**Ketonuria Is Associated with Changes to the Abundance of** *Roseburia* **in the Gut Microbiota of Overweight and Obese Women at 16 Weeks Gestation: A Cross-Sectional Observational Study**


Received: 18 July 2019; Accepted: 6 August 2019; Published: 8 August 2019

**Abstract:** The gut microbiome in pregnancy has been associated with various maternal metabolic and hormonal markers involved in glucose metabolism. Maternal ketones are of particular interest due to the rise in popularity of low-carbohydrate diets. We assessed for differences in the composition of the gut microbiota in pregnant women with and without ketonuria at 16 weeks gestation. Fecal samples were obtained from 11 women with fasting ketonuria and 11 matched controls. The samples were analyzed to assess for differences in gut microbiota composition by 16S rRNA sequencing. Supervised hierarchical clustering analysis showed significantly different beta-diversity between women with and without ketonuria, but no difference in the alpha-diversity. Group comparisons and network analysis showed that ketonuria was associated with an increased abundance of the butyrate-producing genus *Roseburia.* The bacteria that contributed the most to the differences in the composition of the gut microbiota included *Roseburia*, *Methanobrevibacter*, *Uncl. RF39,* and *Dialister* in women with ketonuria and *Eggerthella*, *Phascolarctobacterium*, *Butyricimonas,* and *Uncl. Coriobacteriaceae* in women without ketonuria. This study found that the genus *Roseburia* is more abundant in the gut microbiota of pregnant women with ketonuria. *Roseburia* is a butyrate producing bacterium and may increase serum ketone levels.

**Keywords:** microbiome; pregnancy; obesity; ketonuria; *Roseburia*

#### **1. Introduction**

Pregnancy is a time of metabolic and hormonal change. Ketogenesis is accelerated in pregnancy, particularly in the third trimester. Ketones are produced from the breakdown of lipids when the mother's metabolic needs can no longer be met by glucose. The body produces three ketone bodies, beta-hydroxybutyrate, acetoacetate, and acetone. Beta-hydroxybutyrate and acetoacetate can be used as energy sources by the mother and the fetus and occur in a 1:1 ratio. Elevated maternal ketone levels have been associated with adverse fetal and childhood outcomes, particularly with regard to intelligence quotient (IQ), although results of these studies have been inconsistent [1–4].

The role of the gut microbiome in the metabolic changes of pregnancy has been an area of increasing interest. We have reported that the composition of the gut microbiome in pregnancy is associated with various metabolic and hormonal markers involved in glucose metabolism [5]. Whether

the gut microbiome actually causes these metabolic changes is yet to be fully determined. However, mice colonized with the microbiome from women in the third trimester of pregnancy develop insulin resistance and increased adiposity, supporting the idea that the gut microbiome itself drives some of the metabolic changes observed in pregnancy [6].

Butyrate is a short-chain fatty acid (SCFA) that is produced by certain bacteria within the microbiome. Known butyrate-producing species include *Faecalibacterium prausnitzii*, *Roseburia* spp., and *Eubacterium rectale* [7]. Butyrate is the main fuel for energy production in colonocytes and studies of metabolism of human and rat colonocytes have shown that butyrate is metabolized to ketone bodies and carbon dioxide [8,9]. In keeping with this finding, mice colonized with *Roseburia* have higher serum levels of the ketone, beta-hydroxybutyrate [10].

There are no studies that have reported an association between the composition of the gut microbiome in pregnancy and maternal ketone levels. We hypothesized that gut microbiome composition is associated with maternal ketone levels and that butyrate-producing bacteria are more abundant in women with higher ketone levels.

#### **2. Materials and Methods**

Women enrolled in the Study of Probiotics IN Gestational diabetes (SPRING study) who supplied a stool sample and fasting urine sample at baseline (<16 weeks gestation) were included in this study. Women taking probiotics in pregnancy prior to 16 weeks gestation and sample collection were excluded from enrolment. This study was approved by the human research ethics committee of the Royal Brisbane and Women's Hospital on the 16th January 2012 (HREC/11/QRBW/467) and The University of Queensland on the 25th January 2012 (201200080). All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki. All women enrolled in the study were either overweight or obese, as defined by pre-pregnancy body mass index (BMI) > 25 kg/m2. Women collected a stool sample and fasting urine sample within a 24-h time period. Women fasted for between 9.5 and 12 h prior to collection of urine. The stool sample was kept in storage at −80 ◦C prior to fecal DNA isolation. Urine samples were immediately tested for the presence of ketones. Urine dipstick tests were performed using SIEMENS Multistix 10 SG reagent strips and measured levels of the ketone, acetoacetate. A ketone level of trace, small, moderate, and large corresponded to an acetoacetate level of 0.5 mmol/L, 1.5 mmol/L, 4 mmol/L, and >= 8 mmol/L, respectively. All women provided dietary information from the start of pregnancy by food frequency questionnaire (Cancer Council Victoria's Dietary Questionnaire for Epidemiological Studies (Version (2)).

Women with any level of ketonuria present were matched with women with no ketonuria. The matching was performed on future GDM status, ethnicity, BMI and age. Fecal samples from each group were then analyzed to assess for differences in the gut microbiota between these two groups. Funding for this study was provided by the National Health and Medical Research Committee (NHMRC1028575) of Australia, the Royal Brisbane and Women's Hospital Foundation, the Mater Foundation and the Australian Diabetes in Pregnancy Society. HB is funded by an NHMRC Early Career Fellowship.

#### *2.1. Fecal DNA Extraction*

Stored stool samples were thawed at 4 ◦C before analysis. Aliquots of 250 mg of stool were removed from each sample for DNA extraction using the repeated bead beating and column (RBB + C) protocol. The aliquots were mixed with the RBB + C lysis buffer and sterile zirconia beads (0.1 and 0.5 mm diameter) and homogenized using a Tissue Lyser II (Qiagen, Chadstone VIC, Australia) for 3 min at 30 Hz. Samples underwent DNA purification using Qiagen AllPrep columns [5,11]. The quality and quantity of DNA was analyzed using the Nanodrop ND 1000 spectrophotometer (NanoDrop Technologies, Thermo Scientific, Scoresby, VIC, Australia) system.

#### *2.2. Fecal Bacterial Identification*

Bacteria within each sample were identified via 16S rRNA Sequencing. PCR amplification for the V6–V8 hypervariable regions of the bacterial 16S rRNA gene was performed using the 926F forward (50-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG AAA CTY AAA KGA ATT GRC GG-30) and 1392R reverse (50-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GAC GGG CGG TGW GTR C-30) primers. Positive (*E. coli* JM109 DNA) and negative (deionized sterile water) controls were included in each PCR run. Nextera XT V2 index kit Sets A and B were used to barcode PCR products and the AMPure XP bead system (Illumina, San Diego, CA, USA) was used for purification. Barcoded DNA underwent quantification, normalization and pooling to develop sequencing libraries which were then sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at the Australian Centre for Ecogenomics at The University of Queensland. The Quantitative insights Into Microbial Ecology (QIIME) v1.9.1 analysis tool was used to join and de-multiplex forward and reverse sequences. Using the Greengenes reference database, the open reference operational taxonomic unit (OTU) picking method was used for taxonomic assignments with a pairwise identity threshold of 97%. Taxonomic units that were present in the negative controls were removed from the analysis along with OTUs with a relative abundance of <0.0001. Prior to downstream analysis, the OTU table was rarefied to 3000 sequences/sample with no samples removed in this step.

