Genetic and epidemiological studies provide evidence supporting the contribution of a transgenerational background of parental obesity to the development of obesity itself and further metabolic risks in the offspring (13, 46–48). Other than understanding the underlying mechanisms through which parental obesity takes its toll on the offspring’s health, an increasing body of research has been raising resolutive approaches. However, most of them rely on preventive measures targeted at the pre-conception and/or gestational period (49). Here, to address the problem once the damage is already set, we investigated the effects of CBD treatment on the offspring as a way to attenuate the negative outcomes of maternal obesity.

An increasing number of studies have addressed the pleiotropic role of the endocannabinoid system on metabolic regulation at the central and peripheral levels. Endocannabinoid regulation of metabolism is extremely relevant to the central nervous system (CNS), especially in the hypothalamus where it plays a pivotal role on energy balance and feeding behaviors, contributing not only for the pathogenicity of obesity but also the development of eating disorders (34, 36–38, 50, 51). Nonetheless its receptors are also expressed in peripheral organs such as the adipose tissue, liver, skeletal muscle, pancreas, kidney, and gastrointestinal tract (52), hence its particularly promising modulation in the context of obesity and metabolic disorders (53, 54). Here, we show that CBD treatment is able to revert a number of metabolic dysfunctions and neuroinflammation arising from maternal consumption of CAF during pregnancy and lactation, including higher visceral adiposity, insulin resistance, endotoxemia, and overexpression of inflammatory markers in the hypothalamus of the offspring.

Besides obesity itself, the consumption of a diet rich in saturated fats and carbohydrates is also associated with the development of metainflammation, a chronic and self-sustained state of low-grade inflammation (55). In this study, we demonstrated that both male and female offspring of obese mothers had higher visceral WAT deposits and fasting glucose levels, followed by elevated plasma cholesterol and triglycerides levels in females and insulin in males. Previous studies with different models of maternal obesity have established that both the gestation and the lactation-suckling periods are critical for WAT development, impacting epigenetic regulation of key genes for energy metabolism–such as dopamine and opioid genes related to food behavior (56) and hypothalamic nutrient sensors (46)–and altering long-term adiposity set points (57, 58). In a model of maternal high-fat diet (HFD), the offspring of obese mothers showed an increased expression and activity of stearoyl-CoA desaturase-1 (SCD1), a key enzyme of fatty acid (FA) metabolism. SCD1 converts saturated FAs, such as palmitate and stearate, to monounsaturated FAs, the predominant substrates for triglyceride synthesis (59). It is important to highlight that CAF, which closely mimics the Western urban eating patterns, is not only high in sugar but also saturated fats, with palmitate being the most predominant FA, hence the prominent impact of this dietary pattern on lipid profile (60, 61).

Interestingly, no effect of maternal obesity was found on total body weight, even though WAT deposition was altered. This finding could be due to a diminished muscle mass that may have compensated for the heavier adiposity. In previous studies, both 3- and 12-weeks old pups from CAF-fed mothers showed equal or lower body weight and lean mass but greater fat accumulation than controls, which has been described as the thin-outside-fat-inside phenotype (62, 63). Also, since in our study the offspring were fed a normal diet after weaning, we were able to show that the metabolic impairments observed were independent of the offspring’s own diet. However, in previous studies, when offspring of CAF-fed dams were given CAF after weaning, there was no increase in body weight at puberty (4 weeks of life), but animals had higher weight at adulthood (16 weeks of life), and no difference in visceral adiposity was reported in male offspring. Thus, differences in body composition seem to be dependent on the post-weaning diet as well as the sex of the offspring (64, 65).

