately available as substrate10. DCI is the conversion product of MI, by an epimerization reaction, which is unidirectional and insulin-dependent11. DCI, differently from MI, is able to determine the intracellular accumulation of glucose, (i.e., glycogen synthesis). So, both stereoisomers are considered to play a key role in the insulin pathway, acting synergistically as insulin-sensitizing 5
ACCEPTED MANUSCRIPT agents through the enhancement of glucose peripheral tissue uptake and glycogen synthesis12. In vivo animal13,14 and human studies15-19 demonstrated that Inositols supplementation during pregnancy improve glucose profile and reduces the adverse effects of hyperglycemia. Moreover, MI supplementation was proven to reduce insulin resistance in post-menopausal women with MS and
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in women with polycystic ovary syndrome, a metabolic and endocrine disorder associated with insulin resistance12. However, to the best of our knowledge, Inositols supplementation effect has never been investigated in pregnancies complicated by MS. Moreover, most of the studies in
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pregnant women focus on the effect of MI supplementation alone, whereas mounting evidence in PCOS studies suggest that the administration of combined MI/DCI, at the physiological plasma
cardiovascular risk parameters20.
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ratio (40:1) ensures better clinical results, such as the reduction of insulin resistance and
Thus our aim was to evaluate the effect of natural compounds, as Myoinositol and D-chiro inositol, used as a mixture, to treat pregnancies complicated by obesity and MS, which represent
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different degrees of a similar metabolic disorder. We hypothesize that MI/DCI treatment improve the abnormal maternal metabolic profile in a pregnant mouse model of either MLS or obesity. The metabolic profile improvement seen with MI/DCI treatment could also translate into positive long-
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term maternal and fetal health. To achieve our aim we used well-characterized mice models of obesity and MS that will allow identifying the best target population for a MI/DCI treatment in
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pregnancies with metabolic abnormalities. We previously reported that heterozygous mice lacking endothelial nitric oxide synthase (eNOS) gene and born to hypertensive mothers21, eNOS-/+, when fed high fat diet (HFD) for at least 4 weeks, develop metabolic like syndrome (MLS) phenotype such as: obesity, glucose intolerance, elevated systolic blood pressure, low HDL, high insulin, and low adiponectin levels compared to their counterpart fed control diet22. Furthermore, wild type C57BL/6J female mice fed obesogenic diet from weaning for 4 consecutive weeks show dietinduced obesity23.
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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Animals Female and male mice, homozygous for disruption of the eNOS gene (eNOS-KO-/-, strain B6.129P2, stock no. 002684) and their age matched wild-type controls (WT, strain C57BL/6J, stock
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no. 000664) were purchased from Jackson Laboratory (Bar Harbor, ME) at 6 weeks of age. The study was approved by the Animal Welfare Committee (AWC) of the University Of Texas Health Science Center, at Houston (UTHSC-H). The mice were housed separately in temperature and
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humidity-controlled quarters with constant 12:12-hours light-dark cycles in the animal care facility
Metabolic-like-syndrome mouse model
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at the (UTHSC-H).
eNOS-KO-/- females were bred with WT males to obtain eNOS+/- heterozygous females. Then a MLS mouse model was achieved by using eNOS-/+ heterozygous offspring manifesting a moderate hypertension phenotype21,24, and fed obesogenic diet for 4 consecutive weeks after
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weaning (Figure 1)22. At 7-8 weeks of age, non-pregnant eNOS-/+ females were bred with WT males. At gestational day 1 (GD1) of pregnancy, determined by the presence of vaginal plug after overnight exposure to male breeders, MLS dams were randomly allocated to receive either a
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mixture of MI/DCI dissolved in water (see next paragraph for concentrations) or plain water, as placebo (control group) (Figure 1).
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Obesity mouse model
To induce the obesity phenotype (Figure 1), WT-C57BL/6J female mice were fed obesogenic diet from weaning for 4 consecutive weeks23. Then, at 7-8 weeks of age, obese females were bred with WT males and starting from GD1, obese mice were randomly allocated to receive either a mixture of MI/DCI dissolved in water (see next paragraph for concentrations) or plain water, as placebo (control group). Obesogenic Diet
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ACCEPTED MANUSCRIPT Both models, eNOS-/+-heterozygous and WT females offspring were allocated to obesogenic diet, high fat diet (HFD, D12492, 60% of fat) until 7-8 weeks of life to induce the MLS and obesity phenotypes respectively. HFD was maintained during the whole pregnancy until animals were
of food left on the grid from the initial weight of food supplied. Inositols Supplementation during pregnancy
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sacrificed at term, on GD18. Daily food consumption was estimated by subtracting the total amount
The MI/DCI mixture corresponds to the physiological plasma ratio of MI and DCI equal to
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40:1, which has been proven to be the most effective20. On GD1, MLS and obese mice were randomly allocated to receive either a mixture of MI/DCI dissolved in water (7.2/0.18 mg/ml
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respectively, based on previous animals13,14 and human15-19 studies) or plain water as placebo. Considering that blood volume during pregnancy physiologically shows 20% increase compared to the non-pregnant status, we adjusted the MI/DCI doses by a 20% increase to the previously established doses used in a non-pregnant obese mouse model (MI: 6 mg/ml)13,14. Myo-inositol and
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D-chiro-inositol were purchased from Sigma Chemicals. The treatment was maintained until term gestation (GD18 of pregnancy), when dams were sacrificed. Pregnant mice were single-housed to be able to carefully evaluate water intake and consequently daily dose of MI/DCI. Daily water
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consumption was estimated by subtracting the total amount of water left in the bottle from the initial amount supplied. On average pregnant mice drink 5 ml/ day, so MI/DCI daily consumption
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was approximately 36/0.9 mg of respectively MI/DCI / day, per mouse. At term gestation, the metabolic profile of the MLS and obese pregnant dams were obtained including: systolic blood pressure (SBP), glucose tolerance test (GTT), maternal gestational weight gain and serum levels of metabolic biomarkers relevant to diabetes and obesity. Damns were then sacrificed, pups and placentas weights and numbers collected. Blood Pressure Measurement SBP measurements were always taken on GD18 in the morning at the same time using a calibrated eight-chamber tail-cuff system (CODA, Kent Scientific, Torrington, CT). Dams were 8
ACCEPTED MANUSCRIPT kept warm using a warming pad. Mice underwent an initial 10 cycles of acclimatization period; BP was then monitored and recorded over 10 new cycles. SBP averaged of the last 10 cycles was used for the final BP measurement and utilized for data analysis25,26. Glucose Tolerance Test (GTT)
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Pregnant dams on GD18 underwent GTT after being fasting for 6 hours. Mice received 1.0 g/kg of glucose I.P. and serial blood glucose levels, via a tail nick at 0, 15, 30, 60, and 120 min, were immediately determined with the Accu-Chek Aviva Blood Glucose Meter System (Roche
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Diagnostics) after glucose administration. Glucose levels and the area under the curve (AUC) were compared between MLS and obese pregnant mice treated with MI/DCI and identical non-treated
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ones27. Serum Metabolic Panel
Blood samples were obtained as soon as animals were sacrificed by heart puncture. Serum levels of metabolic biomarkers relevant to diabetes and obesity such as glucagon, insulin, leptin,
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ghrelin, Gastric Inhibitory Peptide (GIP), Glucagon-like Peptide 1 (GLP-1) and resistin were measured by multiplex elisa assay (Bio-Rad). Statistical Analysis
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Results are expressed as mean ± SEM. All the data were analyzed using unpaired t-test with SigmaPlot 12. The analysis was done comparing MLS MI/DCI-treated pregnant mice versus MLS
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non-treated (placebo) pregnant mice and obese MI/DCI-treated pregnant versus obese non-treated (placebo) pregnant mice. Numbers of pregnant mice per group were: MLS MI/DCI-treated, n= 9; MLS placebo-treated, n=8; Obese MI/DCI-treated, n= 8; Obese placebo-treated, n=6. (Figure 1).
