With this study, we demonstrated the efficacy of a combined multitarget treatment approach in ameliorating the peripheral and central metabolic alterations induced by a high-fructose high-fat diet (HFHFD) which leads to complicated obesity. Rats exposed to a STD or HFHFD diet were studied to identify the phenotypic alterations induced by the HFHF diet, analyzing changes in BW, biochemical plasma parameters, and the protein expression of key homeostatic regulatory signaling systems in various brain areas related to food intake regulation and cognition, such as the HYPO, HIPPO, and PFC. In parallel, the effect of the chronic administration of the GLP-1R agonist LIG and the PPARα agonist/CB1 antagonist OLHHA, and their combination was studied. Currently, multiple studies are investigating the combined action of drugs targeting CB1 and PPARα receptors [9], or GLP-1 and CB1 receptors [19], as well as interactions between GLP-1R and PPAR receptors [20,21]. However, no evidence is currently available about treatments combining these three receptors simultaneously.

Several studies have demonstrated the efficacy of LIG for BW reduction, in both clinical and preclinical research [12,13], as well as its ability to improve the peripheral parameters associated with obesity, including blood lipids and hepatic enzymes [22,23]. At the same time, previous research conducted in our laboratory demonstrated that OLHHA administration is effective in reducing BW and metabolic syndrome in obesity models [17].

In this study, a severe obesity model, characterized by the consumption of high-fat foods and exposure to drinking fructose, was used to mimic the conditions of excessive food intake observed in morbid obesity. Our results showed that HFHF consumption increased BW gain, blood lipids, and hepatic enzymes [16]. However, contrary to our expectations, treatment with LIG did not significantly reduce BW, although it improved the plasma lipidic profile, possibly due to the use of a submaximal dose of this GLP-1 agonist. Consistently, previous studies have shown BW reductions with LIG in rats, but at higher doses [24,25,26,27]. On the other hand, in agreement with previous findings, OLHHA reduced BW and plasma bilirubin. Most surprisingly, treatment with the combination of the two drugs was the most effective strategy in this obesity model, significantly reducing BW and improving several plasma biochemical parameters (triglycerides, LDL, ALT, and AST), suggesting that the simultaneous targeting of PPARα, CB1, and GLP-1R receptors may be the key for effective obesity treatment.

One of the first interests of our study was to investigate the expression of the ECS in the various brain regions. In our model, we detected regio-specific alterations to the expression of the ECS in response to diet exposure. In all the analyzed areas, we evidenced significant alterations in the enzymes responsible for endocannabinoid synthesis and degradation, along with disruptions in the ratio synthesis/degradation of both 2-AG and AEA. Endocannabinoid signaling in the HYPO is pivotal for the balance of anorexigenic and orexigenic signals [28], and disruption in the levels of endocannabinoids can be directly related to obesity and metabolic syndrome. Mainly, endocannabinoid action on feeding in the HYPO is mediated by CB1 receptors, which are expressed at both pre-and post-synaptic levels in pro-opiomelanocortin (POMC) neurons (mediating satiety) and NPY/Agouti-related peptide (Agrp) ones (mediating hunger). Disrupted CB1 expression in the HYPO, as observed in animals exposed to a HFHFD, causes the altered modulation of the cross-talk among these neurons, provoking increased hunger signals and altered brain and peripheral metabolism [28]. On the other hand, the ECS in the HIPPO is implied in mediating the hedonic aspects of eating but also in learning and memory: alterations in ECS signaling that we detected in this region are compatible with previous results obtained by Massa et al. [29], showing that a HFHFD is associated with disruptions in endocannabinoids which can alter the important processes of synaptic transmission and plasticity that influence cognition. Similarly, various studies reported ECS alterations in the PFC in response to palatable food consumption and abstinence [30,31,32,33,34]. Disruptions of the ECS in this region can compromise processes of synaptic plasticity and neurotransmitter dynamic balance, resulting in altered cognitive behavior [35,36].