#### *2.3. Statistical Analysis*

Median and interquartile ranges (IQR) were used to present the data as bacterial abundance was not normally distributed. Non-parametric statistical methods were used and a *p* value of <0.05 was considered statistically significant. Sample profiles were analyzed via the online Calypso software tool [12] and results are presented at the genus level of taxonomic assignment. Chao1 and Shannon indices were used for comparison of alpha diversity (within sample diversity) and the Bray-Curtis dissimilarity index was used to assess beta diversity (between sample diversity). Network analysis was performed to identify positive and negative correlations between bacterial taxa for both patients with and without ketonuria. Genera associated with samples from women with and without ketonuria were identified using Spearman's rho correlation coefficients with 1000-fold permutations. The strength of the color of the node reflects the significance of the association with either group and results are reported as significant if the false discovery rate (FDR) was <0.05. The size of the node reflects the abundance of the genus. Group comparisons were performed on genus level using the Wilcoxon Rank test, with no genera passing the statistical threshold for multiple testing, which is likely a reflection of the overall number of participants in this sub-study.

#### **3. Results**

Eleven women with ketonuria at 16-weeks gestation were matched with 11 women without ketonuria (Table 1). There were no differences in baseline BMI, maternal age, ethnicity, fasting blood glucose levels, future GDM status, or carbohydrate intake between the groups.


**Table 1.** Participant characteristics.


**Table 1.** *Cont.*

Data presented as median (IQR). BMI, body mass index; GDM, gestational diabetes mellitus; g, grams.

#### *Comparison of Gut Microbiome Composition*

There was no difference in the alpha diversity at genus level (Chao1, Shannon index) between the two groups (see Figure 1A,B). There was also no difference between the two groups in beta diversity at genus level with unsupervised hierarchical clustering principle coordinates analysis (PCoA)(Bray-Curtis dissimilarity index) (See Figure 2A), but with supervised redundancy analysis (RDA) there was a significant difference (*P* < 0.0001; RDA analysis, see Figure 2B). Analysis of the variance in the beta-diversity displayed no significant difference between the groups (see Figure 2C).

**Figure 1.** Alpha diversity of the gut microbiota at genus level between women with and without ketonuria. (**A**) Alpha diversity as assessed with the Chao1 index; (**B**) alpha diversity, as assessed with the Shannon index.

**Figure 2.** Beta diversity of the gut microbiota at genus level between women with and without ketonuria. (**A**) Unsupervised hierarchical clustering analysis by PCoA; (**B**) supervised clustering analysis by RDA and (**C**) variance analysis by Anosim analysis. Black circles/squares, ketonuria; white circles, no ketonuria.

Presence of urinary ketones was associated with an increased abundance of the butyrate-producing genus *Roseburia* in the network analysis. The brightness of the nodes is related to the level of significance of the association. *Roseburia* is the brightest of the nodes associated with ketonuria (see Figure 3); however, the butyrate-producer *Faecalibacterium* is also associated with ketonuria as is the acetate/propionate producer *Dialister*. In women who did not have ketonuria at 16 weeks, the abundance of *Adlercreutzia*, *Bifidobacterium*, *Dorea,* and *Collinsella* was higher (see Figure 3). Group comparisons revealed a statistically significantly higher abundance of *Roseburia* in women with ketonuria (see Figure 4A), and *Dialister* and *Faecalibacterium* abundance tended to be higher in the women with ketonuria (*P* = 0.066 and *P* = 0.076 respectively). In the women without ketonuria, *Adlercreutzia* abundance trended to be higher (*P* = 0.066), but that of *Bifidobacterium*, *Dorea,* and *Collinsella* was not significantly higher (*P* = 0.15; *P* = 0.14; and *P* = 0.17), respectively.

**Figure 3.** Network analysis of gut microbiota composition between women with and without ketonuria at genus level. Purple circles, ketonuria; green circles, no ketonuria; lines indicate positive correlations between the abundances of the bacteria. The size of the circle indicates the overall abundance of the genus and the brightness of the color indicates the degree to which the genus is associated with the group.

**Figure 4.** Bacterial genera that are specifically associated with the presence or absence of ketonuria. (**A**) Abundance of *Roseburia* between the groups. (**B**) sPLS-DA analysis of the contribution of bacteria genera to the differences between the gut microbiota between the groups. Black bars, ketonuria; white bars, no ketonuria. \*\* *p* < 0.01.

The bacteria that contribute the most to the differences in the composition of the gut microbiota include *Roseburia*, *Methanobrevibacter*, *Uncl. RF39,* and *Dialister* in the women with ketonuria and *Eggerthella*, *Phascolarctobacterium*, *Butyricimonas,* and *Uncl. Coriobacteriaceae* in the women without ketonuria (see Figure 4B). Predicted bacterial functions that were increased in women with ketonuria included riboflavin metabolism, lipid biosynthesis, carbon fixation pathways in prokaryotes, zeatin biosynthesis, adipocytokine signaling pathway, biotin metabolism, folate biosynthesis, prenyltransferases, and peroxisome (See Supplementary Figure S1). In women without ketonuria, bacterial functions ascorbate and aldarate metabolism; electron transfer carriers, phosphotransferase system PTS; aminobenzoate degradation, drug metabolism cytochrome P450; limonene and pinene degradation; chlorocyclohexane and chlorobenzene degradation; and styrene degradation were predicted to be more abundant. These results were all statistically significant (*p* < 0.05) on simple testing, but not after correction for multiple comparisons.

#### **4. Discussion**

This study shows that *Roseburia* is more abundant in the stool samples of women with fasting ketonuria at 16 weeks gestation. *Faecalibacterium* and *Dialister* species tended higher in the stool samples of women with ketonuria; however, this increased abundance did not reach statistical significance. Other studies have shown an association between the gut microbiota and hormonal and metabolic markers of glucose metabolism in pregnancy [5]. Our study expands on these findings and suggests that the microbiota may affect the capacity to produce ketone bodies during fasting.

*Roseburia* and *Faecalibacterium* are both genera of obligate gram-positive anaerobic bacteria. These bacteria ferment carbohydrates in the colon to produce SCFAs, particularly butyrate. The presence of both *Roseburia* and *Faecalibacterium* species in the gut has been associated with human metabolism outside of pregnancy. In two large metagenome-wide association studies, concentrations of butyrate-producing bacteria, *Roseburia intestinalis* and *Faecalibacterium prausnitzii* were lower in patients with type 2 diabetes mellitus when compared with those with normal carbohydrate metabolism [13,14]. The abundance of *F. prausnitzii* has also been found to be significantly lower in obese patients when compared with lean subjects [15]. In contrast, *Roseburia* has been found to be significantly higher in patients with higher BMI [16]. *Roseburia* abundance is linked to overall carbohydrate intake with individuals with lower carbohydrate intake having lower *Roseburia* abundance [17]. In our study, carbohydrate intake was not significantly different between women with ketonuria (137.2 (95.7–171.2) g/day) and those without ketonuria (155.9 (122.0–170.1) g/day; *P* = 0.46), but this may be due to the small sample size. *Roseburia* spp. contain genes involved in riboflavin metabolism and folate biosynthesis indicating how the increases in the abundance in the predicted function analysis may be related to increased *Roseburia* abundance. In addition, *Dialister* spp. and *Faecalibacterium* spp. also express genes for riboflavin metabolism, folate biosynthesis, and biotin metabolism, which could further contribute to the predicted functional differences between the groups.