RESULTS Food and water intake Daily food intake was not different between MLS-MI/DCI-treated and MLS-placebo mice (MI/DCI: 6.0 ± 0.9 vs placebo: 6.5 ± 0.7 g), nor between Obese MI/DCI-treated and Obese placebo9
ACCEPTED MANUSCRIPT treated (MI/DCI: 6.3 ± 0.6 vs placebo: 6.0 ± 0.7 g). Daily water intake was not different between MLS-MI/DCI-treated and MLS-placebo mice (MI/DCI: 5.1 ± 0.9 vs placebo: 4.9 ± 0.7 ml), nor between Obese MI/DCI-treated and Obese placebo-treated (MI/DCI: 4.8 ± 0.9 vs placebo: 5.0 ± 0.7 g).
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Systolic Blood Pressure Measurement
At term gestation, mean SBP was lower in MLS pregnant mice treated with MI/DCI compared to placebo group (MI/DCI: 138.52 ± 6.48 vs. placebo: 157.03 ± 7.79 mmHg, p=0.04)
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(Figure 2a). In the obese pregnant dams, there was no difference in SBP between MI/DCI-treated and placebo damns (MI/DCI: 143.18 ± 7.6 vs. placebo: 150.09 ± 7.9), (Figure 2b).
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Glucose tolerance
The glucose levels in the GTT were lower in the MLS pregnant mice treated with MI/DCI compared to placebo-treated mice at all time periods except for the 15 minutes value (Figure 3a). To confirm, the AUC for GTT was lower in MLS mice MI/DCI-treated group (MI/DCI: 17512.5 ±
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3984.4 vs. placebo: 29687.14 ± 8258.7; p= 0.003). In contrast, in the obese pregnant dams, glucose levels in the GTT and the AUC were not different between MI/DCI-treated and placebo-treated dams (MI/DCI: 23573.6 ± 4758.2vs. placebo: 25410 ± 5764.4) (Figure 3b).
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Serum Metabolic Profile
Serum leptin levels were lower in the MLS MI/DCI-treated mice compared with the placebo
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-treated group (MI/DCI: 16985 ± 976.4 pg/dl vs. placebo: 24181.9 ± 3128.2 pg/dl, p= 0.045). No other differences were seen in the serum levels of the remaining metabolic biomarkers studied in the MLS pregnant mice (Table 1). No difference was found between obese MI/DCI-treated and non-treated mice in any of the investigated metabolic biomarkers (Table 1). Weights Gestational weight gain was not different between MLS-MI/DCI-treated and placebo mice (MI/DCI: 11.53 ± 0.9 vs placebo: 11.6 ± 0.7 g), but was lower in the obese MI/DCI-treated dams compared to the placebo treated mice (MI/DCI: 10.9 ± 0.5 vs. placebo: 12.6 ± 0.6 g, p=0.04). 10
ACCEPTED MANUSCRIPT There were no differences between the pregnant MLS MI/DCI-treated and placebo groups respectively in fetal weights and placental weights, as reported in Table 1. Likewise, fetal and placental weights did not differ between MI/DCI-treated and placebo obese pregnant mice (Table
obese either treated or non-treated MI/DCI. A summary of all experiments results is reported in Figure 6. COMMENT
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1). Furthermore no differences were found regarding number of pups born from dams with MLS or
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We found that MI/DCI supplementation in pregnant mice with MLS phenotype improved blood pressure, glucose tolerance and leptin levels. However, in obese pregnant mice, MI/DCI
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treatment prevented maternal increased weight gain associated with a HFD but did not affect maternal glucose tolerance, blood pressure and the metabolic markers related to diabetes. Our findings therefore, support the rationale for the use of combined MI/DCI supplementation in pregnancies complicated by MS, but not by obesity alone in these animal
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models. Inositols are FDA approved supplements28 with a potential role in improving metabolic profile in MS. It is well established that MS traits, such as obesity and insulin resistance during pregnancy have negative impact on the pregnancy and on the metabolic and cardio-vascular future
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health of the mother and the offspring4-7,29. Therefore, the potential of a new ‘‘nonpharmacological’’ approach as a preventive and therapeutic agent is clinically relevant, since
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appropriate preventive strategies for MS in pregnancy have not yet been identified. Previous human studies focused either on gestational diabetes prevention/therapy15-19 or on MS associated with menopause30, 31 or PCOS32-34, whereas to the best of our knowledge this is the first study to investigate Inositols combination effect in a MLS and obese pregnant mouse model. Four human studies reported on the efficacy of myo-inositol supplementation during pregnancy to prevent gestational diabetes in patients at risk due to family history, obesity or PCOS15-18. In addition, one prospective randomized trial demonstrated a therapeutic role of MI in women with diagnosed gestational diabetes showing that MI in second trimester reduced homeostatic model 11
ACCEPTED MANUSCRIPT assessment resistance and adiponectin concentrations compared to controls19. Similarly, in nonpregnant women with PCOS32-34 and in postmenopausal women30,31, MI was shown to reduce insulin resistance and improve cardiovascular risk parameters (reduction in BP, total and LDLcholesterol, triglycerides and enhancement of the HDL-cholesterol). Our study reveals that Inositol
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supplementation improved SBP and glucose tolerance, in pregnancies complicated by MS but not in obese pregnancies.
