Maternal metformin treatment decreases fetal inflammation in a rat model of obesity and metabolic syndrome

Research

www.AJOG .org

OBSTETRICS

Maternal metformin treatment decreases fetal inflammation in a rat model of obesity and metabolic syndrome Neeraj Desai, MD; Amanda Roman, MD; Burton Rochelson, MD; Madhu Gupta, MBBS, MS; Xiangying Xue, MD; Prodyot K. Chatterjee, PhD; Hima Tam Tam, MD; Christine N. Metz, PhD OBJECTIVE: Obesity and metabolic syndrome are associated with systemic inflammation and increased perinatal morbidity. Metformin improves metabolic and inflammatory biomarkers in nonpregnant adults. Using in vivo and in vitro models, we examined the effect of metformin on maternal and fetal inflammation. STUDY DESIGN: Female Wistar rats (6-7 weeks old) were fed a normal diet (NORM) or a high-fat/high-sugar diet (HCAL) for 5-6 weeks to induce obesity/metabolic syndrome. After mating with NORM-fed male rats, one-half of the HCAL-fed female rats received metformin (300 mg/kg, by mouth daily). All dams continued their respective diets until gestational day 19, at which time maternal and fetal outcomes were assessed. Maternal and fetal plasma and placentas were analyzed for metabolic and inflammatory markers. Cultured human placental JAR cells were pretreated with vehicle or metformin (10 mmol/L-2.5 mmol/L) before tumor necrosis factor a (TNF-a; 50 ng/mL), and supernatants were assayed for interleukin6 (IL-6).

RESULTS: HCAL rats gained more prepregnancy weight than NORM rats (P ¼ .03), had higher levels of plasma insulin and leptin, and exhibited dyslipidemia (P < .05). Fetuses that were exposed to the HCAL diet had elevated plasma IL-6, TNF-a, and chemokine (C-C motif) ligand 2 levels (P < .05) and enhanced placental TNF-a levels (P < .05). Maternal metformin did not impact maternal markers but significantly decreased diet-induced TNF-a and chemokine (C-C motif) ligand 2 in the fetal plasma. Finally, metformin dosedependently reduced TNF-aeinduced IL-6 and IkBa levels in cultured placental JAR cells. CONCLUSION: Diet induced-obesity/metabolic syndrome during pregnancy significantly enhanced fetal and placental cytokine production; maternal metformin reduced fetal cytokine levels. Similarly, metformin treatment of a placental cell line suppressed TNF-aeinduced IL-6 levels by NFkB inhibitor.

Key words: cytokine, inflammatory metabolic syndrome, metformin, obesity

Cite this article as: Desai N, Roman A, Rochelson B, et al. Maternal metformin treatment decreases fetal inflammation in a rat model of obesity and metabolic syndrome. Am J Obstet Gynecol 2013;209:136.e1-9.

M

etabolic syndrome is defined as a cluster of symptoms that are associated with the development of serious health conditions, such as type 2 diabetes mellitus and cardiovascular disease.1 The National Heart, Lung, and

Blood Institute in collaboration with the American Heart Association has identified components of metabolic syndrome: obesity (primarily abdominal), insulin resistance, atherogenic dyslipidemia (elevated triglycerides, reduced

From the Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, HofstraNorth ShoreeLIJ School of Medicine, Hempstead (Drs Desai, Roman, Rochelson, and Tam Tam), and the Centers for Immunology and Inflammation and Patient-Oriented Research, Feinstein Institute for Medical Research, North ShoreeLIJ Health System, Manhasset (Drs Gupta, Xue, Chatterjee, and Metz), NY. Received Jan. 17, 2013; revised March 30, 2013; accepted May 1, 2013. Support for this research was provided by the Oxenhorn family and the Feinstein Institute for Medical Research. The authors report no conflict of interest. Presented, in part, in oral format at the annual meeting of the Society for Maternal-Fetal Medicine, Dallas, TX, Feb. 6-11, 2012, and the 57th Annual Scientific Meeting of the Society for Gynecologic Investigation, Orlando, FL, March 25, 2010. Reprints: Christine N. Metz, PhD, Investigator and Professor, The Feinstein Institute for Medical Research, Hofstra-North Shore-LIJ School of Medicine, 350 Community Dr., Manhasset, NY 11021. [email protected]. 0002-9378/$36.00  ª 2013 Published by Mosby, Inc.  http://dx.doi.org/10.1016/j.ajog.2013.05.001

136.e1 American Journal of Obstetrics & Gynecology AUGUST 2013

high-density lipoprotein [HDL] cholesterol), hypertension, and a proinflammatory/prothrombotic state.2 The prevalence of metabolic syndrome in the United States is 16% among women who are <40 years old. Overweight and obese women are approximately 5 and 17 times, respectively, more likely than normal weight women to exhibit metabolic syndrome.3 Obesity during pregnancy has been implicated in increased susceptibility to gestational diabetes mellitus, preeclampsia, stillbirth, cesarean deliveries, and poor maternal wound healing.4,5 Consumption of high-calorie diets by humans and animals can lead to the development of obesity and metabolic dysfunction.6-11 In pregnancy, high-calorie diets promote numerous factors that are associated with increased maternal and fetal morbidity and death, which include obesity, inflammation, aberrant lipid metabolism, and insulin resistance.12-14

Obstetrics

www.AJOG.org In nonpregnant obese individuals, antidiabetic drugs may reduce the risks of metabolic syndrome and insulin resistance. Specifically, metformin is one drug that can decrease the adverse consequences of obesity-related insulin resistance by improving hepatic insulin sensitivity. It has been given to pregnant women outside of the United States since the 1970s15 and is now increasing in acceptance as an alternative treatment of infertility16,17 and diabetes mellitus in pregnancy in the United States.18 Finally, metformin has been shown to exert antiinflammatory activity.19,20 Thus, the advantage of metformin for treating insulin resistance is its potential to moderate metabolic dysfunction and inflammation. Because metformin modifies both insulin resistance and inflammation, we investigated whether metformin modulated maternal and fetal metabolic dysfunction in an acute diet-induced obesity model in pregnant rats.