As expected, treatment with LIG in HFHFD-fed rats was not effective in restoring ECS alterations in any brain area. On the other hand, thanks to its activity on CB1 and PPARα receptors, OLHHA was partially effective in recovering alterations noticed in the HYPO, HIPPO, and PFC. However, once more, the combined treatment (OLHHA+LIG) was the most effective in restoring ECS protein expression to levels comparable to standard-fed rats in all the brain areas analyzed, suggesting a synergic action of the two drugs.

The HFHFD induced significant alterations in the expression of cerebral GLP-1R: compatibly with previous studies [37], we detected a significant increase in the GLP-1R expression in the HYPO. Conversely, the same animals reported a significantly lower expression of GLP-1R in the HIPPO and PFC. Moreover, HFHFD-fed animals displayed a significant increase in NPY1-R expression, which may drive overeating [38]. Also, significant alterations were noticed in MOR transmission both in the HYPO and PFC [39], sustaining the fact that a HFHFD induces alterations in reward signaling, which sustains palatable food consumption.

Interestingly, only the combined treatment with OLHHA+LIG significantly increased the expression of GLP-1R in HFHFD-fed animals, enhancing GLP-1 activity in all the analyzed brain areas, suggesting that this receptor activation is fundamental for its pharmacological action. NPY1-R normal expression, which was boosted by the diet, was dramatically reduced only by the combined treatment, thus removing the hyperphagic NPY-dependent signal. This pharmacological action may justify the BW loss noticed in these animals [40]. No similar effect was shown in animals exposed to a HFHFD and treated with LIG or OLHHA alone, evidencing, once more, the positive effects of the combined treatment.

Another goal of our study was to determine whether a HFHFD could result in cerebral insulin resistance. Starting with the HYPO, we noticed signs compatible with insulin resistance in HFHFD-fed animals, which provoked an increased expression of IRβ, chronic activation of PI3K, and decreased activity of GSK3β. Chronic hyperinsulinemia, commonly observed in obesity, can lead to the persistent stimulation of hypothalamic insulin receptors and disrupt normal insulin signaling, causing metabolic dysregulation and appetite imbalances, contributing to overeating and sustaining BW gain [41,42]. Moreover, increased PI3K signaling in the HYPO can derive from leptin resistance, which is common in HFHFD-fed rats [43]. Disrupted GSK3β in HFHFD-fed animals directly depends on aberrant PI3K signaling and compromises whole-body energy balance and glucose metabolism [44]. The HFHFD also provoked changes in the expression of proteins partaking to the insulin pathway in cognitive areas such as the HIPPO and PFC, where we noticed a decreased expression of IRβ and IRS-1 and a decreased activation of PI3K and AKT, leading globally to a decrease in insulin signaling [41], which has been previously associated with a deficit in memory and learning and disrupted food reward signaling [45,46,47,48,49].

Surprisingly, the administration of LIG was not effective in reverting any of the alterations noticed in insulin pathway protein expression; despite LIG being widely used for the therapy of diabetes [14], it had no efficacy in reverting signs of brain insulin resistance in our model. In this case, the use of a submaximal dose should not have an impact because LIG generally has a glucose-regulating action and increases insulin sensitivity at lower doses compared to the ones necessary for BW reduction [15]. Similarly, little to no significant improvements were detected with OLHHA administration. Conversely, the combined administration of the two drugs (OLHHA+LIG) re-established normal IRS-1 signaling in all the analyzed areas, normalized IRβ and PI3K expression in the HYPO, and restored AKT activation and GSK3β expression in the PFC.

Additionally, the downstream proteins involved in insulin pathways, such as mTOR and ERK1/2, were severely disrupted by the HFHFD exposure. In the HYPO, mTOR signaling is fundamental for regulating energy balance, glucose, lipid metabolism, and hepatic function [50,51], and, all over the brain, it has a pivotal role in promoting synaptic function, cell proliferation, and autophagy [52,53,54]. Similarly, ERK1/2 function as metabolic sensors and regulate insulin sensibility and neuronal energy balance [55]. Moreover, ERK is linked to several cognitive functions, including memory, learning, and attention, and dysfunctions in its pathway have been associated with the development of neurodegenerative diseases [56,57]. Disruptions in mTOR signaling provoked by HFHFD exposure were restored only by combined treatment with OLHHA+LIG, whereas ERK1/2 protein expression and activation were normalized by the administration of all treatments (LIG, OLHHA, and OLHHA+LIG).