The lack of effect in the obese pregnant mice could be due to the short period of HFD
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exposure leading only to weight gain but not to metabolic abnormalities severe enough to reveal effects of MI/DCI supplementation. In fact, according to the literature 4 weeks of HFD exposure
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should be sufficient to develop a diet-induced obesity phenotype23, however several animal studies reported a longer period of HFD exposure, up to 12 weeks, in order to manifest metabolic abnormalities and an Inositol effect13. Moreover, the lack of effect in obese pregnant mice could be due to different mechanistic pathways involved. We speculate that MI/DCI treatment improves
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glucose tolerance in MLS pregnant mice but not in the obese ones possibly involving NO pathway, which is known to be defective in our MLS model, but not in the obese one21,35. The exact molecular mechanisms of action of MI and DCI have not yet been fully elucidated. It has been
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shown that potential mechanisms by which the Inositols might act is by improving endothelial function in decreasing radical oxidative stress, enhancing endothelial nitric oxide synthase and NO
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activity36. This eNOS-/+ heterozygous mouse manifests MLS probably due to the lack of eNOS associated with an obesogenic diet during pregnancy. The metabolic abnormalities seen in this model of MLS might be a consequence of an endothelial dysfunction leading to hyperinsulinemia, insulin resistance and increased blood pressure. Thus, this mouse model of MLS seems very well suited to benefit from Inositols supplementation, which aims at restoring endothelial function. Indeed in our obese pregnant mouse model, obesogenic diet alone and for a short period of time does not seem to lead to such an endothelial dysfunction for which Inositol supplementation could show a target effect. 12
ACCEPTED MANUSCRIPT In our study leptin levels were improved by MI/DCI treatment in MLS pregnant mice, which is consistent with previous studies that showed decreased circulating leptin concentration after 16 weeks of MI treatment in women with PCOS12,37. The exact mechanism by which MI/DCI reduces leptin level remains unclear. Leptin controls energy balance and body weight by regulating
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neuronal activity in the hypothalamus38. It is possible that MI/DCI improved leptin resistance through either reductions in circulating leptin levels or up-regulations of receptors mediating leptin’s action to the brain, which needs further investigation. Furthermore in our study, maternal
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gestational weight gain was significantly reduced in the obese mice after MI/DCI treatment, in agreement with previous studies on gestational diabetes patients15,39. In both, obese and MLS
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models, pups and placentas weights did not differ between MI/DCI-treated and placebo animals. These findings are in contrast to others who found a reduction in hyperglycemia-related complications, such as fetal macrosomia after MI supplementation in women at risk for gestational diabetes for a family history of type 2 diabetes16 or elevating fasting glucose levels15; however
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again this could be due to a difference in the duration of HFD or to the fact that we evaluated the average weights of litters and placentas independently from their genotype (eNOS-/+ heterozygous, 25%, or wild type, 75%).
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Study strengths are: 1) to the best of our knowledge this is the first time that combined Inositols (MI/DCI) have been evaluated as a mixture rather than single compounds during
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pregnancy, 2) and furthermore being used in a MLS mouse model during pregnancy. Indeed, dietinduced obesity could be considered a less severe degree of the same metabolic disease that ultimately leads to MS. Therefore, the thorough investigation of Inositol in the presence of a spectrum of components of MS, rather than obesity alone, allows for the identification of the best target population for MI/DCI treatment. Moreover MI and DCI have shown different concentrations at the tissue level40,41 and different roles in the whole cell11,20 leading to the conclusion that both of them should be used in a synergistic action for the purpose of better treatment efficacy in metabolic diseases20. Unfer and colleagues performed dose–response studies identifying the physiological 13
ACCEPTED MANUSCRIPT plasma ratio of MI and DCI equal to 40:1, which was proven to be the most effective in PCOS overweight patients with metabolic abnormalities
20, 41-43
, but it has never been studied in
pregnancies with metabolic abnormalities. The main weakness of our study is that we did not investigate the effect of MI/DCI on lipid
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profile, which is an important feature of MS. We also did not evaluate the nitric oxide pathway as well as the inositol phosphoglycans intracellular mediators as possible mechanisms involved in the MI/DCI actions to elucidate our results. Moreover, in our study we did not plan to evaluate the
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combined MI/DCI effect in physiologic uncomplicated pregnancies, nor we planned to evaluate the effect of MI or DCI alone.
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Future studies need to be performed to investigate the effect of MI/DCI treatment on longterm maternal health and in the offspring born to MS and obese pregnancies in order to identify a possible role of Inositols on metabolic fetal programming. Undoubtedly, further studies are warranted to fully elucidate the molecular pathways triggered by MI and DCI, and to define the
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ideal timing (pre-conceptional, pregnancy and/or post-partum) dose, and combination of Inositol
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stereoisomers of such a supplementation.
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Chiossi G, Costantine MM, Tamayo E, Orise P, Hankins GD, Saade GR, Longo M. . Effect
of age and gender on the progression of adult vascular dysfunction in a mouse model of fetal programming lacking endothelial nitric oxide synthase. Am J Physiol Heart Circ Physiol. 2011
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Whitesall SE, Hoff JB, Vollmer AP, D'Alecy LG. Comparison of simultaneous
measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. Am
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J Physiol Heart Circ Physiol. 2004 Jun;286(6):H2408-15. Epub 2004 Feb 12. Feng M, Whitesall S, Zhang Y, Beibel M, D'Alecy L, DiPetrillo K. Validation of volume-
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pressure recording tail-cuff blood pressure measurements. Am J Hypertens. 2008 Dec;21(12):128891. Epub 2008 Oct 9. 27.
Barros RPA, Gabbi C, Morani A, Warner M, Gustafsson JA. Participation of ERα and
ERβ in glucose homeostasis in skeletal muscle and white adipose tissue. Am J Physiol- Endocrinol Metabolism. July 2009 Vol. 297 no. 1, E124-E133 28.
G. Carlomagno, V. Unfer, Inositol safety: clinical evidences, Eur. Rev. Med. Pharmacol.
Sci. 15 (2011) 931-936.
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Kaul P, Savu A, Nerenberg KA, et al. Impact of gestational diabetes mellitus and high
maternal weight on the development of diabetes, hypertension and cardiovascular disease: a population level analysis. Diabet Med. 2015;32 (2):164-173. 30.
A. Santamaria, D. Giordano, F. Corrado, B. et al. One-year effects of myo-inositol
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supplementation in postmenopausal women with metabolic syndrome, Climacteric 15 (2012) 490495. 31.
D. Giordano, F. Corrado, A. Santamaria, S. Quattrone, B. Pintaudi, A. Di Benedetto, R.
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D’Anna, Effects of myo-inositol supplementation in postmenopausal women with metabolic syndrome: a perspective, randomized, placebo-controlled study, Menopause 18 (2011) 102-104. Unfer V, Carlomagno G, Dante G, Facchinetti F. Effects of myo-inositol in women with
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PCOS: a systematic review of randomized controlled trials. Gynecol Endocrinol 2012; 28(7):50915 33.
Genazzani AD, Lanzoni C, Ricchieri F, Jasonni VM. Myo-inositol administration positively
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affects hyperinsulinemia and hormonal parameters in overweight patients with polycystic ovary syndrome. Gynecol Endocrinol 2008;24(3):139-44 34.
Facchinetti F, Bizzarri M, Benvenga S et al. Results from the International Consensus
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Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS. Eur J Obstet Gynecol Reprod Biol. 2015 Oct 3;195:72-76. Dmitriy N. Atochin, Paul L. Huang. Endothelial nitric oxide synthase transgenic models of
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endothelial dysfunction. Pflugers Arch. 2010 November; 460(6): 965–974. Published online 2010 August 10. 36.