M ATERIALS

AND

M ETHODS

Animals The Institutional Animal Care and Use Committee (IACUC) approved all animal studies before animal experimentation (IACUC #2010-031). Female Wistar rats (6-7 weeks old; Taconic Farms, Germantown, NY) were acclimatized initially with free access to standard rat chow and water for at least 72 hours. Rats were assigned randomly to 1 of 2 ad libitum diets: (1) control rat diet or normal-fed (NORM; n ¼ 10) or (2) high-calorie diet (HCAL; n ¼ 20) for 5 weeks. The HCAL diet consisted of 33% ground commercial rat diet, 33% full fat sweetened condensed milk, 7% sucrose, and 27% water, as previously described.13 After 5-6 weeks on their diets, lean and acutely obese female rats (maintained on their respective diets) were mated with NORM-fed male Wistar rats. On gestation day 1, one-half of the dams that were fed the HCAL diet received metformin (300 mg/kg, by mouth daily). All dams continued their respective diets (with/without metformin) throughout gestation. Rats were weighed immediately before diet, before mating, and then on gestational days 1, 5, 12, and 19. Maternal plasma (nonfasting)

was collected before mating and on gestational day 19 by retroorbital bleeding. On gestational day 19, dams were euthanized by CO2 inhalation followed by exsanguination by cardiac puncture with heparinized needles/syringes. Fetuses that were delivered by cesarean section were euthanized by decapitation, and blood (nonfasting) was collected into heparinized capillary tubes. Maternal weight gain, placental weight, and fetal outcomes (fetal weights and litter sizes) were assessed. Maternal and fetal blood (pooled from each dam) was centrifuged, and plasma was isolated. Maternal and fetal plasma and placentas (free of decidua) were flash frozen in liquid nitrogen and stored at e80 C.

Assessment of fetal rat plasma and placental cytokines After a brief centrifugation step, fetal plasma samples were analyzed for cytokines with the use of customized rat cytokine 5-plex kits (chemokine [C-X-C motif] ligand 1 [CXCL1]; chemokine [C-C motif] ligand 2 [CCL2]; interleukin6 [IL-6]; interleukin-1 beta [IL-1b]; and tumor necrosis factor alpha [TNF-a]; Meso Scale Discovery, Rockville, MD), according to the manufacturer’s directions. This particular group of cytokines/chemokines is representative of the proinflammatory state that is associated with obesity and metabolic syndrome and could be assessed with multiplex formats. After reading on the Sector Imager 2400 plate reader (Meso Scale Discovery), raw data were measured as electrochemiluminescence signals that were detected by photo-detectors and analyzed with the use of the Discovery Workbench software (version 3.0; Meso Scale Discovery). A 4-parameter logistic fit curve that was generated for each cytokine with these standards was used to determine the concentration of the individual cytokines in each sample (sensitivity, <3 pg/mL for each cytokine). Frozen rat placentas were homogenized with freshly prepared lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris [pH 7.5], 0.25 % Tx-100, 10 mmol/L NaF, and phosphatase/protease inhibitor cocktail [Thermo Scientific, Waltham, MA]). After centrifuging was completed, cell-

Research

free homogenates were analyzed for CXCL1, CCL2, IL-1b, IL-6, and TNF-a, with the Meso Scale Discovery technology described earlier. Placental cytokine data were normalized for protein concentrations, as determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA).

Assessment of maternal plasma cytokines, adipokines, lipids, and metabolic hormones Maternal nonfasting plasma samples were analyzed for CXCL1, IL-1b, IL-6, insulin, leptin, and TNF-a levels with the use of Luminex XMAP technology (Millipore, St. Louis, MO). In addition, maternal nonfasting plasma samples were assayed for HDL cholesterol, lowdensity lipoprotein cholesterol, and triglyceride levels by the Core Laboratories of the North Shore-LIJ Health System. In vitro studies with the use of a human placental cell line Human placental choriocarcinoma JAR cells (ATCC, Manassas VA) were cultured in 96-well plates (1  105 cells/well) in Roswell Park Memorial Institute medium that contained 10% fetal bovine serum and 1% penicillin-streptomycinglutamine. JAR cells were pretreated with either vehicle or metformin (0.012.5 mmol/L; Calbiochem, San Diego, CA) before stimulation with recombinant human TNF-a (0-50 ng/mL). After overnight stimulation, IL-6 levels in cell-free culture supernatants were measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Cell viability was determined after the JAR cells were treated with vehicle or metformin for 24 hours with the sulforhodamine B cytotoxicity assay method.21,22 To evaluate mechanisms of inhibition, JAR cells were pretreated with an NFkB inhibitor, Bay 11-7082 (20 mmol/L; Calbiochem) for 15 minutes before TNF-a stimulation (50 ng/mL). After overnight stimulation, IL-6 levels were assessed as described earlier. Western blotting was performed with JAR cell lysates that were prepared after treatment with TNF-a (in the presence and absence of metformin) using an antibody that is specific for IkBa (Cell Signaling

AUGUST 2013 American Journal of Obstetrics & Gynecology

136.e2

Research

Obstetrics

Technology, Danvers, MA), which is the inhibitor of NFkB nuclear localization and activation.

