The diagnosis of glucose intolerance during pregnancy or Gestational Diabetes Mellitus (GDM), affects up to 14% of pregnancies globally (1, 2). Although pregnancy is a natural state of insulin resistance, women with GDM face a worsened level of insulin resistance coupled with glucose intolerance (3–8). This disease can then further be exacerbated by a lack of beta cell expansion in the pancreas, leading to reduced insulin secretion (9–11). This failure to adapt to the metabolic demands of pregnancy is associated with an increased risk of type 2 diabetes, GDM in subsequent pregnancies, hypertension, cardiovascular disease, increased body mass index, and cesarean delivery (12). Moreover, GDM induces adverse outcomes in the fetus, including increased risk for preterm birth, macrosomia, and type 2 diabetes (12).

Recently, studies have found that women with GDM have a heightened risk for developing metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD), during pregnancy and post-partum (13–15). Women with MASLD were three times more likely to develop GDM (16, 17). Furthermore, compared to their healthy, pregnant counterparts, women with GDM display alterations in hepatic serum metabolites such as lysophosphatidylcholine, glycerophospholipids, monoacylglycerol, serine, proline, leucine, and isoleucine (18, 19). These metabolites are involved in lipid and amino acid metabolism (19, 20), and such alterations of these metabolites may help explain the metabolic impact of GDM (21–23), but a mechanistic link between GDM and MASLD has yet to be determined.

MASLD, characterized by a greater than 5% of hepatocytes containing lipid, is the most common form of chronic liver disease (24, 25). MASLD is linked to several metabolic disorders including type 2 diabetes, cardiovascular disease, and obesity (24, 26). Although the specific cause of MASLD is unknown, there are a number of processes that influence this disease, such as alterations in lipid metabolism and mitochondrial function (27–30). The hepatic mitochondrion is responsible for the breakdown of different substrates via glycolysis and fatty acid oxidation which then feed into the TCA cycle and activates ATP synthesis via the electron transport chain, a process known as respiration (31–34). Further, functional mitochondria are in a constant cycle of turnover via biogenesis, autophagy, and mitophagy. During MASLD, mitochondrial respiration, metabolism, activity, and turnover are impaired (35–37). This creates a pool of poorly functioning mitochondria that exhibits inflammation, increases reactive oxygen species generation, and decreases in antioxidant capacity (35). The health of a hepatic mitochondria depends on the interaction between these processes and when impaired significantly contributes to the development and progression of MASLD (32, 37).

As stated previously, there is limited investigation into the mechanistic links between GDM and MASLD. Previous murine models of GDM have shown alterations in maternal hepatic electron transport chain and UCP2, a marker of ROS (38). Offspring of mice with GDM display impaired mitochondrial function and hepatic lipid accumulation (38, 39). The purpose of this study was to evaluate the impact of GDM on hepatic lipid metabolism, mitochondrial function, and inflammation in the maternal liver. It is imperative to understand how GDM and MASLD are connected. By evaluating the liver at varying time points during gestation, we can further understand the natural changes that occur in the liver in a healthy vs. GDM pregnancy. We hypothesized that GDM will significantly increase maternal hepatocellular injury.

Gestational diabetes mellitus is associated with an increased risk of MASLD, but there is a paucity of data mechanistically linking these two conditions. Here we provide novel evidence in an established high fat, high sucrose-fed mouse model of GDM (40–42) that changes in maternal liver lipid metabolism may play an important role in GDM pathology. GDM mice developed hepatic steatosis, the accumulation of lipid in the liver, with increases in hepatocellular inflammation and ballooning degeneration, consistent with MASH (35). The GDM mice also exhibited global downregulation in markers of lipid metabolism, and mitochondrial function, biogenesis, mitophagy, and autophagy with increasing gestation time.

Heightened insulin resistance is the distinguishing feature of GDM that is commonly accompanied by inadequate pancreatic beta cell expansion (51). Obesity is a risk factor for the development of GDM (and MASLD), but it has been found that up to one third of women with GDM are considered lean (BMI < 18.5 kg/m²) (52). To better characterize the condition, GDM is now being classified into subtypes dependent on insulin metabolism. Approximately 50% of women with GDM will have systemic insulin resistance, 35% will display insufficient insulin secretion, and 15% will exhibit variations of altered insulin metabolism (53–55). These data highlight the importance of understanding and investigating the varying pathophysiology of GDM. The mouse model used in the current study exhibited systemic insulin resistance and impaired insulin secretion with attenuated beta cell expansion, making it a preferred translational model to investigate GDM (40–42).