Nascimento NR, Lessa LM, Kerntopf MR, Sousa CM, Alves RS, Queiroz MG, Price J,
Heimark DB, Larner J, Du X, Brownlee M, Gow A, Davis C, Fonteles MC. Inositols prevent and reverse endothelial dysfunction in diabetic rat and rabbit vasculature metabolically and by scavenging superoxide. Proc Natl Acad Sci U S A. 2006 Jan 3;103(1):218-23. Epub 2005 Dec 22.
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Gerli S, Papaleo E, Ferrari A, Di Renzo GC. Randomized, double blind placebo-controlled
trial: effects of myo-inositol on ovarian function and metabolic factors in women with PCOS. Eur Rev Med Pharmacol Sci. 2007 Sep-Oct;11(5):347-54. 38.
de Git KC, Adan RA. Leptin resistance in diet-induced obesity: the role of hypothalamic
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Reece EA, Khandelwal M, Wu YK, Borenstein M. Dietary intake of myo-inositol and
neural tube defects in offspring of diabetic rats. Am J Obstet Gynecol. 1997;176(3):536-539. Nestler JE, Jakubowicz DJ, Reamer P, et al. Ovulatory and metabolic effects of D-chiro-
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inositol in the polycystic ovary syndrome. N Engl J Med 1999;340(17): 1314-20. Heimark D, McAllister J, Larner J. Decreased myo-inositol to chiro-inositol (m/c) ratios and
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increased m/c epimerase activity in PCOS theca cells demonstrate increased insulin sensitivity compared to controls. Endocr J 2014;61 (2):111-17. 42.
Carlomagno G, Unfer V, Roseff S. The D-chiro-inositol paradox in the ovary. Fertil Steril
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Minozzi M, Nordio M, Pajalich R. The Combined therapy myo-inositol plus D-Chiro-
inositol, in a physiological ratio, reduces the cardiovascular risk by improving the lipid profile in
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PCOS patients. Eur Rev Med Pharmacol Sci 2013; 17(4):537-40.
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ACCEPTED MANUSCRIPT TABLE Table 1 - Serum Metabolic Profile (pg/ml) and Fetal & Placental Weights (g) MLS MODEL
OBESE MODEL
Placebo
p
MI/DCI
Placebo
p
GIP
530.1 ± 49.3
674.6 ± 88.0
0.18
412.5 ±111.3
542.1 ± 70.8
0.34
GLP-1
42.6 ± 4.2
45 ±5.9
0.74
34.9 ±6.7
36.6 ± 4.6
0.84
Glucagon
564.5 ± 83.0
461.1 ±75.8
0.37
400.8 ± 48.7
453.1 ± 64.5
0.53
1179.3 ± 136.9 18156.6 ± 1472.1* 15016.1 ± 3459.7
1146.7 ±176.7 22620.4 ± 3290.2 22676.6 ± 15492.8
0.88
999.7 ± 204.9
0.045
0.9 ± 0.01
0.88 ± 0.01
0.11 ± 0.02 7
Ghrelin Mean Fetal Weight Mean Placental Weight Number of pups
21998.2 ±3525.4 10052.3 ±5764.2
0.81
0.9 ± 0.01
0.87 ± 0.01
0.88
0.11 ± 0.01
0.92
0.10 ± 0.01
0.10 ± 0.01
0.99
6.7
0.77
7.3
6.5
0.63
0.64
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Leptin
1042.8 ± 231.3 18914.4 ± 3809.1 42544.8 ± 26978.8
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Insulin
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MI/DCI
0.89 0.66 0.28
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Metabolic-like-Syndrome (MLS), Myo-Inositol (MI), D-chiro-Inositol (DCI), GIP (Gastric Inhibitory Peptide), GLP-1 (Glucagon-like Peptide 1). All serum metabolic marker were measured
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in pg/ml. Data are shown as mean ± SEM. *p = 0.045; MLS MI/DCI-treated pregnant mice versus MLS placebo pregnant mice and Obese MI/DCI-treated pregnant mice versus Obese placebo
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pregnant mice.
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ACCEPTED MANUSCRIPT FIGURES LEGEND Figure 1 - Metabolic Like Syndrome (MLS) and Obesity Mice Models Scheme MLS mouse model: Maternal (Mat, eNOS-/+) heterozygous females (grey mouse), with mild hypertension, were placed on high fat diet (HFD) for 4 weeks, in order to develop a Metabolic like
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Syndrome (MLS) phenotype. Obesity mouse model: Wild type CB57 females (white mouse) were placed on high fat diet (HFD) for 4 weeks, in order to induce an obese phenotype. Both animal models were then bred with wild type normotensive males and starting from gestational day 1 were
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allocated either to Myoinositol/D-chiroinositol (MI/DCI) treatment or water as placebo, until term gestation when the experiments were performed, as displayed in the scheme.
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Figure 2 - Average systolic blood pressure (SBP, mmHg) in MLS and Obese pregnant mice SBP in Metabolic like Syndrome (MLS) pregnant mice, (Fig. 2a), and Obese mice (Fig. 2b) at term gestation (GD18) with (in red) and without (in blue) MI/DCI treatment. Data are shown as mean ± SEM. Significance is indicated on the figure (*p= 0.04); SBP was higher in MLS mice treated with
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MI/DCI versus non treated damns; no difference in SBP is seen in Obese mice. Figure 3 - Glucose Tolerance Test (GTT, mg/dl) in MLS and Obese pregnant mice Glucose levels in Metabolic like Syndrome (MLS) pregnant mice (3a) and obese pregnant mice
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(3b) at term gestation with and without MI/DCI treatment. Glucose levels were significantly lower in MI/ DCI treated mice with MLS compared to non-treated animals at all time periods except for
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the 15 min. In obese mice GTT was not different between MI/DCI-treated and non-treated animals. Significance is indicated on the figure *p<0.05. Figure 4 - Results Summary
Pregnant mice with MLS phenotype treated with Myoinositol/ D-chiroinositol during pregnancy improve blood pressure, glucose tolerance and leptin levels compared to non-treated mice. In obese mice instead, MI/DCI treatment prevented maternal increased weight gain associated with a high fat diet but did not affect maternal insulin resistance and blood pressure.
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S0002-9378(16)30254-X
DOI:
10.1016/j.ajog.2016.05.038
Reference:
YMOB 11127
To appear in:
American Journal of Obstetrics and Gynecology
Received Date: 5 March 2016 Revised Date:
18 May 2016
Accepted Date: 24 May 2016
Please cite this article as: Ferrari F, Facchinetti F, Ontiveros AE, Roberts RP, Saade MM, Blackwell SC, Sibai BM, Refuerzo JS, Longo M, The effect of combined Inositols supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity, American Journal of Obstetrics and Gynecology (2016), doi: 10.1016/j.ajog.2016.05.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The effect of combined Inositols supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity
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Francesca Ferrari MD1,2, Fabio Facchinetti MD2, Alejandra E. Ontiveros MD1, Robyn P. Roberts MD1, Mia M. Saade1, Sean C. Blackwell MD1, Baha M. Sibai MD1, Jerrie S. Refuerzo MD1, Monica Longo MD, PhD1 1
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Division of Maternal Fetal Medicine, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Texas Health Science Center at Houston, Houston, Texas; 2 Department of Obstetrics & Gynecology, University of Modena and Reggio Emilia, Modena, Italy Corresponding Author:
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Francesca Ferrari, M.D. Division of Maternal Fetal Medicine Department of Obstetrics & Gynecology, University of Modena and Reggio Emilia, Via del Pozzo 71 41124 Modena, Italy Office: +39 059 4225334 E-Mail: [email protected]
The authors report no conflict of interest
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Financial support: Partially supported by the Larry Gilstrap Center
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Presented as oral presentation # 69 at the 36th Annual Meeting of the Society of Maternal Fetal Medicine, held in Atlanta, February 1-6, 2016.