Statistical analyses Prestudy power analysis estimated the need for approximately 6 rats in each group for comparison. The negative control group (NORM) was designated to be those dams that received the normal diet. In addition, only lean dams that gained <1 SD above the overall mean body weight percent increase for this group were included in the analyses. This allowed us to maintain an optimal control group for analysis by excluding animals with a weight gain that approached the mean weight gain (within 1 SD) of the HCAL-fed group. Maternal and fetal outcome data (weight gain, fetal weight, placental weight, litter size, and maternal lipids) were analyzed, and the medians were compared with the use of nonparametric tests. Maternal and fetal cytokine, insulin, and leptin data were normalized by a log transformation; the geometric means were compared with the use of the Student t test, and significance was determined to be a probability value of < .05. Two separate comparisons were performed in parallel. First, we analyzed the difference between the NORM- vs HCAL-fed groups. Preconception data (weight gain, insulin, and leptin) were combined for all dams that received either NORM or HCAL diets because, at this point, there were no additional differences in treatment (ie, metformin was not started). Then, we examined the effect of treatment on exposure to the HCAL diet (HCAL þ metformin vs HCAL alone). For the JAR cell data, IL-6 levels among the various TNF-a and metformin concentrations vs vehicle alone were analyzed with analysis of variance. Pairwise comparisons of various concentrations of TNF-a or metformin to vehicle-treated cells (no metformin) were performed; significance was determined to be a probability value of < .05.

R ESULTS HCAL diet promotes weight gain and metabolic dysregulation HCAL-fed rats gained significantly more weight during the prepregnancy period

www.AJOG.org than NORM diet-fed rats (61.2%  15.7% vs 47%  10.4%; P ¼ .03; Figure 1). Although HCAL-fed dams gained more weight than NORM-fed dams during pregnancy, pregnancy weight gain (unadjusted and adjusted for litter size) for the 2 groups was not significantly different (Figure 1). Consistent with the observed weight increase that is associated with the HCAL diet, nonfasting plasma leptin levels were significantly higher in HCAL-fed animals after 5-6 weeks of diet (preconception; 2795  980 pg/mL vs 9687  4092 pg/mL; P < .0001; Table 1). In addition, at gestational day 19, HCALfed dams had significantly higher plasma triglycerides, lower HDL concentrations, and elevated cholesterol:HDL ratios (P < .05; Table 1). The HCAL diet also significantly increased plasma insulin levels when measured on gestational day 19 (P < .05; Table 1). Surprisingly, on gestational day 19, maternal plasma cytokine levels were not different in HCAL dams compared with control dams (Table 1). Rats that were fed the HCAL diet had elevated plasma leptin levels compared with NORM-fed rats when assessed before pregnancy; as predicted, pregnancy was associated with a

significant increase in circulating leptin levels in NORM-fed dams (Table 1). By contrast, leptin levels in the HCAL-fed rats did not significantly increase over gestation (Table 1). Maternal metformin administration to HCAL-fed dams during pregnancy did not significantly affect maternal weight gain, lipid profiles, plasma insulin, leptin levels, or circulating cytokine levels (Table 1).

HCAL diet promotes fetal and placental inflammation that is reduced by maternal metformin treatment Although fetal weights on gestational day 19 were similar among the HCAL and NORM groups, HCAL-fed dams had slightly smaller placental weights (P ¼ .07; Table 2). The average fetal weight of animals that were exposed to the HCAL þ metformin diet was 1.38  0.35 g], which was not significantly different from those that were exposed to HCAL alone (1.37  0.29 g). Placental weight was higher on average among HCAL þ metformin dams (0.39  0.06 g) when compared with HCAL-alone dams (0.35  0.08 g), but this was not significant (Table 2). Few fetal

FIGURE 1

High-fat/high-sugar diet promotes obesity

Weight over the prepregnancy period after 5-6 weeks of high-fat/high-sugar (HCAL) diet or control (NORM ) diets (Days e42 to 0; left panel ) and weight throughout pregnancy gestational day (GD ) 1-19 (right panel ); dams maintained their control (NORM ) diet (shaded squares) or HCAL diet (in the presence [open circles] or absence [exes] of metformin [MET ] treatment (300 mg/kg daily by mouth throughout gestation) or NORM diet. Data are shown as mean weight (g). The asterisk indicates a probability value of < .05 that compares the percentage of change in weight gain on day 0 between NORM vs HCAL-fed rats (Mann-Whitney test; n ¼ 8-19 per group). Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

136.e3 American Journal of Obstetrics & Gynecology AUGUST 2013

Obstetrics

www.AJOG.org

Research

TABLE 1

Maternal circulating blood analytes pre- and post-conception Diet Analyte

Normal (control)

High-fat/high-sugar

High-fat/high-sugar D metformin

P valuea

1101.2  647 (10)

1700.8  1212.6 (19)

—

A: .13

2795  980.41 (10)

9687.8  4092.4 (19)

—

A: .0001

34.6  13 (8)

22.5  11.5 (10)

Preconception (gestational day 0) Insulin, pg/mLb Leptin, pg/mL

b

Gestation day 19 High-density lipoprotein, mg/dLb Triglycerides, mg/dL

379  152 (8)

b

Insulin, pg/mLb Leptin, pg/mL

b

Interleukin-6, pg/mL

b

Tumor necrosis factor a, pg/mLb Chemokine (C-C motif) ligand 2, pg/mL

b

Interleukin-1b, pg/mL

b

597  208 (10)