Studies have found various links between butyrate-producing bacteria and serum ketone levels. Butyrate is the main energy source for colonocytes. In vitro studies of human and rat colonic cells show that the metabolism of butyrate involves oxidation and that part of the oxidized butyrate is converted to ketone bodies, acetoacetate, and beta-hydroxybutyrate [8,9]. A link between *Roseburia* and serum ketones has also been seen in mice. When mice were fed a diet with a high content of plant polysaccharides and colonized with a 'core' community of bacterial species including *Roseburia intestinalis*, they had higher levels of serum beta-hydroxybutyrate when compared with mice on the same diet, colonized with the same 'core' community without *Roseburia intestinalis* [10]. It is uncertain as to how these results can be extrapolated to humans, as the human gut microbiota is highly diverse and it is unclear if the presence of a single species would elicit a similarly large effect. Furthermore, dietary intake varies between individuals and across cultures.

A different mechanism for the link between butyrate and ketone levels has been hypothesized. Intraperitoneal administration of butyrate into mice led to increased serum beta-hydroxybutyrate levels and fibroblast growth factor 21 (FGF21) levels [18]. The hormone FGF21 stimulates fatty acid metabolism in the liver leading to increased ketogenesis. The authors postulated that the increase

in serum ketone levels was due to butyrate increasing FGF21 levels via induction of FGF21 gene expression in the liver. In humans, FGF21 is also expressed in the liver and is a downstream target of the transcription factor peroxisome proliferator-activated receptor alpha (PPARα). PPARα is a major regulator of lipid metabolism in the liver and is activated by both fasting and by consumption of ketogenic diets [19]. It is not known whether serum butyrate induces FGF21 gene expression in humans and what level of butyrate would be required to do so. In the current study, butyrate levels in the circulation were not measured and it is not clear if the increased abundance of *Roseburia* results in higher levels in the circulation.

*Adlercreutzia* abundance trended to be higher in those without ketonuria. *Adlercreutzia* is an obligate anaerobic coccobacillus and has been linked to various inflammatory conditions including inflammatory bowel disease, primary sclerosing cholangitis and multiple sclerosis [20–22]. Studies linking *Adlercreutzia* to human metabolism are lacking. One study did find a reduced abundance of *Adlercreutzia* in patients with HIV who developed diabetes compared with those who did not develop diabetes [23]. *Eggerthella*, *Phascolarctobacterium*, *Butyricimonas,* and *Uncl. Coriobacteriaceae* were the bacteria that contributed most to the differences in the composition of the gut microbiota in women without ketonuria. In a metagenome-wide association study of GDM, *Phascolarctobacterium* was more abundant in women with GDM whereas *Eggerthella* was more abundant in healthy controls [24]. In the same study, when assessing metagenomic linkage groups, *Methanobrevibacter smithii* was enriched in healthy controls. In our study, *Methanobrevibacter* was one of the bacteria that contributed most to the differences in the composition of the gut microbiota in women with ketonuria.

Maternal ketonuria has been associated with adverse fetal and childhood outcomes, particularly reduced childhood IQ [1,2]. However, these studies had disparate methodologies, with inconsistent results [3,4]. Ketone production occurs more rapidly in pregnancy, particularly in the third trimester [25]. It is felt to be due to increased maternal lipid metabolism and reduced glucose levels due to glucose being transported to the fetus for energy [26,27]. A maternal diet that is low in carbohydrate will also result in increased maternal ketone levels. The fetus utilizes ketones for energy and also as an important precursor for brain tissue [28]. It would therefore appear necessary for the fetus to be exposed to some level of ketones, however whether a high level of ketone exposure *per se* is harmful to the fetus is unclear. Our results suggest that ketone production in pregnancy may be more complex than a simple metabolic switch from glucose to lipid metabolism when maternal glucose supply is low. The gut microbiota may have a role to play via other metabolic pathways.

Limitations of the study included that urine ketone levels were only measured at one time point in the first trimester. Women in the control group may have had ketonuria at other times from when their samples were collected. A strength of the study is that women fasted for at least 9.5 h prior to the collection of the urine and ketogenesis is more pronounced with increased duration of the fasting state. Our sample size was limited which may have reduced the power of the study, particularly in relation to any differences in dietary intake. Lastly, circulating SCFA levels were not measured, which could indicate whether circulating butyrate levels are different in women with and without ketonuria.

#### **5. Conclusions**

*Roseburia* is more abundant in the microbiome of pregnant women with ketonuria. *Roseburia* is a butyrate-producing bacteria and studies have shown a link between both *Roseburia* and butyrate in the colon and elevated serum ketone levels. This study is further evidence of such a link and the first time that this link has been seen in pregnancy. Increased butyrate production by the gut microbiota may alter signaling in the host and thereby contribute to overall metabolic health in pregnancy. Larger studies of the microbiome in pregnant women with and without ketonuria at multiple time points in the pregnancy, with detailed dietary data needs to be done to further understand the relationship between *Roseburia* and maternal metabolism.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/11/8/1836/s1, Figure S1: Predicted differential bacterial function in women with and without ketonuria.

**Author Contributions:** Conceptualisation H.R., M.D.N., H.B., Methodology H.R., M.D.N., H.B., Formal Analysis M.D.N., L.G.-A., Data Curation H.R., Original Draft Preparation H.R., Writing H.R., M.D.N., H.B., L.C., L.G.-A., L.C., H.D.M., Supervision M.D.N., H.B., L.C., Funding Acquisition L.C., H.D.M.