Word count: Manuscript: 3090/3000; abstract: 442/500
Figure 3 can be included in the print issue of the Journal.
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ACCEPTED MANUSCRIPT Condensation Inositols supplementation improves metabolic profile and blood pressure only in pregnancies
Short Title
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Inositols effect in pregnancies with metabolic syndrome and obesity
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complicated by metabolic syndrome.
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ACCEPTED MANUSCRIPT ABSTRACT Background: Myoinositol (MI) and D-chiro-inositol (DCI) improve insulin resistance in women with obesity, gestational diabetes and in post-menopausal women with metabolic syndrome (MS). We previously reported that offspring born to hypertensive dams lacking endothelial nitric oxide
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synthase (eNOS) and fed high fat diet (HFD) develop metabolic like syndrome (MLS) phenotype. Objective: To investigate the effect of a mixture of MI/DCI supplementation during pregnancy on the maternal metabolic profile in pregnancies complicated by MLS and obesity using a pregnant model.
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Study Design: Female heterozygous eNOS-/+ mice with moderate hypertension were placed on
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HFD for 4 weeks to induce a MLS phenotype. Similarly, wild type (WT) C57BL/6 mice were placed on high fat diet (HFD) for 4 weeks to induce a murine obesity model. Mice were then bred with WT males. On gestational day 1 (GD1), dams were randomly allocated to receive either a mixture of MI/DCI in water (7.2/0.18 mg/ml respectively) or water as control (placebo). At term,
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(GD18), maternal weights, systolic blood pressure (SBP) and glucose tolerance test (GTT) were obtained. Dams were then sacrificed; pups and placentas were weighed and maternal blood collected. Serum levels of metabolic biomarkers relevant to diabetes and obesity (ghrelin, GIP,
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GLP-1, glucagon, insulin, leptin, resistin) were measured by multiplex elisa assay (Bio-Rad). Analysis was done comparing MLS-MI/DCI-treated versus MLS-non-treated mice and obese-
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MI/DCI-treated
versus
obese-non-treated
mice.
Results: Mean SBP was lower in MLS pregnant mice treated with MI/DCI compared to placebo (p=0.04), whereas there was no difference in SBP between treated and placebo obese pregnant mice. Pregnant MLS mice treated with MI/DCI showed lower glucose values during the GTT and in area under the curve (AUC, MI/DCI: 17512.5 ± 3984.4 vs. placebo: 29687.14 ± 8258.7; p=0.003), but no differences were seen in the obese pregnant mice. Leptin serum levels were lower in the MLS-MI/DCI-treated mice compared with the placebo group (MI/DCI: 16985 ± 976.4 pg/dl vs. placebo: 24181.9 ± 3128.2 pg/dl, p= 0.045). No other differences were seen in any of the 3
ACCEPTED MANUSCRIPT remaining serum metabolic biomarkers studied in MLS and in obese pregnant mice. Maternal weight gain was not different in the pregnant MLS dams, while it was lower in the obese MI/DCItreated dams compared to the placebo group (MI/DCI: 10.9 ± 0.5 g vs 12.6 ± 0.6 g, p=0.04). Fetal and placental weights did not differ between MI/DCI-treated and non-treated pregnant dams with
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MLS and obesity.
Conclusion: Combined Inositols treatment during pregnancy improves blood pressure, glucose levels at GTT and leptin levels in pregnant dams with MLS phenotype, but not in obese pregnant
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dams. In addition, Inositols treatment was associated with lower gestational weight gain in the
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obese, but not in the MLS pregnant dams.
Key words: Inositol, Metabolic syndrome, Obesity, Insulin Resistance, Blood Pressure, eNOS
KO
mice
and
C57BL/6J
mice,
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Pregnancy,
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ACCEPTED MANUSCRIPT INTRODUCTION Metabolic syndrome (MS) and obesity are growing causes of morbidity and mortality worldwide. MS is a major risk factor for cardiovascular disease (CVD) and is defined by the National Institutes of Health (NIH) by having at least three of the following conditions:
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hypertension, elevated fasting plasma glucose, central obesity, elevated triglycerides or low highdensity lipoprotein cholesterol1,2. MS etio-pathogenesis and long-term consequences are still largely unknown and preventive strategies have not yet been identified3. The impact of MS and obesity
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during pregnancy is substantial: indeed, all metabolic changes that develop during pregnancy have well-known effects not only on maternal and fetal health during pregnancy but they also act as a
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catalyst for future health throughout later life4,5. Epidemiological and animal studies have shown that pregnancies complicated by MS and obesity are at risk for premature CVD, gestational diabetes and preeclampsia and predispose the offspring to an increased risk of cardiovascular and metabolic disease later in life6-8.
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Inositols are a family of simple carbohydrates naturally found in several foods. Inositols exist in 9 possible stereoisomers, 2 of which are predominantly found in eukaryotic cells: Myoinositol (MI) and D-chiro inositol (DCI) 9. The exact molecular mechanisms of action of MI and DCI have not
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yet been fully elucidated, however it is well established that MI and DCI have different roles as mediators of insulin, which lead to different functions within the cells. MI is the precursor of
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inositol triphosphate, a second messenger, responsible for the regulation of many hormones such as insulin, thyroid-stimulating hormone, and follicle-stimulating hormone. The activation of phospholipids-containing MI by insulin, activates glucose-transporters, such as GLUT-4, and increases the cell membrane permeability to glucose, which gets into the cell and is immediately available as substrate10. DCI is the conversion product of MI, by an epimerization reaction, which is unidirectional and insulin-dependent11. DCI, differently from MI, is able to determine the intracellular accumulation of glucose, (i.e., glycogen synthesis). So, both stereoisomers are considered to play a key role in the insulin pathway, acting synergistically as insulin-sensitizing 5
ACCEPTED MANUSCRIPT agents through the enhancement of glucose peripheral tissue uptake and glycogen synthesis12. In vivo animal13,14 and human studies15-19 demonstrated that Inositols supplementation during pregnancy improve glucose profile and reduces the adverse effects of hyperglycemia. Moreover, MI supplementation was proven to reduce insulin resistance in post-menopausal women with MS and
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in women with polycystic ovary syndrome, a metabolic and endocrine disorder associated with insulin resistance12. However, to the best of our knowledge, Inositols supplementation effect has never been investigated in pregnancies complicated by MS. Moreover, most of the studies in
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pregnant women focus on the effect of MI supplementation alone, whereas mounting evidence in PCOS studies suggest that the administration of combined MI/DCI, at the physiological plasma
cardiovascular risk parameters20.
