DIETARY INTERVENTIONS TARGETING GLUCOSE METABOLISM AND HYPERINSULINEMIA: A NEW TRANSLATIONAL PERSPECTIVE FOR THE MANAGEMENT OF ACUTE INTERMITTENT PORPHYRIA

Acute Intermittent Porphyria (AIP) represents the most severe form of hepatic porphyrias, an inherited disorder caused by genetic mutations in the hydroxymethylbilane synthase (HMBS) gene, encoding the porphobilinogen deaminase (PBGD) enzyme and reducing hepatic heme availability. In stressful conditions, as fasting, which stimulates the demand of heme production, the delayed heme biosynthesis leads to the accumulation of porphyrins ’precursors, involved in neurotoxicity, and hyperactivity of ALAS1, the first rate-limiting enzyme of the heme pathway. AIP clinical manifestations presents in 0.5-1% of HMBS-mutation carriers and includes severe neurovisceral/neuropsychiatric acute attacks, which are managed with hemin administration and Givosiran, both directed against the hepatic ALAS1 activity. For mild AIP cases, glucose represents an alternative strategy to downregulate ALAS1. Nonetheless, current therapies showed several limitations as biochemical/clinical relapse, raise in transaminases and renal adverse events, thereby encouraging researchers to develop safer therapeutics for the treatment of refractory symptomatology. Recently, it has emerged that AIP mice developed glucose intolerance and hyperinsulinemia during fasting. In this model, hepatic glycogen storages were not exploited to normalize glycemia and alternative mechanisms as ketogenesis were aberrantly activated to face with energy demand. In addition, AIP patients with clinically stable disease or highly porphyrins’ excreters may develop similar metabolic disturbances, which may alter the response to glucidic therapy. In these subset of patients, hyperinsulinemia was associated with a protection against the acute symptomatology, possibly due to the ALAS1 downregulation mediated by insulin. Despite the protection from the acute episodes, stable AIPs (>90%) did not benefit of any therapeutic treatment albeit they are at risk to develop metabolic-associated disorders, as diabetes, chronic kidney disease and liver cancer due to the high exposure to toxic porphyrins, opening the possibility to introduce insulin-sensitizers as a novel class for AIP management. Therefore, aims of this study were to assess whether nutritional interventions may correct metabolic dysfunctions observed in AIP, possibly providing the proof-of-concept that metabolic and hepatic assessment of AIP patients, which still do not represent a routine clinical practice, may address towards a personalized medicine. For this purpose, we tested insulin-sensitizing compounds, which have recently shown attractive results for the management of diabetes and metabolic syndrome in preclinical models and patients. Firstly, the study deepened the role of an insulin-mimetic, the α-lipoic acid (α-LA), in PBGD-silenced hepatocytes and in a genetic AIP murine model, as it showed beneficial effects in porphyria cutanea tarda in previous findings. α-LA enhanced hepatocellular heme pool, glycolysis, and ATP production in in vitro model during stressful conditions. Similarly, α-LA administration in AIP mice increased hepatic heme, ATP content and citrate synthase activity during fasting, supporting that this molecule may provide substrates for heme biosynthesis by stimulating Krebs cycle and ameliorate the two major energy-yielding pathways, glycolysis and mitochondrial respiration. Furthermore, it rescued hyperinsulinemia in fasted AIP mice, leading to a great improvement of hepatic glucose metabolism and systemic energetic balance. Indeed, the aberrant activation of insulin signaling, observed during caloric restriction in the livers of AIP mice, was recovered after the α-LA supplementation. Still, AIP+α-LA induced glycogenolysis in fasted state and increased the glucose-sensing Glut2 protein levels in the liver, supporting that α-LA may promote the hepatic glucose export during fasting in order to provide glucidic reserves to the other organs as physiologically occurs. After glucose injection, AIP mice receiving α-LA improved GTT and glucose uptake on PET/CT scans in the liver and brain, skeletal muscle and white adipose tissue (WAT). From PET/CT results, it has emerged that AIP mice enhanced brown adipose tissue (BAT) activation, used as a mean of heat production from glucidic/lipidic sources, after cold stress compared to Wt, probably as an adaptive mechanism to meet whole-body energy requests. Interestingly, AIP+ α-LA did not show a higher BAT metabolic activity, similar to Wt, thus sustaining the improvement of AIP energy status and that the insulin-mimic may avoid energy dissipation attempting to preserve energy reserves. We then focused on the potential efficacy of probiotics, as dietary consumption of fermented-foods has been proposed as an effective solution to achieve health benefits for common metabolic disorders, in which adiposity represent one of the main risk factors. Nonetheless, their applications in rare disorders has been scarcely investigated. Here, we examined heat-killed probiotic Bifidobacterium animalis subsp. lactis CECT 8145 (BLP1), the BLP1-derived postbiotic lipoteichoic acid (LTA), which is responsible of many of the BLP1 health cares, and the alive probiotic Bacillus coagulans (B. coagulans), whose spores resist to heat temperatures, more than other probiotic strains, during manufacturing processes of fortified foods. These compounds mainly modify intestinal microbial composition and stimulate fat disposal in the liver and WAT. Lipid breakdown and heat dissipation are mediated by BAT, resulting in an improvement of body weight and insulin sensitivity. Here, we found that the use of probiotics (heat-killed or alive) or postbiotic ameliorated glucose tolerance, hyperinsulinemia, lean/mass ratio and muscular energy utilization. However, they boosted a potentially maladaptive BAT activation, which was observed in AIP mice after cold stress, supporting that, although biochemical and glucidic response were improved, a careful monitoring of their energetic status after the prolonged supplementation with these diets need to be considered. As a translational approach, we firstly provided a metabolic and hepatic characterization in a series of AIP patients (n=14), who were stratified according to recurrence of the attack as following: a) stable AIPs (n=9) who were asymptomatic highly excreters, and b) active AIPs (n=5) who manifested >2 annual attacks. AIP patients underwent bioimpedance analysis, which provided information about energetic status of AIP. We found that stable AIPs had higher % metabolic active cells in skeletal muscle compared to active AIPs, correlating with %lean mass fraction, suggesting that most of the muscle mass in AIP patients pushed their metabolic activity. Similar to AIP models, stable AIPs had altered OGTT curve paralleled by higher insulin levels and HOMA-IR. Therefore, stable AIP patients showed both signs of early hyperinsulinemia and muscle hypermetabolism, possibly being part of an adaptive para-physiologic mechanism to prevent energy substrate deficiency. Mild-moderate fibrosis was non-invasively detected in 5/9 stable AIP cases but not in active ones. Liver stiffness values were significantly correlated with urinary ALA, suggesting a direct role of this porphyrin to induce liver damage, and showed a positive correlation with insulin levels and HOMA-IR, corroborating that metabolic and hepatic screening should be introduced for monitoring and management of AIP.