**Funding:** This research was funded by the National Health and Medical Research Committee (NHMRC1028575) of Australia, the Royal Brisbane and Women's Hospital Foundation, the Mater Foundation and the Australian Diabetes in Pregnancy Society. HB is funded by an NHMRC Early Career Fellowship.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Leptin and Nutrition in Gestational Diabetes**

**Antonio Pérez-Pérez 1,\*, Teresa Vilariño-García 1, Pilar Guadix 2, José L. Dueñas <sup>2</sup> and Víctor Sánchez-Margalet 1,\***


Received: 18 May 2020; Accepted: 30 June 2020; Published: 2 July 2020

**Abstract:** Leptin is highly expressed in the placenta, mainly by trophoblastic cells, where it has an important autocrine trophic effect. Moreover, increased leptin levels are found in the most frequent pathology of pregnancy: gestational diabetes, where leptin may mediate the increased size of the placenta and the fetus, which becomes macrosomic. In fact, leptin mediates the increased protein synthesis, as observed in trophoblasts from gestational diabetic subjects. In addition, leptin seems to facilitate nutrients transport to the fetus in gestational diabetes by increasing the expression of the glycerol transporter aquaporin-9. The high plasma leptin levels found in gestational diabetes may be potentiated by leptin resistance at a central level, and obesity-associated inflammation plays a role in this leptin resistance. Therefore, the importance of anti-inflammatory nutrients to modify the pathology of pregnancy is clear. In fact, nutritional intervention is the first-line approach for the treatment of gestational diabetes mellitus. However, more nutritional intervention studies with nutraceuticals, such as polyphenols or polyunsaturated fatty acids, or nutritional supplementation with micronutrients or probiotics in pregnant women, are needed in order to achieve a high level of evidence. In this context, the Mediterranean diet has been recently found to reduce the risk of gestational diabetes in a multicenter randomized trial. This review will focus on the impact of maternal obesity on placental inflammation and nutrients transport, considering the mechanisms by which leptin may influence maternal and fetal health in this setting, as well as its role in pregnancy pathologies.

**Keywords:** nutrition; polyphenolic compounds; bioactive compounds; leptin resistance; obesity; inflammation; gestational diabetes mellitus; Mediterranean diet

#### **1. Introduction**

Gestational diabetes (GDM) is a hyperglycemic state that is recognized for the first time during pregnancy [1], and its pathophysiology is not fully clarified yet. GDM is one of the most common complications in pregnancy, affecting 3–8% of all pregnancies [2]. This prevalence has increased in recent decades (≥20% of pregnancies in some parts of the world), both in developed and developing countries, due to increased average age of pregnant women and increased obesity [3], one of the greatest public health challenges of the 21st century. Although the GDM phenotype is highly heterogeneous [4], half of its prevalence can be explained by overweight and obesity [5]. Indeed, obese women have an increased risk of GDM compared to women of normal weight [4]. Moreover, in women with GDM, pre-pregnancy obesity, excessive gestational weight gain and poor glycemic control are also linked with others pregnancy complications, such as gestational hypertension and preeclampsia [6]. It has even been reported that excessive gestational weight gain was the variable with the greatest effect on the

probability of a newborn with macrosomia (a conditions associated with an increased risk of perinatal mortality and neonatal morbidity). GDM also increases, in turn, the risk, in both mother and offspring, of developing type 2 diabetes, metabolic syndrome and obesity [7,8]. Therefore, obesity during pregnancy is an important risk factor for adverse health outcomes both in the mother and offspring, and imposes substantial economic burdens. Therefore, prevention of obesity would be directly related to a lower risk of GDM. Impaired glucose homeostasis in GDM is also related to higher production of reactive oxygen species (ROS), consequently depleting the anti-oxidative status. This is why, to restrain the spread of epidemic excess weight, women must receive a comprehensive intervention before, during, and after pregnancy. Lifestyle changes, including nutrition initiated during early pregnancy, have been unsuccessful overall in preventing GDM in at-risk obese women [9]. Now, there is a consensus regarding the need for effective interventions targeting obesity and lifestyle that reduce the metabolic burden earlier in life, well before motherhood, and it underlines that such interventions may benefit both the mother and the future offspring. Although energy restriction leading to weight loss is a successful dietary intervention for improving obesity-associated metabolic disorders, other dietary interventions, such as those leading to a reduction in adipose tissue inflammation regardless of weight loss, have not been explored in detail. In this sense, leptin, produced by adipocytes, is a key regulator of appetite and is present in elevated concentrations in obesity [10]. Therefore, new research into nutritional mechanisms that restore leptin metabolism and signals of energy homeostasis may inspire new treatment options for obesity-related disorders such as GDM. In this review, we wanted to address the current insights and emerging concepts on potentially valuable nutrients and food components to modulate leptin metabolism. Moreover, obesity is associated with a chronic low-grade inflammation in the adipose tissue [11], and several dietary food components, such as phenols, peptides, and vitamins, are able to decrease the grade of inflammation and improve leptin sensitivity by upor down-regulation of leptin-related genes. Others food components, such as saturated fatty acids should be avoided, since they may worsen chronic inflammation, subsequently increasing the risk for pathological complications. Finally, given the crucial role that the placenta plays in mediating pregnancy outcomes, it is important to consider the impact of micronutrient supplementation on the mechanisms associated with placental function, as well as maternal and fetal homeostasis.

#### **2. Leptin**

The hormone leptin, discovered in 1994 [12], critically regulates body weight and metabolism at central level in the brain [13], and disruption of leptin/leptin receptor (LEPR) signaling results in morbid obesity and severe metabolic disease [14,15]. In individuals of normal weight, the brain responds to increased plasma leptin levels by reducing food intake and increasing energy expenditure [16,17]. Leptin and leptin receptors are highly expressed in the preoptic area (POA), in the arcuate nucleus (ARC) of the hypothalamus as well as in other regions, such as the lateral hypothalamus, ventromedial hypothalamus and dorsomedial hypothalamus (DMH) [18]. There, it regulates energy homoeostasis and the neuroendocrine function, among other functions [19]. In these regions, leptin signaling is mediated by the JAK2/Stat3 pathway, in which several negative regulators of JAK2, including SOCS3 and PTP1B, have been reported to promote obesity [20,21], supporting the notion that JAK2 inhibitory molecules increase risk for leptin resistance and obesity. Therefore, hyperleptinemia and hypothalamic inflammation in diet-induced obesity may activate a common negative regulator of leptin signaling, SOCS3 or PTP1B, and contribute to central leptin resistance. In fact, up-regulation of SOCS3 in proopiomelanocortin (POMC) neurons leads to impairment of STAT3 signaling, with consequential leptin resistance and obesity, as well as glucose intolerance [22]. It has also been reported that mice with whole body or neuron-specific deletion of PTP1B are hypersensitive to leptin, and are resistant to diet-induced obesity [23]. Importantly, obesity is associated with impaired adipose sympathetic nerve transmissions [24,25], but the underlying mechanism is poorly understood. In this context, the leptin resistance at a central level may prevent negative feedback on the anti-inflammatory action of the sympathetic nervous system (SNS) [15,26]. That is why leptin is now considered one of the adipokines

responsible for the inflammatory state found in obesity that could predispose to GDM. Surprisingly, Sh2b1 (an SH2 and PH domain-containing adaptor protein) [27,28] has emerged as an endogenous sensitizer for leptin action on the sympathetic nervous system (SNS) and energy expenditure, perhaps by enhancing JAK2 activation [29]. In this way, the LepR Sh2b1 neuron mediates leptin stimulation of the SNS and supports the preservation of adipose SNS against degeneration [30].

Apart from the JAK-2/Stat-3 pathway, activation of the MC4R signaling pathway by proopiomelanocortin (POMC)-derived melanocyte stimulating hormone (MSH) peptides also represents a critical convergence point in the control of body weight. The leptin–melanocortin pathway (MC4R pathway) integrates parallel inputs from the orexigenic peptides ghrelin, neuropeptide Y (NPY) and agouti-related peptide (AgRP), and activation of the MC4R pathway dominantly counteracts these orexigens. Limited efficacy of lifestyle intervention in individuals with mutations in gene-encoding components of this pathway demonstrates its importance in the control of body weight homeostasis [31,32].