During healthy gestation, several metabolic adaptations occur such as elevated glucose utilization, decreases in fatty acid oxidation in the liver, and subsequent systemic insulin resistance (56, 57). As pregnancy progresses into its later stages, adipose tissue undergoes increased lipolysis to ensure sufficient circulating free fatty acids (58). GDM appears to extend these adaptations beyond what is metabolically necessary, aligning with MASLD development. Here, we found in our model of GDM that maternal liver triglycerides were significantly elevated at day 17.5 compared to control diet fed mice, while serum triglycerides were markedly decreased ( Figure 1 ). Similarly, NAS, which includes steatosis, was markedly increased at day 0, 6.5, and 17.5 in GDM compared to CD with elevations occurring as gestation processed. Our data showed that markers of lipid breakdown (Ampk, pAMPK/AMPK) and export (Mttp) decreased as gestation progresses, in both groups. These data suggest that increased hepatic lipid uptake occurs in GDM, concomitant with significant decreases in lipid breakdown and export. This outcome was accompanied with decreased levels of FAS and ACC, markers of hepatic de novo lipogenesis (DNL), which is the formation of fats in the liver from other sources such as sugar (59). Decreased markers of hepatic DNL correspond with other short-term high fat diet feedings studies that show similar reductions (60–64). Overall, these data suggest GDM significantly decreases in lipid breakdown and export, eliciting the formation of lipid accumulation in the liver.

The mitochondrion is the main residence for lipid import and break down. The dysfunction of this organelle is an important feature of MASLD development (27, 28). With our findings of altered lipid storage and export, we hypothesized that our model of GDM would display significant alterations in hepatic mitochondrial function, aligning with MASLD progression. Citrate synthase and β-HAD activities, enzymes that catalyze the TCA cycle and fatty acid β-oxidation within the mitochondria, respectively, were both significantly decreased in GDM mice compared to control mice by day 17.5 (65). Interestingly, early in GDM pregnancy, markers of mitochondrial activity (citrate synthase, β-HAD), biogenesis (Tfam), autophagy (Atg5), and mitophagy (BNIP3, Bnip3, Pink1) were either significantly elevated or trending. Similar outcomes were seen with inflammation (Il1-β, Tnf-α, Tgf-β) and antioxidant defense (Sod2, Gpx1). This information suggests that early pregnancy is a critical window where transcriptional hepatic adaptations to GDM first emerge. In line with these findings, work from our group have previously shown that mitochondrial dysfunction actually precedes MASLD development (28), and findings from others suggest that alterations in plasma lipidomics in early pregnacy predict GDM development (21–23). It is known that pregnancy requires great metabolic flexibility, and substrate and additional energy requirements by the fetus, placenta, and uterus take priority (66–68). Regardless of GDM or control conditions, we found that all measures of mitochondrial turnover, lipid metabolism, inflammation, and antioxidant defense were attenuated by day 17.5 of pregnancy. Early pregnancy displays the largest alterations in hepatic mRNA and protein markers between GDM and CD, but it is not until later in pregnancy that the liver cannot compensate, and it develops overt metabolic characteristics of MASLD exhibited by increased liver triglycerides, elevated NAS (steatosis, inflammation, and ballooning), and attenuated mitochondrial activity.

One of the strengths of this study is the use of an established diet-fed mouse model to develop GDM. This model closely mimics key metabolic features of human GDM, such as insulin resistance and impaired insulin secretion. Furthermore, this is the first study to evaluate the impact of GDM on hepatic markers of mitochondrial activity, biogenesis, mitophagy, autophagy, inflammation, antioxidant defense, and lipid metabolism throughout gestation. Despite these strengths, there are limitations. While the HFHS-fed mouse model does recapitulate GDM pathophysiology, it is important to note that pregnancy in a human is much lengthier and likely more complex, limiting the translation of these findings from rodent GDM to human GDM. Secondly, the present study does not include any non-pregnant mice as a control group. Although previous literature suggests that control-diet fed non-pregnant mice would not display any hepatic metabolic alterations over time (69–72), direct comparisons with this experimental setting would help strengthen the conclusions.

In conclusion, our data demonstrates that HFHS feeding in a GDM model leads to increased maternal hepatic triglyceride accumulation, elevated NAS, and reduced mitochondrial activity, accompanied by increases in hepatic inflammation and antioxidant defense. Pregnancy itself acts as a powerful metabolic switch, reprogramming nutrient utilization and energy balance to support fetal development. This metabolic shift appears strong enough to override liver adaptations to high-fat diet-induced GDM, ensuring continued development of the fetus and support organs but leading to hepatic lipid increases. To our knowledge, this is the first study to evaluate and characterize alterations in hepatic mitochondrial and lipid metabolism during both normal gestation and in a GDM model. These findings highlight the significant impact of gestation and GDM on liver health and MASLD risk.