Cannabidiol (CBD) treatment was able to mitigate most of the metabolic dysfunctions caused by maternal obesity, reducing visceral fat content and IR in males, plasma triglyceride levels in females, and plasma LPS in both sexes. CB1 receptor activation is generally considered a powerful orexigenic signal; thus, the endocannabinoid system’s inhibition is beneficial for treating obesity and related metabolic diseases. Since CBD is an allosteric modulator of CB1 receptors, inhibiting its activation by endogenous ligands or exogenous agonists might trace the pathway through which CBD attenuates peripheral disturbances arising from maternal obesity (66). CB1-KO mice maintained on a normocaloric, standard diet have been shown to have a decreased body weight gain over time, which was associated with increased energy expenditure and elevated β(3)-adrenergic receptor and uncoupling protein-1 (UCP1) mRNA levels in the brown adipose tissue, suggestive of enhanced peripheral sympathetic activation and thermogenesis (66).

Diets high in saturated FAs, such as the Western diet, increase the uptake and storage of sphingolipids and their essential fractions, such as ceramide, sphingosine, sphinganine, and sphingomyelin. Interestingly, previous data suggest that phytocannabinoids and other agonists of CB1 or CB2 receptors can modulate sphingolipid concentrations in specific organs under the increased availability of FAs in the diet. CBD significantly lowered the concentration of sphingolipids in the adipose tissue (67), the skeletal muscle (31), and the brain by increasing catabolism, inhibiting salvage and/or de novo synthesis, which restores the tissue’s insulin sensitivity and, therefore, attenuates IR (68). Other than that, other mechanisms are proposed for the beneficial effects of CBD on metabolic disorders in peripheral organs, such as the protective effect of CBD on adipose-derived stem cells against endoplasmic reticulum stress and its complications related to IR and diabetes (69) and attenuation of oxidative stress and inflammatory response, associated with an improved n-6/n-3 polyunsaturated fatty acids (PUFAs) ratio in the white and red skeletal muscle (70), indicating a narrow relationship between the endocannabinoid system and hormonal and energetic balance.

It is worth noting that the sexual dimorphisms observed in this study regarding glucose metabolism and IR corroborate with what has been demonstrated in different models of obesity and maternal obesity (71). Female sex hormones play a fundamental role in dimorphic insulin signaling since estrogens increase insulin sensitivity in metabolic tissues and upregulate insulin transcription in pancreatic beta cells as well as GLUT4 synthesis in adipose tissue and muscle (72). Female mice fed an HFD showed reduced susceptibility to developing obesity-induced IR and WAT inflammation when compared to HFD-fed males. Meanwhile, HFD-fed males treated with estradiol presented the same protective effect as females, indicating that the dimorphic effects of obesity on IR may be due to estrogen-mediated reductions in WAT inflammation (73). Furthermore, a recent study has shown that an androgen-driven gut microbiome may also be responsible for the increased susceptibility to IR in males since gut microbiome depletion abolishes sex-biased glucose metabolism in HFD-fed mice (74).

A growing body of evidence suggests that males are more sensitive to intrauterine hyperglycemia as well; hence both animal and human studies show the same pattern of higher risk for obesity and IR in male offspring of obese/diabetic mothers. In a model of maternal high-sucrose diet (HSD), female HSD offspring were shown to be more glucose intolerant, while male counterparts were more insulin resistant (75). Furthermore, human cohort and prospective studies have shown a strong correlation between offspring metabolic impairments and maternal diabetes for males but not for females (76–78). This relationship may be explained by the fact that, during preimplantation, the male embryo is believed to have a greater ability to adapt to the adverse environment and, as a result, has a higher sensitivity to programming influences (79).