Therefore, leptin can act as metabolic switch connecting the nutritional status of the body to high energy-consuming processes. This is especially important in pregnancy, where leptin not only modulates satiety and energy homoeostasis in the mother [13,33], but it is also produced by the placenta, which responds to the environment attempting to maintain fetal viability. This placental production of leptin is one of the major sources of higher levels of maternal circulating leptin other than maternal gain of fat mass [34]. Thus, the effects of placental leptin on the mother may contribute to endocrine-mediated alterations in energy balance, such as the mobilization of maternal fat, which could further aggravate the insulin resistance associated with pregnancy and the onset of GDM [35,36]. In fact, obese pregnant women have significantly elevated plasma leptin concentrations compared with nonobese pregnant women throughout pregnancy [1]. Moreover, maternal obesity is also associated with changes in the placental function and structure, which likely impact fetal growth and development. For example, obesity has been associated with several changes related mainly with placental size, hypervascularization, higher branching capillaries of the villi (chorangiosis) [37,38] and increased glycogen deposits, among others. Increased macrophage infiltration is also evident in the placenta of obese women, suggesting an exaggeration of the inflammatory state which occurs in normal pregnancy [39]. However, it is unclear which histological changes are due to the pathophysiology and which are compensatory adaptations to this disease. Regardless, alterations in placental nutrient and hormone transporter capacity have been demonstrated in human and animal models of obesity, and are hypothesized as a mechanism leading to an accelerated fetal growth trajectory and macrosomia [1]. In this sense, we have demonstrated that the increased expression of aquaporin-9 (AQP9) (or others aquaglyceroporins) observed in placentas from obese women with GDM could be mediated by hyperleptinemia, suggesting an increase in the transport of glycerol to the fetus and thus contributing to the increased energy intake requirements in the macrosomic fetus in GDM [40]. Leptin has also been identified as a critical trophic factor that influences the development of the hypothalamic projections [40]. Alterations in the pattern of leptin secretion (premature peak, excess, or deficiency) during neonatal life could have significant adverse effects on hypothalamic development and metabolic phenotype [41] (Figure 1).

Finally, one of the peripheral functions of leptin is a regulatory role in the interplay between energy metabolism and the immune system, which is, in part, responsible for the inflammatory state associated to obesity [42]. Several inflammatory mediators produced by inflammatory cells also regulate leptin expression and promote the development of chronic inflammation [43]. In this regard, leptin effects include the inflammation and the modulation of innate and adaptive immunity [44,45]. Therefore, proinflammatory leptin actions might also have significant implications in the pathogenesis of GDM [29,46].

Taken all together, since hypothalamic inflammation results in central leptin resistance and hepatic insulin resistance [47], blocking the peripheral and central inflammation induced by a high fat diet could have the potential to treat obesity and GDM. Therefore, novel therapies incorporating effective natural agents (macro and micronutrients), particularly agents with the dual properties of preventing inflammation and controlling body weight by improving leptin sensitivity, might be an alternative intervention targeting obesity and GDM.

**Figure 1.** Effects of bioactive food compounds on the leptin resistance associated with obesity and gestational diabetes.

Leptin levels are increased in gestational diabetes with obesity (1). The high plasma leptin levels may be potentiated by leptin resistance at central level, in which SOCS3 and PTPB are induced by leptin and involving in a negative feed-back loop. The resulting effect is a decrease in the leptin-induced activation of the JAK2/STAT-3 signaling, leading to a reduction in the central effects of leptin (2). Leptin also impacts the placenta itself in an autocrine/paracrine fashion. The integration of numerous signaling by intracellular regulatory pathways such as MAPK, PI3K and JAK-STAT has been demonstrated to increase the size of the placenta and to affect placental nutrient transport and fetal growth (macrosomia) (3). Bioactive food compounds such as polyphenols might reduce circulating leptin levels, partly decreasing leptin expression in the placenta from women with GDM. The resulting effect is a decrease in the leptin resistance at a central level and optimal placental nutrients transport.

#### **3. Nutrients and Bioactive Food Components Useful for Counteracting Hyperleptinemia and Leptin Resistance in GDM**

Since the landmark Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study in 2010, an international consensus on diagnostic criteria has not been reached nearly a decade later [47]. However, there is now a consensus regarding the need for effective interventions targeting obesity. In this regard, since first-line medical (pharmaceutical) therapy has recently been called into question [48] and the socioemotional component of nutrition therapy for GDM has a dominant influence on adherence [49], other dietary strategies should be further investigated in detail. For example, since diet plays an important role in inflammation, and both obesity and GDM are considered a state of chronic inflammation, a healthy and active life style, which includes a diet rich in fruits, vegetables, non-sugared foods, non-ultra-processed foods associated with a higher prevention of inflammatory diseases should be incorporated into the diet [50]. However, a high consumption of red, processed meat, saturated or trans-fat, ultra-processed food based on refined ingredients or alcohol associated with pro-inflammatory processes should be avoided [35,36].

Since obese women and women with GDM show high circulating leptin levels and are hence considered leptin-resistant [51,52], we have identified nutritional strategies to counteract leptin resistance in both obesity and GDM. In this context, several micro- and macro-nutrients and bioactive food components might have the ability to increase leptin sensitivity and to reverse leptin resistance in obesity and GDM.

#### *3.1. Polyphenolic Compounds*

Polyphenolic compounds such as flavonoids represent an important bioactive component in plants (fruits, vegetables, legumes, tea, etc.) with some specific parts of these foods richer in flavonoids than others (for example the peel of certain fruits) [53]. The accumulation of flavonoids often occurs in plants subjected to abiotic stresses. This fact has made their determination an attractive field in food science and, in recent years, an increased number of studies have analyzed their potential benefits in human health.

As mentioned above, both the impact of nutritional status on the immune system, specially T cells [54,55] and the role of leptin as a mediator of inflammation [42], as well as a link between energy stores and the immune response, have been proposed [56,57]. In this sense, the (poly)phenols might modulate both the leptin effect and circulating leptin levels using different experimental approaches. For example, it has been reported that flavonoids have anti-inflammatory properties and thus have an important role in the control of several immune cells and immune mechanisms that are important in the inflammatory processes. More specifically, certain flavonoids (myricetin, quercetin, procyanidins) can inhibit multiple central kinases that are involved in multiple signaling pathways related to inflammation [58], such as phosphoinositol kinase, protein kinase C (PKC), phosphatidylinositol kinase and tyrosine kinase or cyclin-dependent kinase-4 [59]. Besides, flavonoids can modulate these protein kinases via inhibition of transcription factors (e.g., NF-κB and AP-1) [60]. Intriguingly, both insulin and leptin share several signaling pathways, such as mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathway, which may also activate several protein kinases involved in signal transduction during the inflammation process, via NF-κB. In this context, our group have demonstrated that the increase in placental leptin expression is mediated by NFκB signaling [61]. Therefore, in GDM, associated with insulin resistance, hyperinsulinemia and hyperleptinemia [62,63], flavonoids might downregulate the synergistic interaction between insulin and leptin signaling in the inflammatory processes. In addition, flavonoids might also decrease leptin expression in the placenta of women with GDM.