2  0.7 (8)

Cholesterol/high-density lipoprotein ratiob

19.3  9.1 (9) 689  265 (9)

3.8  2.1 (10)

A: .03/B: .55 A: .007/B: .39

4.5  2.6 (9)

A: .03/B: .62

895.2  444.3 (6)

1715.4  838.8 (6)

1621.3  719.9 (6)

A: .026/B: .82

4070.2  1274.5 (6)

8825.2  1372.5 (6)

10690.1  4017.9 (6)

A: .0004/B: .37

ND

ND

ND

ND

ND

ND

119.9  29.7 (6)

96.3  28.5 (6)

89.9  44.2 (6)

A: .23/B: .58

74  26.7 (6)

106.4  51.6 (6)

199.3  179.4 (6)

A: .68/B: .10

ND, no data. a

A indicates normal vs high-fat/high-sugar diet; B indicates high-fat/high-sugar þ metformin diet; b Data are given as mean  SD. Values in parentheses indicates sample sizes.

Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

resorptions/fetal deaths were observed in all groups. When compared with the fetal pups from the NORM-diet group, the fetal pups from the HCAL-diet group had elevated plasma CCL2, IL-6, and TNF-a levels (P < .05; Figure 2). There was no significant effect of HCAL diet exposure on fetal plasma CXCL1 or IL-1b levels (Figure 2). Maternal metformin treatment significantly decreased HCAL-induced CCL2 and TNF-a levels in the fetal plasma

(Figure 2). Likewise, HCAL-induced fetal plasma IL-6 levels were reduced by maternal metformin administration; however, this effect was not significant (Figure 2). Placental CCL2, CXCL1, IL-1b, and IL-6 levels were not significantly different between the HCAL-diet and NORM-diet groups (Table 3). However, TNF-a levels were significantly elevated (P < .05) in the placentas that were obtained from the HCAL-diet group when compared with NORM-fed group (Table 3).

Although metformin treatment reduced HCAL-induced placental TNF-a levels, the effect was not quite significant (P ¼ .06; Table 3).

Metformin reduces TNF-aeinduced IL-6 production by JAR cells through the NFkB pathway Using the placental JAR cell line, we observed that TNF-a treatment (0.1-50 ng/mL) enhanced IL-6 production in a dose-dependent manner (Figure 3, A). Metformin treatment

TABLE 2

Fetal and placental characteristics at gestational day 19 Diet Characteristic

Normal (control) 1.45  0.21 (8)

Fetal weight, gb

12.4  1.9 (8)

Fetuses/litter, nb Placental weight, g

b

Dams with 1 fetal resorptions, n

High-fat/high-sugar 1.37  0.29 (10) 11.6  2.9 (10)

High-fat/high-sugar D metformin 1.38  0.35 (9) 11.85  2.7 (9)

P valuesa A: 0.19/B: 0.78 A: 0.65/B: 0.96

0.41  0.04 (8)

0.35  0.08 (10)

0.39  0.06 (9)

A: 0.07/B: 0.16

1 (8)

3 (10)

2 (9)

A: NS /B: NS

NS, not significant. a

A indicates normal vs high-fat/high-sugar diet; B indicates high-fat/high-sugar þ metformin diet; b Data are given as mean  SD. Values in parentheses represent sample sizes.

Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

AUGUST 2013 American Journal of Obstetrics & Gynecology

136.e4

Research

Obstetrics

www.AJOG.org

FIGURE 2

Diet-induced maternal obesity increases fetal inflammation: effect of maternal metformin

Fetal plasma cytokines were measured on gestational day 19 after exposure to maternal normal control diet (NORM ) or high-fat/high sugar diet (HCAL; with/without metformin [MET] treatment). A, Chemokine (C-C motif) ligand 2 (CCL2 ); B, chemokine (C-X-C motif) ligand 1 (CXCL1); C, interleukin (IL)-1b; D, IL-6; and E, tumor necrosis factor a (TNFa). Data are shown as cytokine levels (picograms/milliliters) from fetal plasma (pooled from each individual dam); the bars indicate the average cytokine levels; the asterisks indicate a probability value of < .05, in a comparison of NORM vs HCAL and HCAL þ metformin vs HCAL groups (Mann-Whitney test; n ¼ 4-7 per group). Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

(2.5 mmol/L for 18 hours) did not affect JAR cells viability (Figure 3, B). When JAR cells were pretreated with metformin at concentrations of 0.01-2.5 mmol/L,

TNF-aeinduced IL-6 production was significantly reduced in a dose-dependent manner. With 500 mmol/L metformin, TNF-aeinduced IL-6 production was

reduced by 41.9% (P < .05; Figure 3, C); with higher doses of metformin (1 and 2.5 mmol/L), IL-6 production was reduced by 53.6% and 76.4%,

TABLE 3

Placental analytes at gestational day 19 Dieta Analytes

Normal (control) High-fat/high-sugar High-fat/high-sugar D metformin P valueb

Chemokine (C-C motif) ligand 2, pg/mg

15.0  2.6 (4)

17.4  3.1 (5)

21.5  13.4 (7)

A: .26/B: .81

Chemokine (C-X-C motif) ligand 1, pg/mg 34.3  16.7 (4)

32.1  15.9(5)

33.5  14.0 (7)

A: 1.0/B: .73

Interleukin-1b, pg/mg

12.0  2.2 (4)

14.5  2.5 (5)

13.0  3.0 (7)

A: .19/B: 1.0

Interleukin-6, pg/mg

32.1  7.2 (4)

40.5  9.8(5)

31.8  7.2 (7)

A: .16/B: .16

6.2  1.3 (4)

9.1  1.3 (5)

6.8  1.5 (7)

Tumor necrosis factor a, pg/mg a

A: .0317/B: .0635

Data are given as mean  SD; b A indicates normal vs high-fat/high-sugar diet; B indicates high-fat/high-sugar þ metformin diet. Values in parentheses represent sample sizes.

Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

136.e5 American Journal of Obstetrics & Gynecology AUGUST 2013

Obstetrics

www.AJOG.org

FIGURE 3

Metformin reduces TNFaeinduced IL-6 production by placental cells

A, TNFa treatment of JAR cells induces IL-6 production in a dose-dependent manner. IL-6 data are shown as mean IL-6 (picograms/milliliters)  SD. One asterisk indicates a probability value of < .05; 2 asterisks indicate a probability value of < .001 (Mann-Whitney test). B, JAR cells were treated with metformin (0-2.5 mmol/L); after an overnight incubation, cell viability was assessed by the sulforhodamine B method. Data are shown as mean OD570nm (cell viability)  SD. C, JAR cells were treated with media plus vehicle or metformin (0-2.5 mmol/L) followed by TNFa (50 ng/mL). After an overnight stimulation, IL-6 levels in the culture supernatants were determined by enzyme-linked immunosorbent assay. Data are shown as mean IL-6 (picograms/milliliters)  SD. One asterisk indicates a probability value of < .05; 2 asterisks indicate a probability value of < .001 (MannWhitney test). D, JAR cells were treated with vehicle or TNFa  NFkB inhibitor, Bay 11-7082 (Bay11), and IL-6 production was determined by enzyme-linked immunosorbent assay. Data are shown as mean IL-6 (picograms/milliliters)  SD. The asterisk indicates a probability value of < .05. E, JAR cells were treated with vehicle or TNFa (with/without metformin; 2.5 mmol/L). After 30 minutes, IkBa levels compared with glyceraldehyde 3-phosphate dehydrogenase (GAPDH; control) levels in the cell lysates were determined by Western blotting methods. Density values indicate band densities of IkBa levels (corrected for loading with the use of GAPDH). IL-6, interleukin-6; TNFa, tumor necrosis factor a. Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

respectively (P < .001; Figure 3, C). Similar to the suppression observed with metformin, treatment of JAR cells with Bay 11, an NFkB inhibitor, significantly reduced TNF-aeinduced IL-6 production (Figure 3, D). To confirm that metformin reduced TNF-ae induced IL-6 production through the NFkB pathway, we examined the effect of metformin on the degradation of IkBa (the inhibitor of NFkB activation)

using JAR cells. We found that metformin (2.5 mmol/L) significantly reduced TNF-aeinduced IkBa degradation (Figure 3, E).

C OMMENT Consumption of the HCAL diet by rats significantly promoted greater prepregnancy weight gain when compared with normal-fed rats (Figure 1) along with significantly increased leptin levels

Research

(Table 1). Continuation of the HCAL diet into pregnancy induced maternal metabolic dysregulation, with elevated insulin levels and abnormal circulating lipids (Table 1). Coincident with this maternal metabolic dysregulation, fetuses that were exposed to the HCAL diet in utero exhibited higher circulating cytokines (CCL2, IL-6, and TNF-a; Figure 2) and elevated placental TNF-a levels (Table 3). Maternal metformin administration during pregnancy promoted normalization of HCAL-induced systemic fetal cytokine levels (Figure 2), and, although not significant, maternal metformin administration reduced HCAL-induced placental TNF-a levels (P ¼ .0635; Table 3). In vitro data revealed that metformin suppressed TNF-a-stimulated inflammation with the use of a human placental cell line, in a dose-dependent fashion (Figure 3, C) through the NFkB pathway (Figure 3, D and E). The animal model used herein was based on that described by Holemans et al13 who reported that a high-fat, high-sugar “cafeteria” diet that was given to prepregnant and pregnant rats resulted in obesity and insulin resistance. We were able to duplicate these findings and study a model that is characteristic of the metabolic state in obese human mothers. These women also have lowgrade inflammation, as evidenced by higher concentrations of C-reactive protein and IL-6.23 The concept that inflammatory mediators play a role in the development of type 2 diabetes mellitus began in the early 1990s when several groups showed that TNF-a induced insulin resistance.24,25 Later studies implicated additional inflammatory mediators that included IL-6 and CCL2.26,27 However, TNF-a remained the potential link between obesity and insulin resistance.28,29 Schmatz et al30 makes a convincing argument that the addition of pregnancy to the altered immunologic state that is associated with obesity/metabolic syndrome is a factor that contributes to the adverse outcomes that have been observed in this population. The placenta plays a central role in pregnancy, and placental trophoblasts are believed to mediate the maternal