Knowing the link between obesity and neurodegenerative diseases and the association between brain insulin resistance and tauopathies [58], we analyzed the expression of total tau and its phosphorylated forms, along with the expression of CDK5 and catalytic subunits, p35 and p25, which are involved in the process of tau phosphorylation. The HFHFD changed total tau expression in all the brain areas analyzed and provoked a significant increase in its phosphorylated form, AT8, in both the PFC and HIPPO, suggesting a potential disruption in tau homeostasis and the onset of tau hyperphosphorylation, which could be indicative of early-stage neurodegenerative processes or increased vulnerability to cognitive decline, accordingly to previous evidence [59,60]. Significant alterations in tau observed in the HFHFD were accompanied by a disruption in CDK5 and its catalytic subunits p35 and p25 in all the brain regions analyzed, suggesting the involvement of this pathway in tau phosphorylation [61]. Rising evidence suggests that GLP-1R agonists may act with neuroprotective effects [62,63]; however, in this study, we report no pharmacological activity of LIG in restoring tau or CDK5-associated protein expression. Little to no effect was observed with the administration of OLHHA; however, once more, the combination treatment (OLHHA+LIG) was effective in restoring some of the alterations in the expression of these proteins in all the brain areas analyzed.

As expected, HFHFD exposure resulted in glial and microglial activation [64,65], with increased GFAP in the HIPPO and PFC and increased IBA-1 expression in the HYPO and PFC. As previously published [66], in the hippocampus these alterations were coupled with the activation of NF-kB, suggesting neuroinflammation-based disruptions of the hippocampal function supporting the cognitive alterations associated with obesity. Surprisingly, HFHFD-fed animals display increased BDNF and TrkB expression in the HYPO: knowing that BDNF collaborates in the control of energy and glucose balance, we suppose that its increment is trying to compensate for the aberrant variations induced by the HFHFD, which were discussed previously [67]. Conversely, a reduced expression of TrkB was noticed in the HIPPO, sustaining the hypothesis of impaired neuronal survival in animals fed with a HFHFD [68]. Once more, rats treated with LIG displayed no significant change in protein expression due to pharmacological treatment. Interestingly, changes occurring in the HIPPO were significantly restored only by the OLHHA treatment, whereas alterations due to the HFHFD in the HYPO were successfully restored by the administration of the combination of the two drugs (OLHHA+LIG).

Moreover, we demonstrate that the peripheral parameters present strong correlations with brain protein expression, meaning that peripheral biomarkers could potentially serve as non-invasive indicators of altered neurological processes or brain health status in obese patients, thus favoring special medical interventions to prevent the onset of cognitive impairment.

Finally, with this study, we demonstrated that exposure to a HFHFD for 8 weeks is sufficient to induce BW gain and increase plasma triglycerides, LDL, and hepatic parameters. A HFHFD provoked disruptions in the expression of the proteins regulating food intake, the ECS, insulin pathways, and inflammation and resulted in altered tau expression and phosphorylation in various brain areas, which is indicative of signs of neurodegeneration. Based on our results, the administration of the GLP-1R agonist LIG alone or a double PPARα agonist/CB1 antagonist (OLHHA) with anorexigenic activity is not sufficient to completely revert the alterations noticed, both at the peripheral and central level. On the other hand, the combined treatment with both compounds (OLHHA+LIG) resulted in the most effect in normalizing BW, plasma parameters, and ameliorating aberrant brain protein expression. These findings align with previous research, underlying that CB1 antagonism can potentiate GLP-1R agonist activity [69,70,71,72].

Of course, some limitations must be accounted for in this study: it was developed only in male rats, lacking a genre perspective. Moreover, even though the results obtained in the brain suggest the presence of cognitive impairment in HFHFD animals, no behavioral tests have been conducted to support this hypothesis. Furthermore, more studies may be needed to clarify the molecular mechanism of action justifying the synergy of the OLHHA+LIG administration.