It has also been reported that high cAMP levels inhibit leptin expression by human chorionic gonadotropin (hCG), and these increased levels of cAMP have been associated with anti-inflammatory functions [64]. In this sense, flavonoids have also demonstrated the potential to block cAMP degradation and prolong cAMP signaling [65]. Finally, flavonoids may have an impact on cell activation, signaling transduction and cytokine production in several immune cells. For instance, flavonoids have been shown to inhibit maturation of dendritic cells (DCs) by suppressing the expression of CD83 and CD80, which would translate into an inhibitory effect in the secretion of pro-inflammatory cytokines [66,67]. These effects are contrary to leptin, which promotes the switch towards Th1 cell immune responses by increasing interferon-γ (IFN-γ) expression and facilitates Th17 responses. All these findings position flavonoids as modulators of immune response and, very specifically, as inhibitors of transcription factors, involved in the expression of different pro-inflammatory genes such as the leptin gene [60].

On the other hand, it is well known that the imbalance between the oxidative and anti-oxidative systems plays a crucial role in the pathogenesis of several human diseases such as obesity and diabetes, among others [68–70]. In this context, flavonoids are also potent antioxidants that are able to scavenge free radicals and decrease their formation. For example, grape juice by-products are a source of phenolic compounds with demonstrated antioxidant activities [71]. In these, the main phenolic compounds include flavones (luteolin) [71,72], flavonols (myricetin, fisetin, quercetin and kaempferol derivatives) [70,73], anthocyanins (cyanidin-3-glucoside) [74], flavan-3-ols (catechin and epicatechin monomers and proanthocyanidins) [75,76], stilbenes (resveratrol), and phenolic acids [74]. The most studied of these is resveratrol, which has been demonstrated to diminish circulating leptin levels and to reduce intake [77] by increasing phospho-STAT3 content in the hypothalamus, with no changes in SOCS3. This suggest that resveratrol might improve the leptin sensitivity in obesity [78,79]. Proanthocyanidins (PACs), known as condensed tannins, are found in a wide variety of fruits (e.g., berries), in addition to grapes, and other sources such as flowers, seeds of some plants, nuts or barks [80,81]. Plant and food-derived PACs are also attracting attention due to their ability to prevent chronic diseases [82]. For example, PACs from grape seeds and blackberry–blueberry fermented beverages have shown high anti-inflammatory and antioxidant activity in vitro [83] and have shown a broad therapeutic health effect against diabetes mellitus and obesity. In this regard, obesity and related complications such as GDM are linked with higher susceptibility to oxidative stress and the administration of grape seed extracts has shown an improvement in the oxidative status in obese people by inhibiting lipid peroxidation and avoiding ROS production [84]. Moreover, PACs can reduce inflammation by decreasing the oxidative stress or other indirect mechanisms [85]. In this sense, PACs from grape seed extract modulate IL-6, TNF-α and adiponectin gene expression in adipose tissue, thus, reducing the diet-induced low-grade inflammation [86]. PAC-rich extracts have also proved to be involved in obesity modulation (even at low doses) through the suppression of food intake and the increase in energy expenditure [87], possibly by mediating leptin levels. However, the mechanism underlying this effect of grape seed PACs has not been fully elucidated. Finally, grape seed extract improved the insulin resistance index as well as the plasma glucose and insulin levels in diet-induced obese animal models [88], although there are discrepancies in this regard.

Other bioactive food compounds have also been proven to be able to reduce circulating leptin levels in obesity. Myricetin, a bioflavonoid abundant in others fruits (e.g., berries), as well as tea and vegetables, has been shown to reduce hyperleptinemia and to favor insulin action via PI3-kinase pathway activation, and translocation of glucose transporter subtype 4 (GLUT4) to the cell membrane [89]. Accumulating evidence also suggests that propolis extracts (rich in flavonoids and cinnamic acid derivatives) have therapeutic effects on obesity by controlling adipogenesis, adipokine secretion, food intake, and energy expenditure. Particularly, considering the anorectic activity of leptin, propolis has potential to attenuate feeding and subsequently prevent obesity [90]. Moreover, various reports in animal and cellular models have demonstrated that propolis and its derived compounds improve insulin secretion and insulin sensitivity by modulating oxidative stress, the accumulation of advanced glycation end products (AGEs), and adipose tissue inflammation, all of which contribute to insulin resistance or defects in insulin secretion [91,92]. For example, several flavonoids in propolis, such as quercetin, chrysin, luteolin, amentoflavone, luteolin 7-O-glucoside and daidzein, have been found to have therapeutic effects in diabetic animal models by different mechanisms [93,94]. It has also been reported that propolis mitigates metabolic dysfunction through normalization of intestinal microflora [95]. Therefore, propolis intake might have beneficial effects for metabolic disorders such as GDM, attributable to flavonoids and natural phenols. However, propolis might have adverse effects on patients and, therefore, monitoring of biological effects should be carried out.

The polyphenols of olives and olive leaves also have numerous beneficial effects on human health, such as antioxidant capacity, hypoglycemic [96] and anti-inflammatory [97], as well as a coadjuvant role in the treatment of obesity [98]. In this sense, oleuropein, responsible for the bitter taste of olive leaves and drupes, and its derived form, the most abundant phenolic compounds present in olives and olive oils, are well known for their hypoglycemic property; possibly by the potential of affecting glucose-induced insulin release and/or increasing peripheral glucose uptake [99]. This hypoglycemic effect is also attributed, at least in part, to the antioxidant activity of oleuropein [100]. Moreover, it has been reported that oleuropein down-regulates leptin mRNA levels in epididymal adipose tissue and reduces serum leptin levels [101]. That is why the prophylactic use of oleuropein has been proposed in the reduction in complications resulting from oxidative stress in obesity and diabetes [99]. Other major phenolic components present—not only in olive extracts but in fruits (e.g., grapes), such as luteolin

and luteolin-4 -O-β-D-glucopyranoside—have been shown to inhibit the formation of AGEs and, thus, might delay the development of diabetic complications [96]. However, these effects have been tested in animals and it is necessary to perform studies in humans in order to confirm the benefits attributed to polyphenols from olives in GDM.

#### *3.2. Polyunsaturated Fatty Acids (PUFAs)*

The developing fetus requires substantial amounts of fatty acids to support rapid cellular growth and activity, and especially, metabolic derivatives of the essential fatty acids such as linolenic acid (Ω-3) and linoleic acid (Ω-6) and polyunsaturated fatty acids (PUFAs) are crucial [102]. The most biologically important PUFAs are docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) [102]. In this context, DHA appears to be crucial to the fetus and infant for early neural development (brain and visual system) [103]. However, modern dietary trends have led to an imbalance in the consumption of PUFAs, with deficiency in Ω-3 and increasing Ω-6 PUFA intake which far exceeds nutritional requirements, promoting the pathogenesis of many prevalent human diseases including GDM [104].