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ratio (40:1) ensures better clinical results, such as the reduction of insulin resistance and
Thus our aim was to evaluate the effect of natural compounds, as Myoinositol and D-chiro inositol, used as a mixture, to treat pregnancies complicated by obesity and MS, which represent
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different degrees of a similar metabolic disorder. We hypothesize that MI/DCI treatment improve the abnormal maternal metabolic profile in a pregnant mouse model of either MLS or obesity. The metabolic profile improvement seen with MI/DCI treatment could also translate into positive long-
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term maternal and fetal health. To achieve our aim we used well-characterized mice models of obesity and MS that will allow identifying the best target population for a MI/DCI treatment in
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pregnancies with metabolic abnormalities. We previously reported that heterozygous mice lacking endothelial nitric oxide synthase (eNOS) gene and born to hypertensive mothers21, eNOS-/+, when fed high fat diet (HFD) for at least 4 weeks, develop metabolic like syndrome (MLS) phenotype such as: obesity, glucose intolerance, elevated systolic blood pressure, low HDL, high insulin, and low adiponectin levels compared to their counterpart fed control diet22. Furthermore, wild type C57BL/6J female mice fed obesogenic diet from weaning for 4 consecutive weeks show dietinduced obesity23.
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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Animals Female and male mice, homozygous for disruption of the eNOS gene (eNOS-KO-/-, strain B6.129P2, stock no. 002684) and their age matched wild-type controls (WT, strain C57BL/6J, stock
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no. 000664) were purchased from Jackson Laboratory (Bar Harbor, ME) at 6 weeks of age. The study was approved by the Animal Welfare Committee (AWC) of the University Of Texas Health Science Center, at Houston (UTHSC-H). The mice were housed separately in temperature and
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humidity-controlled quarters with constant 12:12-hours light-dark cycles in the animal care facility
Metabolic-like-syndrome mouse model
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at the (UTHSC-H).
eNOS-KO-/- females were bred with WT males to obtain eNOS+/- heterozygous females. Then a MLS mouse model was achieved by using eNOS-/+ heterozygous offspring manifesting a moderate hypertension phenotype21,24, and fed obesogenic diet for 4 consecutive weeks after
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weaning (Figure 1)22. At 7-8 weeks of age, non-pregnant eNOS-/+ females were bred with WT males. At gestational day 1 (GD1) of pregnancy, determined by the presence of vaginal plug after overnight exposure to male breeders, MLS dams were randomly allocated to receive either a
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mixture of MI/DCI dissolved in water (see next paragraph for concentrations) or plain water, as placebo (control group) (Figure 1).
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Obesity mouse model
To induce the obesity phenotype (Figure 1), WT-C57BL/6J female mice were fed obesogenic diet from weaning for 4 consecutive weeks23. Then, at 7-8 weeks of age, obese females were bred with WT males and starting from GD1, obese mice were randomly allocated to receive either a mixture of MI/DCI dissolved in water (see next paragraph for concentrations) or plain water, as placebo (control group). Obesogenic Diet
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of food left on the grid from the initial weight of food supplied. Inositols Supplementation during pregnancy
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sacrificed at term, on GD18. Daily food consumption was estimated by subtracting the total amount
The MI/DCI mixture corresponds to the physiological plasma ratio of MI and DCI equal to
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40:1, which has been proven to be the most effective20. On GD1, MLS and obese mice were randomly allocated to receive either a mixture of MI/DCI dissolved in water (7.2/0.18 mg/ml
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respectively, based on previous animals13,14 and human15-19 studies) or plain water as placebo. Considering that blood volume during pregnancy physiologically shows 20% increase compared to the non-pregnant status, we adjusted the MI/DCI doses by a 20% increase to the previously established doses used in a non-pregnant obese mouse model (MI: 6 mg/ml)13,14. Myo-inositol and
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D-chiro-inositol were purchased from Sigma Chemicals. The treatment was maintained until term gestation (GD18 of pregnancy), when dams were sacrificed. Pregnant mice were single-housed to be able to carefully evaluate water intake and consequently daily dose of MI/DCI. Daily water
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consumption was estimated by subtracting the total amount of water left in the bottle from the initial amount supplied. On average pregnant mice drink 5 ml/ day, so MI/DCI daily consumption
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was approximately 36/0.9 mg of respectively MI/DCI / day, per mouse. At term gestation, the metabolic profile of the MLS and obese pregnant dams were obtained including: systolic blood pressure (SBP), glucose tolerance test (GTT), maternal gestational weight gain and serum levels of metabolic biomarkers relevant to diabetes and obesity. Damns were then sacrificed, pups and placentas weights and numbers collected. Blood Pressure Measurement SBP measurements were always taken on GD18 in the morning at the same time using a calibrated eight-chamber tail-cuff system (CODA, Kent Scientific, Torrington, CT). Dams were 8
ACCEPTED MANUSCRIPT kept warm using a warming pad. Mice underwent an initial 10 cycles of acclimatization period; BP was then monitored and recorded over 10 new cycles. SBP averaged of the last 10 cycles was used for the final BP measurement and utilized for data analysis25,26. Glucose Tolerance Test (GTT)
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Pregnant dams on GD18 underwent GTT after being fasting for 6 hours. Mice received 1.0 g/kg of glucose I.P. and serial blood glucose levels, via a tail nick at 0, 15, 30, 60, and 120 min, were immediately determined with the Accu-Chek Aviva Blood Glucose Meter System (Roche
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Diagnostics) after glucose administration. Glucose levels and the area under the curve (AUC) were compared between MLS and obese pregnant mice treated with MI/DCI and identical non-treated
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ones27. Serum Metabolic Panel
Blood samples were obtained as soon as animals were sacrificed by heart puncture. Serum levels of metabolic biomarkers relevant to diabetes and obesity such as glucagon, insulin, leptin,
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ghrelin, Gastric Inhibitory Peptide (GIP), Glucagon-like Peptide 1 (GLP-1) and resistin were measured by multiplex elisa assay (Bio-Rad). Statistical Analysis
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Results are expressed as mean ± SEM. All the data were analyzed using unpaired t-test with SigmaPlot 12. The analysis was done comparing MLS MI/DCI-treated pregnant mice versus MLS
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non-treated (placebo) pregnant mice and obese MI/DCI-treated pregnant versus obese non-treated (placebo) pregnant mice. Numbers of pregnant mice per group were: MLS MI/DCI-treated, n= 9; MLS placebo-treated, n=8; Obese MI/DCI-treated, n= 8; Obese placebo-treated, n=6. (Figure 1).