AUGUST 2013 American Journal of Obstetrics & Gynecology

136.e6

Research

Obstetrics

immune response for both mother and fetus.31 Higher levels of TNF-a that are released into maternal circulation in late pregnancy were found to be more predictive of insulin resistance than placental hormones.32 Likewise, placentas of patients with gestational diabetes mellitus differentially express more genes that are involved in the regulation of inflammation than those from control subjects.33 In a primate obesity model of pregnancy, no increase in circulating maternal cytokine levels were observed after a high-fat diet, yet placentas exhibited increased inflammation.14,34 Consistent with these observations, we found that placental TNF-a levels were elevated significantly among the HCALfed dams (when compared with control dams) in the absence of detectable maternal inflammation (Table 3). Leptin shares both structural and functional similarities with IL-6 and has been implicated in regulating feeding/ satiety, adiposity, fetal growth, insulin resistance, and inflammation.35-38 Consumption of the HCAL diet before conception significantly increased circulating leptin levels when compared with NORM-fed rats (Table 1). The placenta is an important source of leptin in pregnancy and contributes to the circulating maternal concentration in late pregnancy.39 Although we observed a significant increase in maternal circulating leptin levels in the NORM-fed rats during pregnancy (Table 1), we did not observe an increase in maternal plasma leptin levels after the obesogenic (HCAL) diet (although it was elevated above NORM-fed rat leptin levels), nor did we observe a significant change in placental size (Table 2). We did not assess the placentas for histologic changes, nor did we evaluate placental leptin levels. Observations that revealed the antiinflammatory effects of metformin,40,41 which included possible protection against vascular endothelial injury,19,42 strongly supported examining the effect of metformin on obesity-associated inflammation and metabolic dysfunction during pregnancy. Maternal metformin treatment was initiated on gestational day 1 because implantation in the rat occurs on day 543; steady state metformin

www.AJOG.org

FIGURE 4

Potential regulation of NFkB signaling pathway by metformin

Proposed mechanism for the inhibition of placental inflammation by metformin in the setting of obesity/metabolic syndrome (characterized by enhanced NFkB activation). CCL2, chemokine (C-C motif) ligand 2; IL-6, interleukin-6; TNFa, tumor necrosis factor a. Desai. Maternal metformin decreases diet-induced fetal inflammation. Am J Obstet Gynecol 2013.

levels were expected to be achieved within 1-2 days.44 Metformin freely crosses the placenta45,46 and has been found in the umbilical artery and vein at twice the concentration of that in the maternal serum.45,46 These observations supporting higher concentrations of metformin in the fetal compartment may explain the disparate observations in cytokine responses between dams and fetal pups. An alternative explanation for these differences may be the timing of the measurement, the lack of assay sensitivity, or a limited or inadequate exposure to the HCAL diet. In previous studies, longer periods of preconceptional diet administration increased both maternal fat deposition and adipokine levels.47-49 Regardless, additional support for the antiinflammatory role of metformin was confirmed by our in vitro model that showed that metformin treatment reduced TNF-aeinduced IL-6 production by placental JAR cells. Obesity contributes to placental

136.e7 American Journal of Obstetrics & Gynecology AUGUST 2013

inflammation, in part through its regulation of the NFkB pathway.50 We found that Bay 11, an inhibitor of NFkB activation, reduced TNF-aeinduced IL-6 production similar to that observed after metformin treatment (Figure 3, C and D). In addition, metformin treatment of JAR cells suppressed TNF-ae induced degradation of IkBa, the natural inhibitor of NFkB nuclear localization and activation (Figure 3, E). Previous reports showed that metformin decreases proteasome activity,51,52 which mediates IkBa degradation53 (required for NFkB nuclear localization/activation). In addition, metformin inhibits TNF-aeinduced NFkB activation using human umbilical vein endothelial cells.42 Therefore, we propose a model for the regulation of the NFkB signaling pathway by metformin within the placenta (Figure 4). It should be noted that metformin may affect additional pathways that are related to inflammatory responses (eg, oxidative stress54 and AMPK/PTEN/mTOR55); however, these

Obstetrics

www.AJOG.org pathways were not analyzed for this study. The current recommendation is to discontinue metformin at the time of pregnancy diagnosis.56 There are several international groups that continue metformin throughout pregnancy15 because it has not been associated with malformations,57 lower birthweights, or deficiencies in growth, motor, or social development in the first year of life.58 Likewise, we observed no adverse effects of metformin on fetal weight gain, fetal death/resorptions, or litter size. We acknowledge several limitations of this study. The HCAL diet was provided for only 5-6 weeks before pregnancy and represents an acute diet-induced obesity model; a longer period might better reflect chronic obesity. In addition, the NORM and HCAL diets (with or without metformin) were provided ad libitum, and we did not assess exact intakes. It is important to note that our final analyses used “lean-NORM” rats as controls. Although we did observe significantly elevated circulating insulin levels in the HCAL-fed dams on gestational day 19 when compared with normal-fed rats, we did not evaluate maternal insulin sensitivity. However, enhanced insulin resistance was observed by Holemans et al,13 who used a similar model. Finally, as with any animal study, these findings are difficult to extrapolate to humans. Still, the model may provide us with a better understanding of the pathophysiologic changes that are observed in obesity and metabolic syndrome and the use of metformin for obesity-related metabolic syndrome during pregnancy. Researchers are just beginning to address how maternal diet, obesity, insulin resistance, and inflammation impact the developing fetus and the offspring. However, the underlying diseases that are responsible for adverse fetal outcomes and programming changes for the offspring are still not understood completely.59 Based on our observations that show the attenuation of HCAL-dieteinduced cytokine levels in the placenta and fetal circulation by maternal metformin administration future studies might focus on the

mechanism(s) that are involved in vivo and how modulation of placental-fetal inflammatory changes with the use of metformin or other treatments could reduce maternal and neonatal complications and adverse consequences for the offspring over a lifetime.