In addition, our results showed that CBD effectively reverted the increase in plasma LPS levels in both male and female CAF offspring. Obesogenic diets change the gut microbiota composition by altering the Firmicutes: Bacteroidetes ratio, the two most detected bacterial phyla in rodents as well as humans. In normal-weight animals this relation is characterized by a high ratio of Bacteroidetes to Firmicutes, while the opposite is found in obese counterparts (80, 81). It has been proposed that Firmicutes bacteria are more effective in extracting energy from food than Bacteroidetes, thus promoting a more efficient absorption of calories and boosting weight gain (82, 83). This imbalance resulting from obesogenic diets can be traced back to the overabundance of refined sugars and fats as well as the low intake of vegetables, fruits and dietary fibers (84–87). The simultaneous collapse of the gut barrier with increased permeability allows high levels of LPS, Gram negative bacteria’s most potent immunogenic component, to reach the bloodstream and initiate a diffuse inflammatory process named endotoxemia (88). The structural components of LPS are recognized by B cells via cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4), thus leading to nuclear factor kappa-B (NFkB) activation and the release of pro-inflammatory cytokines, such as TNFα and IL1β (89). During pregnancy, these pro-inflammatory mediators, together with LPS itself, can interact with the placenta and cause a range of disturbances, including immature blood vessels, hypoxia, increased inflammation, autophagy, and altered stress markers (90, 91). In that sense, several models of maternal immune activation rely on prenatal exposure to LPS, resulting in a myriad of altered physiological and neurological outcomes in the offspring (92, 93). The data seen here indicates that the endotoxemia caused by obesogenic diets affects not only the pregnant mother but can be seen in the offspring later on, independently of the offspring’s own diet. These findings corroborate with previous studies demonstrating that maternal obesity during gestation and/or lactation negatively impacts the offspring’s gut microbiota (94). On the other hand, CBD has rescued this damage on the offspring, lowering plasma LPS to control levels. Even though we have not performed gut-specific analysis, previous studies in different pre-clinical and clinical models lead us to infer that the effects observed may be due to the influence of CBD on gut microbiota composition (95–97) and/or a protective effect on maintaining gut barrier integrity (98–102).

Regarding the alterations provoked by maternal overnutrition on CNS neuroinflammation, here we show that the treatment with CBD was able to rescue hypothalamic inflammation by reducing gene expression of TNFα and IL6 in the offspring of obese dams. The hypothalamus is one of the main homeostatic centers of the CNS and, therefore, needs to be effectively responsive to fluctuations in peripheral systems. However, due to its naturally increased permeability in order to better receive and respond to signals coming from metabolic organs, the hypothalamus is also one of the first brain regions to suffer with systemic disruptions, resulting in neuroinflammation (103, 104). In the present study, we have demonstrated molecular alterations that are trademarks of neuroinflammation in the hypothalamus of the offspring born from CAF-fed dams. In both sexes, the expressions of TNFα and IL6 were increased in CAF-Veh animals, while the expression of IL1β was increased only in females. These findings corroborate with previous data from a different model of maternal obesity that demonstrates that mice born from mothers fed a HFD diet have increased expression of these inflammatory markers in the hypothalamus compared to the offspring of lean parents (105).

Tumor necrosis factor-alpha (TNFα), IL6, and IL1β are well-known pro-inflammatory cytokines involved in microglial and astrocytic activation in the entire nervous tissue. However, especially in the hypothalamus, they have a remarkable role in the modulation of hypothalamic feeding circuits. It has been previously demonstrated that HFD and high-carbohydrate diets stimulate orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons to produce advanced glycation end products, which activate TNFα, enhancing microglia reactivity. This scenario results in the dysfunction of anorexigenic neurons, altering the appetite-regulatory circuits (106). In addition, Proopiomelanocortin (POMC) neurons, which present anorexigenic activities, also suffer a significant impact from maternal obesity (107). The melanocortin system plays an important role on the regulation of appetite, energy expenditure, and metabolism, therefore, impairments in the POMC and melanocortin 4 receptor (MC4R) pre- and post-translational processing are forerunners for the development of obesity (108, 109). Decreased activity in POMC cells has been shown to be associated with increased food intake and obesity (107) and has been demonstrated in the offspring of obese mothers (110–113). When observing the precise localization of NPY and POMC in the hypothalamus of the offspring of obese mothers, Ornellas and collaborators found that NPY nerve fibers from the ARC to the periventricular nucleus and around the third ventricle were increased, while POMC were diminished in the same areas (105). Variations in the reactivity and/or distribution of hypothalamic astrocytes also seem to affect synaptic organization and POMC responsiveness to glucose, which is associated with energy and metabolic imbalances (114, 115).