RESULTS Food and water intake Daily food intake was not different between MLS-MI/DCI-treated and MLS-placebo mice (MI/DCI: 6.0 ± 0.9 vs placebo: 6.5 ± 0.7 g), nor between Obese MI/DCI-treated and Obese placebo9
ACCEPTED MANUSCRIPT treated (MI/DCI: 6.3 ± 0.6 vs placebo: 6.0 ± 0.7 g). Daily water intake was not different between MLS-MI/DCI-treated and MLS-placebo mice (MI/DCI: 5.1 ± 0.9 vs placebo: 4.9 ± 0.7 ml), nor between Obese MI/DCI-treated and Obese placebo-treated (MI/DCI: 4.8 ± 0.9 vs placebo: 5.0 ± 0.7 g).
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Systolic Blood Pressure Measurement
At term gestation, mean SBP was lower in MLS pregnant mice treated with MI/DCI compared to placebo group (MI/DCI: 138.52 ± 6.48 vs. placebo: 157.03 ± 7.79 mmHg, p=0.04)
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(Figure 2a). In the obese pregnant dams, there was no difference in SBP between MI/DCI-treated and placebo damns (MI/DCI: 143.18 ± 7.6 vs. placebo: 150.09 ± 7.9), (Figure 2b).
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Glucose tolerance
The glucose levels in the GTT were lower in the MLS pregnant mice treated with MI/DCI compared to placebo-treated mice at all time periods except for the 15 minutes value (Figure 3a). To confirm, the AUC for GTT was lower in MLS mice MI/DCI-treated group (MI/DCI: 17512.5 ±
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3984.4 vs. placebo: 29687.14 ± 8258.7; p= 0.003). In contrast, in the obese pregnant dams, glucose levels in the GTT and the AUC were not different between MI/DCI-treated and placebo-treated dams (MI/DCI: 23573.6 ± 4758.2vs. placebo: 25410 ± 5764.4) (Figure 3b).
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Serum Metabolic Profile
Serum leptin levels were lower in the MLS MI/DCI-treated mice compared with the placebo
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-treated group (MI/DCI: 16985 ± 976.4 pg/dl vs. placebo: 24181.9 ± 3128.2 pg/dl, p= 0.045). No other differences were seen in the serum levels of the remaining metabolic biomarkers studied in the MLS pregnant mice (Table 1). No difference was found between obese MI/DCI-treated and non-treated mice in any of the investigated metabolic biomarkers (Table 1). Weights Gestational weight gain was not different between MLS-MI/DCI-treated and placebo mice (MI/DCI: 11.53 ± 0.9 vs placebo: 11.6 ± 0.7 g), but was lower in the obese MI/DCI-treated dams compared to the placebo treated mice (MI/DCI: 10.9 ± 0.5 vs. placebo: 12.6 ± 0.6 g, p=0.04). 10
ACCEPTED MANUSCRIPT There were no differences between the pregnant MLS MI/DCI-treated and placebo groups respectively in fetal weights and placental weights, as reported in Table 1. Likewise, fetal and placental weights did not differ between MI/DCI-treated and placebo obese pregnant mice (Table
obese either treated or non-treated MI/DCI. A summary of all experiments results is reported in Figure 6. COMMENT
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1). Furthermore no differences were found regarding number of pups born from dams with MLS or
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We found that MI/DCI supplementation in pregnant mice with MLS phenotype improved blood pressure, glucose tolerance and leptin levels. However, in obese pregnant mice, MI/DCI
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treatment prevented maternal increased weight gain associated with a HFD but did not affect maternal glucose tolerance, blood pressure and the metabolic markers related to diabetes. Our findings therefore, support the rationale for the use of combined MI/DCI supplementation in pregnancies complicated by MS, but not by obesity alone in these animal
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models. Inositols are FDA approved supplements28 with a potential role in improving metabolic profile in MS. It is well established that MS traits, such as obesity and insulin resistance during pregnancy have negative impact on the pregnancy and on the metabolic and cardio-vascular future
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health of the mother and the offspring4-7,29. Therefore, the potential of a new ‘‘nonpharmacological’’ approach as a preventive and therapeutic agent is clinically relevant, since
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appropriate preventive strategies for MS in pregnancy have not yet been identified. Previous human studies focused either on gestational diabetes prevention/therapy15-19 or on MS associated with menopause30, 31 or PCOS32-34, whereas to the best of our knowledge this is the first study to investigate Inositols combination effect in a MLS and obese pregnant mouse model. Four human studies reported on the efficacy of myo-inositol supplementation during pregnancy to prevent gestational diabetes in patients at risk due to family history, obesity or PCOS15-18. In addition, one prospective randomized trial demonstrated a therapeutic role of MI in women with diagnosed gestational diabetes showing that MI in second trimester reduced homeostatic model 11
ACCEPTED MANUSCRIPT assessment resistance and adiponectin concentrations compared to controls19. Similarly, in nonpregnant women with PCOS32-34 and in postmenopausal women30,31, MI was shown to reduce insulin resistance and improve cardiovascular risk parameters (reduction in BP, total and LDLcholesterol, triglycerides and enhancement of the HDL-cholesterol). Our study reveals that Inositol
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supplementation improved SBP and glucose tolerance, in pregnancies complicated by MS but not in obese pregnancies.
The lack of effect in the obese pregnant mice could be due to the short period of HFD
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exposure leading only to weight gain but not to metabolic abnormalities severe enough to reveal effects of MI/DCI supplementation. In fact, according to the literature 4 weeks of HFD exposure
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should be sufficient to develop a diet-induced obesity phenotype23, however several animal studies reported a longer period of HFD exposure, up to 12 weeks, in order to manifest metabolic abnormalities and an Inositol effect13. Moreover, the lack of effect in obese pregnant mice could be due to different mechanistic pathways involved. We speculate that MI/DCI treatment improves
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glucose tolerance in MLS pregnant mice but not in the obese ones possibly involving NO pathway, which is known to be defective in our MLS model, but not in the obese one21,35. The exact molecular mechanisms of action of MI and DCI have not yet been fully elucidated. It has been
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shown that potential mechanisms by which the Inositols might act is by improving endothelial function in decreasing radical oxidative stress, enhancing endothelial nitric oxide synthase and NO
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activity36. This eNOS-/+ heterozygous mouse manifests MLS probably due to the lack of eNOS associated with an obesogenic diet during pregnancy. The metabolic abnormalities seen in this model of MLS might be a consequence of an endothelial dysfunction leading to hyperinsulinemia, insulin resistance and increased blood pressure. Thus, this mouse model of MLS seems very well suited to benefit from Inositols supplementation, which aims at restoring endothelial function. Indeed in our obese pregnant mouse model, obesogenic diet alone and for a short period of time does not seem to lead to such an endothelial dysfunction for which Inositol supplementation could show a target effect. 12
ACCEPTED MANUSCRIPT In our study leptin levels were improved by MI/DCI treatment in MLS pregnant mice, which is consistent with previous studies that showed decreased circulating leptin concentration after 16 weeks of MI treatment in women with PCOS12,37. The exact mechanism by which MI/DCI reduces leptin level remains unclear. Leptin controls energy balance and body weight by regulating
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neuronal activity in the hypothalamus38. It is possible that MI/DCI improved leptin resistance through either reductions in circulating leptin levels or up-regulations of receptors mediating leptin’s action to the brain, which needs further investigation. Furthermore in our study, maternal
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gestational weight gain was significantly reduced in the obese mice after MI/DCI treatment, in agreement with previous studies on gestational diabetes patients15,39. In both, obese and MLS
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models, pups and placentas weights did not differ between MI/DCI-treated and placebo animals. These findings are in contrast to others who found a reduction in hyperglycemia-related complications, such as fetal macrosomia after MI supplementation in women at risk for gestational diabetes for a family history of type 2 diabetes16 or elevating fasting glucose levels15; however
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again this could be due to a difference in the duration of HFD or to the fact that we evaluated the average weights of litters and placentas independently from their genotype (eNOS-/+ heterozygous, 25%, or wild type, 75%).