The placenta may play a key role in the regulation of fatty acid availability via the release of placental-derived leptin, a potent stimulator of lipolysis [105]. In fact, maternal circulating total fatty acid concentrations increase during pregnancy, enhancing placental access to fatty acids. However, as mentioned above, GDM is associated with oxidative stress and placental inflammation [106], and impaired placental fatty acid transport has been reported [107].

Given that PUFAs exhibit both anti-oxidative and anti-inflammatory activities, maternal dietary DHA and EPA supplementation has been proposed as a potential therapeutic intervention for this placenta-related disorder. For example, maternal dietary supplementation with Ω-3 PUFAs during pregnancy exerts beneficial effects such as reduced inflammation by either disrupting proinflammatory eicosanoid generation or promoting the generation of anti-inflammatory forms [108]. Moreover, it has been also reported that dietary supplementation with Ω-3 PUFAs modulates the activity of key transcription factors (peroxisome proliferator-activated receptors (PPARs) and/or nuclear factor κB (NF-κB)) and the G-protein-coupled receptor (GPR120) involved in inflammatory signaling [109–111]. Consequently, dietary supplementation with Ω-3 PUFAs might reduce risk of pregnancy complications [112] as well as the adipose tissue inflammation via GPR120-mediated suppression of macrophage proinflammatory cytokine secretion, including leptin [113]. Indeed, DHA and EPA have been shown to reduce circulating leptin levels activating the adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway [114]. Another beneficial effect of the Ω-3 PUFAs on GPR120 is the increase in the translocation of intracellular vesicles containing GLUT4, which enhances glucose uptake by adipocytes [111]. Oleic acid, a monounsaturated fatty acid (MUFA), also reduces hyperleptinemia via down-regulating PPARγ mRNA levels in abdominal visceral white adipose tissue in obese mice [115]. Through modulation of adipokine secretion, these fatty acids also favor insulin sensitivity [116].

Finally, excessive oxidative stress in utero-placental tissues plays a pivotal role in the development of GDM [106]. In this context, Ω-3 PUFAs could potentially limit oxidative damage by reducing ROS generation [117]. However, it would be important to ascertain the potential risks of excessive dietary PUFA intake given the susceptibility of PUFAs to lipid peroxidation, which may exacerbate cellular damage caused by an oxidative insult [118].

All together, these findings highlight the potential benefit of dietary supplementation with PUFAs to limit oxidative damage and inflammation associated with obesity and GDM, although further research in humans is required to clarify whether these fatty acids can prevent GDM and the potential risks associated if they are used as supplements.

#### *3.3. Terpenes*

There is evidences that cafestol and/or its metabolites (kahweol), natural diterpenes extracted from coffee beans, can prevent some chronic diseases such as metabolic disease [119–121]. In this context, it has been demonstrated that cafestol promotes insulin secretion and also increased glucose uptake in muscle cells, similarly to that of antidiabetic rosiglitazone. Moreover, kahweol can activate the AMPK pathway, a central modulator of the metabolism of glucose and lipid that stimulates glucose uptake and inhibits the lipid accumulation. A large number of studies have also shown that cafestol and kahweol have anti-inflammation and antioxidant activity, as well as an inhibitory effect on cell proliferation. More specifically, cafestol blocks the AP-1 pathway to reduce PGE2 production and blocks the PI3K/Akt pathway, promoting apoptosis in tumor cells [122]. All these effects could be beneficial in the placental overgrowth observed in GDM. However, despite the fact that caffeine has been shown to activate STAT-3 via decline of ER stress in the hypothalamus [123], it has been reported that kahweol down-regulated the STAT3 signaling pathway by inhibiting its constitutive phosphorylation and activation [124], which may aggravate the leptin resistance in obesity. Therefore, further research and clinical trials are needed to confirm whether the coffee diterpenes might be used to prevent or treat GDM in humans.

Evidence has been reported regarding the effect of tea preventing obesity and abnormal glucose and lipid metabolism [125]. In this sense, in addition to phenolic components, a major bioactive component of tea extract is teasaponin, a triterpene with significant anti-inflammatory properties. More specifically, teasaponin inhibits proinflammatory cytokines by suppressing NFκB signaling upstream of IKK/IκBα [125]. The anti-inflammatory effects of teasaponin have been associated with an improved glycemic status in animal models. Moreover, teasaponin decreases the expression of hypothalamic proinflammatory cytokines as well as the inflammatory signaling in the mediobasal hypothalamus [125]. This may contribute to improved leptin sensitivity and hypothalamic leptin signaling via p-STAT3. In fact, teasaponin significantly decreases the level of SOCS3, a negative regulator of central leptin signaling in the hypothalamus of high-fat diet-induced obese mice. Therefore, teasaponin has important effects in improving glucose tolerance, central leptin sensitivity, and hypothalamic leptin signaling [125], and it might be a potential candidate as therapeutic intervention for obesity and GDM.

#### *3.4. Probiotics*

Early reports of experimental and human studies regarding the role of gut microbiota promoting gut barrier functions and controlling inflammatory responses have attracted scientific interest. The gut microbiota is highly sensitive to the diet and may be involved in fat accumulation, favoring hydrolysis and absorption of indigestible polysaccharides and, thus, excessive storage of nutrients [126,127]. In fact, a distinctive gut microbiota composition in obesity has been reported in humans [127]. For example, a lower fiber intake has been reported to be associated with reduced gut microbiota diversity and richness, greater abundance of genus associated with type 2 diabetes mellitus [128,129], and genus with known pro-inflammatory capacity [34]. Physiological weight gain during pregnancy [130,131] also influences the gut microbiota composition in parallel with weight gain, favoring a higher number of *Bifidobacterium* spp and a lower proportion of *Staphylococcus* spp [132]. These shift in microbial composition are more pronounced in obese pregnancy and women with overweight gain during pregnancy [130–133]. Therefore, a reasonable strategy to fight GDM might be based on specific probiotics, which might counteract excessive absorption and storage of nutrients by modification of the gut microbiota composition. Probiotics in GDM might balance the effect of aberrant indigenous microbiota and normalize the increased intestinal permeability, as well as the secretion of proinflammatory mediators, including leptin. Therefore, as mentioned in a clinical trial [134], specific probiotics or probiotic foods might be used as dietary adjuncts to reduce the risk of diseases associated with aberrant gut microbiota composition, increased intestinal permeability or altered immunological or metabolic balance such as GDM. In fact, the impact of probiotics on GDM might be more pronounced in a high-risk population (e.g., obesity). Moreover, probiotics supplementation would not only affect the maternal metabolic state, but would also modulate fetal physiology and might have a long-term programming effect on child health [135–137]. However, current knowledge

about gut microbiota and diet response in pregnancy complicated by GDM is limited and future studies that integrate genetics and clinical variables should be taken into account.