REFERENCES 1. Kahn R, Buse J, Ferrannini E, Stern M. The metabolic syndrome: time for a critical appraisal: joint statement from the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care 2005;28: 2289-304. 2. Grundy SM, Brewer HB Jr, Cleeman JI, Smith SC Jr, Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation 2004;109:433-8. 3. Ervin RB. Prevalence of metabolic syndrome among adults 20 years of age and over, by sex, age, race and ethnicity, and body mass index: United States, 2003-2006. Natl Health Stat Report 2009;13:1-7. 4. King JC. Maternal obesity, metabolism, and pregnancy outcomes. Annu Rev Nutr 2006;26: 271-91. 5. Sebire NJ, Jolly M, Harris JP, et al. Maternal obesity and pregnancy outcome: a study of 287, 213 pregnancies in London. Int J Obes Relat Metab Disord 2001;25:1175-82. 6. Buettner R, Parhofer KG, Woenckhaus M, et al. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Mol Endocrinol 2006;36:485-501. 7. Reuter TY. Diet-induced models for obesity and type 2 diabetes. Drug Discov Today Dis Models 2007;4:3-8. 8. Angela M, Gadja M, Pellizzon MA, Ricci MR, Ulman EA. Diet-induced metabolic syndrome in rodent models. Animal Lab News 2007. 9. Jones HN, Woollett LA, Barbour N, Prasad PD, Powell TL, Jansson T. High-fat diet before and during pregnancy causes marked up-regulation of placental nutrient transport and fetal overgrowth in C57/BL6 mice. FASEB J 2009;23:271-8. 10. Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, Heymsfield SB. The metabolic syndrome: prevalence and associated risk factor findings in the US population from the third national health and nutrition examination survey, 1988-1994. Arch Intern Med 2003;163: 427-36. 11. Dhingra R, Sullivan L, Jacques PF, et al. Soft drink consumption and risk of developing cardiometabolic risk factors and the metabolic syndrome in middle-aged adults in the community. Circulation 2007;116:480-8. 12. Zhang C, Schulze MB, Solomon CG, Hu FB. A prospective study of dietary patterns, meat

Research

intake and the risk of gestational diabetes mellitus. Diabetologia 2006;49:2604-13. 13. Holemans K, Caluwaerts S, Poston L, Van Assche FA. Diet-induced obesity in the rat: a model for gestational diabetes mellitus. Am J Obstet Gynecol 2004;190:858-65. 14. Frias AE, Morgan TK, Evans AE, et al. Maternal high-fat diet disturbs uteroplacental hemodynamics and increases the frequency of stillbirth in a nonhuman primate model of excess nutrition. Endocrinology 2011;152: 2456-64. 15. Feig DS, Moses RG. Metformin therapy during pregnancy: good for the goose and good for the gosling too? Diabetes Care 2011;34: 2329-30. 16. Thatcher SS, Jackson EM. Pregnancy outcome in infertile patients with polycystic ovary syndrome who were treated with metformin. Fertil Steril 2006;85:1002-9. 17. Glueck CJ, Phillips H, Cameron D, SieveSmith L, Wang P. Continuing metformin throughout pregnancy in women with polycystic ovary syndrome appears to safely reduce firsttrimester spontaneous abortion: a pilot study. Fertil Steril 2001;75:46-52. 18. Rowan JA, Hague WM, Gao W, Battin MR, Moore MP. Metformin versus insulin for the treatment of gestational diabetes. N Engl J Med 2008;358:2003-15. 19. Isoda K, Young JL, Zirlik A, et al. Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells. Arterioscler Thromb Vasc Biol 2006;26:611-7. 20. Dandona P, Aljada A, Ghanim H, et al. Increased plasma concentration of macrophage migration inhibitory factor (MIF) and MIF mRNA in mononuclear cells in the obese and the suppressive action of metformin. J Clin Endocrinol Metab 2004;89:5043-7. 21. Keepers YP, Pizao PE, Peters GJ, van Ark-Otte J, Winograd B, Pinedo HM. Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. Eur J Cancer 1991;27:897-900. 22. Skehan P, Storeng R, Scudiero D, et al. New colorimetric cytotoxicity assay for anticancerdrug screening. J Natl Cancer Inst 1990;82: 1107-12. 23. Ramsay JE, Ferrell WR, Crawford L, Wallace AM, Greer IA, Sattar N. Maternal obesity is associated with dysregulation of metabolic, vascular, and inflammatory pathways. J Clin Endocrinol Metab 2002;87:4231-7. 24. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesitylinked insulin resistance. Science 1993;259: 87-91. 25. Feinstein R, Kanety H, Papa MZ, Lunenfeld B, Karasik A. Tumor necrosis factoralpha suppresses insulin-induced tyrosine phosphorylation of insulin receptor and its substrates. J Biol Chem 1993;268:26055-8. 26. Fried SK, Bunkin DA, Greenberg AS. Omental and subcutaneous adipose tissues of obese subjects release interleukin-6: depot