Although endocannabinoid signaling has been implicated in the modulation of both food intake and energy expenditure, a complete understanding of its role in the hypothalamus is still lacking. A recent study demonstrated that a HFD diet in CB1 receptor-deficient mice contributes to the offspring’s nutritional programming, resulting in increased susceptibility to metabolic challenges both perinatally and during adulthood (116). Additionally, maternal HFD has been shown to upregulate CB1 hypothalamic expression in the offspring, which was associated with leptin pathway impairment and increased susceptibility to obesity (117–119). Other than that, few studies have evaluated cannabinoid modulation in the context of parental obesity, however, the findings shown here are still in line with different models that show the anti-inflammatory effects of CBD on other neuroinflammatory conditions (95, 120–122). Elevated hypothalamic endocannabinoid content has been associated with higher orexigenic signaling of ghrelin (123–125) and defective leptin signaling, observed in genetic models of obesity such as obese Zucker rats and db/db and ob/ob mice (126, 127). These findings suggest that endocannabinoid mediators contribute to hyperphagia and obesity, which also supports the restorative effects of CBD treatment, once it reduces endocannabinoid signaling, especially through CB1 receptors (128). When it comes to inflammation, effects of CBD via CB2 receptor are more distinguished, since this receptor is more predominantly expressed on immune cells, including glial cells. CB2 expression is upregulated in microglia stimulated with pro-inflammatory cytokines, indicating a significant role of CB2 in the regulation of neuroinflammatory states (129). In line with this, CBD has been shown to exert a CB2-dependant anti-inflammatory effect on microglial inflammation (23, 130).

Astrogliosis is a very well-established marker for obesity-related neuroinflammation (16, 131). Variations in the reactivity and/or distribution of hypothalamic astrocytes seem to affect synaptic organization and responsiveness to peripheral fluctuations, which is associated with energy and metabolic imbalances (114, 115). In animal models of obesity, gene and protein expression of the glial fibrillary acidic protein (GFAP), an astrocyte marker, are commonly higher in obese groups when compared to control animals (131–134). Interestingly, we have found that GFAP gene expression was reduced in the hypothalamus of the offspring of obese mothers. This result may have been induced by an adaptative reprogramming mechanism in response to the exposure to a harmful intrauterine environment during neurodevelopment, indicating that molecular mechanisms that rule maternal obesity-induced neuroinflammation may differ from the ones associated with obesity in the individual itself (135–137). Reduction in astrocyte expression can be deleterious during neurodevelopment since these cells play a pivotal role in synapse maturation, and their reduced expression is related to a range of neurological disorders (138–140). We have observed the same reduction of GFAP expression in CBD-treated CT offspring, however, we cannot affirm that the same detrimental effect applies. The reduction in GFAP expression of CAF offspring is a response to a prenatal immune challenge, while the reduction seen in CT-CBD is more likely to be the result of the anti-inflammatory activity of CBD (141, 142).

Regarding microglial activation, unlike previous studies (143, 144), we have not found any differences in the gene expression of ionized calcium-binding adapter molecule 1 (IBA1) in the hypothalamus of the offspring of obese mothers. However, the expression of IBA1 is related to the proliferation and distribution of microglial cells and not the polarization toward a pro-inflammatory state (145). Furthermore, it has recently been described that prenatal immune stress blunts microglia reactivity throughout life (146), which means that the expression levels of microglial cells may remain at control levels, but their innate reactivity to immune stressors can be defective.

These gene expression patterns are consistent with impaired energy and metabolic regulation in the hypothalamus, which might have originated the peripheral deficits observed in the offspring of obese mothers. Together, these results indicate an intricate interplay between peripheral and central counterparts in both the pathogenicity of maternal obesity and the modulation of the endocannabinoid system by CBD. In this context, the impairment of internal hypothalamic circuitry caused by neuroinflammation runs in tandem with the disruptions of important metabolic processes, which can be attenuated by CBD treatment in both ends.