#### *3.5. Others Bioactive Compounds*

Lycopene is a lipophilic carotenoid which is responsible for the red color in various vegetables and fruits, and is commonly found in tomatoes [138,139]. This carotenoid is known for its antioxidant and anti-inflammatory effects [140], and has been reported to improve diseases with chronic inflammatory backgrounds such as obesity. As mentioned above, hyperleptinemia is associated with pro-inflammatory responses and with the chronic subinflammatory state observed in obesity [141]. In this context, it has been suggested that lycopene supplementation may attenuate the inflammatory response in obesity, at least in part, by minimizing hyperleptinemia [142,143]. Other bioactive compounds of many cruciferous vegetables (e.g., watercress and broccoli) are isothiocyanates (ITCs), characterized by the presence of thiol-reactive chemicals that can modify critical cysteine residues on a variety of cellular proteins [144–146]. As mentioned above, accumulating evidence suggests that PTP1B could be involved in the pathways leading to leptin resistance as a major negative regulator of leptin and insulin signaling. In this sense, ITCs have been found to inactivate PTP1B [147,148], which has a reactive cysteine residue at the catalytic center [147]. Particularly, phenethyl isothiocyanate (PEITC), a relatively nontoxic constituent, in addition to inhibiting cellular PTP1B activity, has been demonstrated to enhance phosphorylation of LEPRb, JAK2, and STAT3 in the hypothalamus, resulting in the stimulation of leptin signaling and significantly reduced food intake [149].

#### *3.6. Micronutrients*

Micronutrients include numerous minerals and vitamins derived from the diet that are essential for cellular metabolism and optimal tissue function. It has been reported that an adequate supply of micronutrients during pregnancy may significantly reduce the risk of developing disorders of pregnancy, including GDM [150]. Throughout the course of pregnancy, there may be increased risk of micronutrient deficiency in response to the requirements of the growing fetus [151]. Therefore, although a healthy diet would be the ideal way to cover the micronutrient requirements, it is possible that the physiological challenge of pregnancy might require additional nutritional support of micronutrients [152]. The majority of supplements on the market contain a wide variety of vitamins (B group vitamins, vitamins C, D, E and folate) and minerals (iron, copper, zinc, iodine, selenium [153]. However, despite the benefits of micronutrients in supporting maternal, placental, and fetal homeostasis during pregnancy [152,154], insignificant evidence and varied results have been noted upon randomized trials of supplementation [155]. Such variability may be linked to variations in specific micronutrient supplement preparations and population contexts [155]. Therefore, the possibility that micronutrients supplementation could prevent complications of pregnancy warrants further investigation with larger trials in this field [151].

#### **4. Mediterranean Diet**

Despite the publication of numerous randomized trials on diet and lifestyle interventions in pregnancy [156], as mentioned above, no clear dietary recommendations have emerged to improve pregnancy outcomes for women with metabolic risk factors, particularly GDM. This can be attributed to the lack of robust evidence on effectiveness of the diet [157]. The traditional diet, "Mediterranean diet", has long been associated with preventive activity against chronic inflammation-associated diseases, which are supported by observational and epidemiological data. The Mediterranean-style diet includes components such as a high intake of nuts, extra virgin olive oil, fruit, vegetables, non-refined grains, legumes and micronutrients, as well as moderate to high consumption of fish and low consumption of processed meat, sugary drinks, fast food, and food rich in animal fat [158]. These key components of this diet might help to control the activity of obesity and GDM as well as other inflammatory pathologies. In fact, the Mediterranean diet has been recently found to reduce the risk of GDM in a

multicenter randomized trial [159]. It is possible that this beneficial effect on GDM could be due to the high intake of dietary polyphenols and micronutrients found in key components of the Mediterranean diet, such as extra virgin olive oil, grapes and nuts, which together activate insulin receptors, increase the uptake of glucose in the insulin-sensitive tissues, stimulate insulin secretion, and reduce insulin and leptin resistance. However, it should be stressed that the Mediterranean diet is a complex matrix of compounds and its biological activity cannot be attributable only to polyphenols and micronutrients. In fact, it is very likely that compounds of this diet could have not only additive, but also synergic or complementary activities to phenolic components. With the growing incidence of obesity and GDM, health effects of the Mediterranean diet and its multiple bioactive components will be of relevance not only in the treatment of obesity and GDM but, perhaps more importantly, also in their prevention. Therefore, a simple, individualized, Mediterranean-style diet in pregnancy could have the potential to reduce gestational weight gain and the risk of GDM. Figure 2 summarizes the 442 preventive effects of nutrients from the Mediterranean diet on leptin resistance.

**Figure 2.** Effects of the Mediterranean diet on the oxidative stress and inflammation associated with obesity and gestational diabetes. Obesity and GDM are linked with higher susceptibility to oxidative stress and inflammation. Adipocyte hypertrophy results in elevated circulation of free fatty acids (FFAs) and increased secretion of leptin, which drives T cells toward a pro-inflammatory phenotype (Th1). These in turn result in immune cell infiltration and the activation of pro-inflammatory signaling pathways. Bioactive food compounds in the Mediterranean diet such as polyphenols exert their anti-inflammatory activity by inhibiting ROS production and inhibiting multiple central kinases that are involved in multiple signaling pathways related to inflammation, such as NF-κB, MAPKs and PI3/Akt signaling pathways.

#### **5. Conclusions**

The high social and economic impact of the growing incidence of obesity and GDM have strongly motivated original investigations and the search for novel and rational preventive strategies. Data reported in the literature, and gathered in this review, show that there is scientific evidence supporting the anti-obesity effect of several bioactive compounds present in the Mediterranean diet. In parallel with the reduction in body fat accumulation, other features which are typical of pregnancy with obesity, such as GDM, increased leptin and insulin resistance, stress oxidative and low-grade inflammation, are also improved by these bioactive compounds. Some of the mechanisms of action underlying these effects have been revealed by preclinical studies. Particularly, the leptin sensitivity effect of polyphenolic compounds (one of the most interesting group of bioactive compounds), as well as the improvement of several comorbidities observed in GDM, has also been detailed. However, despite the publication of numerous studies on diet and lifestyle interventions in pregnancy, no clear dietary recommendations have emerged to improve pregnancy outcomes for women with metabolic risk factors. In this regard, observational evidence on the Mediterranean-style diet intervention in pregnancy and

potential reductions in weight gain and the risk of gestational diabetes should be taken into account. The key components of this diet include, at the very least, a high intake of polyphenolic and other bioactive compounds. It addresses some important benefits by using additional non-pharmacological therapy that is based on natural compounds and, moreover, it would be feasible to implement in pregnant women. It should be pointed out that future studies should investigate which bioactive compounds present in the Mediterranean diet are responsible for their effects, as well as the potential synergies between them. This strategy would be helpful to find new therapeutic interventions to prevent or treat GDM.

**Funding:** This research was funded by Instituto de Salud Carlos III, grant number PI19/01741, (Plan Nacional I+D+I 2017-2020) funded in part by FEDER Funds, to Víctor Sánchez-Margalet and Antonio Pérez-Pérez.

**Acknowledgments:** The present work was partly funded by grants from the Instituto de Salud Carlos III (ISCIII), PS12/00117, and PI15/01535, funded in part by FEDER Funds, to Víctor Sánchez-Margalet.

**Conflicts of Interest:** The author declares no conflict of interest.

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