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Study strengths are: 1) to the best of our knowledge this is the first time that combined Inositols (MI/DCI) have been evaluated as a mixture rather than single compounds during
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pregnancy, 2) and furthermore being used in a MLS mouse model during pregnancy. Indeed, dietinduced obesity could be considered a less severe degree of the same metabolic disease that ultimately leads to MS. Therefore, the thorough investigation of Inositol in the presence of a spectrum of components of MS, rather than obesity alone, allows for the identification of the best target population for MI/DCI treatment. Moreover MI and DCI have shown different concentrations at the tissue level40,41 and different roles in the whole cell11,20 leading to the conclusion that both of them should be used in a synergistic action for the purpose of better treatment efficacy in metabolic diseases20. Unfer and colleagues performed dose–response studies identifying the physiological 13
ACCEPTED MANUSCRIPT plasma ratio of MI and DCI equal to 40:1, which was proven to be the most effective in PCOS overweight patients with metabolic abnormalities
20, 41-43
, but it has never been studied in
pregnancies with metabolic abnormalities. The main weakness of our study is that we did not investigate the effect of MI/DCI on lipid
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profile, which is an important feature of MS. We also did not evaluate the nitric oxide pathway as well as the inositol phosphoglycans intracellular mediators as possible mechanisms involved in the MI/DCI actions to elucidate our results. Moreover, in our study we did not plan to evaluate the
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combined MI/DCI effect in physiologic uncomplicated pregnancies, nor we planned to evaluate the effect of MI or DCI alone.
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Future studies need to be performed to investigate the effect of MI/DCI treatment on longterm maternal health and in the offspring born to MS and obese pregnancies in order to identify a possible role of Inositols on metabolic fetal programming. Undoubtedly, further studies are warranted to fully elucidate the molecular pathways triggered by MI and DCI, and to define the
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ideal timing (pre-conceptional, pregnancy and/or post-partum) dose, and combination of Inositol
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stereoisomers of such a supplementation.
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ACCEPTED MANUSCRIPT TABLE Table 1 - Serum Metabolic Profile (pg/ml) and Fetal & Placental Weights (g) MLS MODEL
OBESE MODEL
Placebo
p
MI/DCI
Placebo
p
GIP
530.1 ± 49.3
674.6 ± 88.0
0.18
412.5 ±111.3
542.1 ± 70.8
0.34
GLP-1
42.6 ± 4.2
45 ±5.9
0.74
34.9 ±6.7
36.6 ± 4.6
0.84
Glucagon
564.5 ± 83.0
461.1 ±75.8
0.37
400.8 ± 48.7
453.1 ± 64.5
0.53
1179.3 ± 136.9 18156.6 ± 1472.1* 15016.1 ± 3459.7
1146.7 ±176.7 22620.4 ± 3290.2 22676.6 ± 15492.8
0.88
999.7 ± 204.9
0.045
0.9 ± 0.01
0.88 ± 0.01
0.11 ± 0.02 7
Ghrelin Mean Fetal Weight Mean Placental Weight Number of pups
21998.2 ±3525.4 10052.3 ±5764.2
0.81
0.9 ± 0.01
0.87 ± 0.01
0.88
0.11 ± 0.01
0.92
0.10 ± 0.01
0.10 ± 0.01
0.99
6.7
0.77
7.3
6.5
0.63
0.64
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Leptin
1042.8 ± 231.3 18914.4 ± 3809.1 42544.8 ± 26978.8
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Insulin
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MI/DCI
0.89 0.66 0.28
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Metabolic-like-Syndrome (MLS), Myo-Inositol (MI), D-chiro-Inositol (DCI), GIP (Gastric Inhibitory Peptide), GLP-1 (Glucagon-like Peptide 1). All serum metabolic marker were measured
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in pg/ml. Data are shown as mean ± SEM. *p = 0.045; MLS MI/DCI-treated pregnant mice versus MLS placebo pregnant mice and Obese MI/DCI-treated pregnant mice versus Obese placebo
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pregnant mice.
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ACCEPTED MANUSCRIPT FIGURES LEGEND Figure 1 - Metabolic Like Syndrome (MLS) and Obesity Mice Models Scheme MLS mouse model: Maternal (Mat, eNOS-/+) heterozygous females (grey mouse), with mild hypertension, were placed on high fat diet (HFD) for 4 weeks, in order to develop a Metabolic like
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Syndrome (MLS) phenotype. Obesity mouse model: Wild type CB57 females (white mouse) were placed on high fat diet (HFD) for 4 weeks, in order to induce an obese phenotype. Both animal models were then bred with wild type normotensive males and starting from gestational day 1 were
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allocated either to Myoinositol/D-chiroinositol (MI/DCI) treatment or water as placebo, until term gestation when the experiments were performed, as displayed in the scheme.
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Figure 2 - Average systolic blood pressure (SBP, mmHg) in MLS and Obese pregnant mice SBP in Metabolic like Syndrome (MLS) pregnant mice, (Fig. 2a), and Obese mice (Fig. 2b) at term gestation (GD18) with (in red) and without (in blue) MI/DCI treatment. Data are shown as mean ± SEM. Significance is indicated on the figure (*p= 0.04); SBP was higher in MLS mice treated with
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MI/DCI versus non treated damns; no difference in SBP is seen in Obese mice. Figure 3 - Glucose Tolerance Test (GTT, mg/dl) in MLS and Obese pregnant mice Glucose levels in Metabolic like Syndrome (MLS) pregnant mice (3a) and obese pregnant mice
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(3b) at term gestation with and without MI/DCI treatment. Glucose levels were significantly lower in MI/ DCI treated mice with MLS compared to non-treated animals at all time periods except for
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the 15 min. In obese mice GTT was not different between MI/DCI-treated and non-treated animals. Significance is indicated on the figure *p<0.05. Figure 4 - Results Summary
Pregnant mice with MLS phenotype treated with Myoinositol/ D-chiroinositol during pregnancy improve blood pressure, glucose tolerance and leptin levels compared to non-treated mice. In obese mice instead, MI/DCI treatment prevented maternal increased weight gain associated with a high fat diet but did not affect maternal insulin resistance and blood pressure.
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