AUGUST 2013 American Journal of Obstetrics & Gynecology

136.e8

Research

Obstetrics

difference and regulation by glucocorticoid. J Clin Endocrinol Metab 1998;83:847-50. 27. Sartipy P, Loskutoff DJ. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A 2003;100: 7265-70. 28. Bastard JP, Maachi M, Lagathu C, et al. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw 2006;17:4-12. 29. Nieto-Vazquez I, Fernandez-Veledo S, Kramer DK, Vila-Bedmar R, Garcia-Guerra L, Lorenzo M. Insulin resistance associated to obesity: the link TNF-alpha. Arch Physiol Biochem 2008;114:183-94. 30. Schmatz M, Madan J, Marino T, Davis J. Maternal obesity: the interplay between inflammation, mother and fetus. J Perinatol 2010;30: 441-6. 31. Creasy RK. Creasy and Resnik’s maternalfetal medicine: principles and practice. 6th ed. Philadelphia: Saunders/Elsevier; 2009. 32. Kirwan JP, Hauguel-De Mouzon S, Lepercq J, et al. TNF-alpha is a predictor of insulin resistance in human pregnancy. Diabetes 2002;51:2207-13. 33. Radaelli T, Varastehpour A, Catalano P, Hauguel-de Mouzon S. Gestational diabetes induces placental genes for chronic stress and inflammatory pathways. Diabetes 2003;52: 2951-8. 34. McCurdy CE, Bishop JM, Williams SM, et al. Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates. J Clin Invest 2009;119:323-35. 35. Fantuzzi G, Faggioni R. Leptin in the regulation of immunity, inflammation, and hematopoiesis. J Leuko Biol 2000;68:437-46. 36. Lappas M, Yee K, Permezel M, Rice GE. Release and regulation of leptin, resistin and adiponectin from human placenta, fetal membranes, and maternal adipose tissue and skeletal muscle from normal and gestational diabetes mellitus-complicated pregnancies. J Endocrinol 2005;186:457-65. 37. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature 1999;401: 73-6.

www.AJOG.org 38. Ogueh O, Sooranna S, Nicolaides KH, Johnson MR. The relationship between leptin concentration and bone metabolism in the human fetus. J Clin Endocrinol Metab 2000;85: 1997-9. 39. Ashworth CJ, Hoggard N, Thomas L, Mercer JG, Wallace JM, Lea RG. Placental leptin. Rev Reprod 2000;5:18-24. 40. Morin-Papunen L, Rautio K, Ruokonen A, Hedberg P, Puukka M, Tapanainen JS. Metformin reduces serum C-reactive protein levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2003;88:4649-54. 41. Diamanti-Kandarakis E, Paterakis T, Alexandraki K, et al. Indices of low-grade chronic inflammation in polycystic ovary syndrome and the beneficial effect of metformin. Hum Reprod 2006;21:1426-31. 42. Hattori Y, Suzuki K, Hattori S, Kasai K. Metformin inhibits cytokine-induced nuclear factor kappaB activation via AMP-activated protein kinase activation in vascular endothelial cells. Hypertension 2006;47:1183-8. 43. Suckow MA, Weisbroth SH, Franklin CL. The laboratory rat. 2nd ed. Boston: Elsevier; 2006. 44. Glucophage (metformin hydrochloride tablets) Label Information. Available at: http:// www.accessdata.fda.gov/drugsatfda_docs/label/ 2008/020357s031,021202s016lbl.pdf. Accessed: March 15, 2010. 45. Charles B, Norris R, Xiao X, Hague W. Population pharmacokinetics of metformin in late pregnancy. Ther Drug Monit 2006;28: 67-72. 46. Vanky E, Zahlsen K, Spigset O, Carlsen SM. Placental passage of metformin in women with polycystic ovary syndrome. Fertil Steril 2005;83: 1575-8. 47. Howie GJ, Sloboda DM, Kamal T, Vickers MH. Maternal nutritional history predicts obesity in adult offspring independent of postnatal diet. J Physiol 2009;587:905-15. 48. Samuelsson AM, Matthews PA, Argenton M, et al. Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming. Hypertension 2008;51:383-92. 49. Bytautiene E, Tamayo E, Kechichian T, et al. Prepregnancy obesity and sFlt1-induced

136.e9 American Journal of Obstetrics & Gynecology AUGUST 2013

preeclampsia in mice: developmental programming model of metabolic syndrome. Am J Obstet Gynecol 2011;204:398.e1-8. 50. Zhu MJ, Du M, Nathanielsz PW, Ford SP. Maternal obesity up-regulates inflammatory signaling pathways and enhances cytokine expression in the mid-gestation sheep placenta. Placenta 2010;31:387-91. 51. Wang S, Xu J, Song P, Viollet B, Zou MH. In vivo activation of AMP-activated protein kinase attenuates diabetes-enhanced degradation of GTP cyclohydrolase I. Diabetes 2009;58:1893-901. 52. Viana R, Aguado C, Esteban I, et al. Role of AMP-activated protein kinase in autophagy and proteasome function. Biochem Biophys Res Comm 2008;369:964-8. 53. Chen Z, Hagler J, Palombella VJ, et al. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev 1995;9:1586-97. 54. Srividhya S, Ravichandran MK, Anuradha CV. Metformin attenuates blood lipid peroxidation and potentiates antioxidant defense in high fructose-fed rats. J Biochem Mol Biol Biophys 2002;6:379-85. 55. Nerstedt A, Johansson A, Andersson CX, Cansby E, Smith U, Mahlapuu M. AMP-activated protein kinase inhibits IL-6-stimulated inflammatory response in human liver cells by suppressing phosphorylation of signal transducer and activator of transcription 3 (STAT3). Diabetologia 2010;53:2406-16. 56. Mathur R, Alexander CJ, Yano J, Trivax B, Azziz R. Use of metformin in polycystic ovary syndrome. Am J Obstet Gynecol 2008;199: 596-609. 57. Gilbert C, Valois M, Koren G. Pregnancy outcome after first-trimester exposure to metformin: a meta-analysis. Fertil Steril 2006;86: 658-63. 58. Glueck CJ, Wang P, Kobayashi S, Phillips H, Sieve-Smith L. Metformin therapy throughout pregnancy reduces the development of gestational diabetes in women with polycystic ovary syndrome. Fertil Steril 2002;77:520-5. 59. Catalano PM, Hauguel-De Mouzon S. Is it time to revisit the Pedersen hypothesis in the face of the obesity epidemic? Am J Obstet Gynecol 2011;204:479-87.