Canagliflozin regulates metabolic reprogramming in diabetic kidney disease by inducing fasting-like and aestivation-like metabolic patterns.

AIMS/HYPOTHESIS: Sodium-glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) are antihyperglycaemic drugs that protect the kidneys of individuals with type 2 diabetes mellitus. However, the underlying mechanisms mediating the renal benefits of SGLT2i are not fully understood. Considering the fuel switches that occur during therapeutic SGLT2 inhibition, we hypothesised that SGLT2i induce fasting-like and aestivation-like metabolic patterns, both of which contribute to the regulation of metabolic reprogramming in diabetic kidney disease (DKD). METHODS: Untargeted and targeted metabolomics assays were performed on plasma samples from participants with type 2 diabetes and kidney disease (n=35, 11 women) receiving canagliflozin (CANA) 100 mg/day at baseline and 12 week follow-up. Next, a systematic snapshot of the effect of CANA on key metabolites and pathways in the kidney was obtained using db/db mice. Moreover, the effects of glycine supplementation in db/db mice and human proximal tubular epithelial cells (human kidney-2 [HK-2]) cells were studied. RESULTS: Treatment of DKD patients with CANA for 12 weeks significantly reduced HbA1c from a median (interquartile range 25-75%) of 49.0 (44.0-57.0) mmol/mol (7.9%, [7.10-9.20%]) to 42.2 (39.7-47.7) mmol/mol (6.8%, [6.40-7.70%]), and reduced urinary albumin/creatinine ratio from 67.8 (45.9-159.0) mg/mmol to 47.0 (26.0-93.6) mg/mmol. The untargeted metabolomics assay showed downregulated glycolysis and upregulated fatty acid oxidation. The targeted metabolomics assay revealed significant upregulation of glycine. The kidneys of db/db mice undergo significant metabolic reprogramming, with changes in sugar, lipid and amino acid metabolism; CANA regulated the metabolic reprogramming in the kidneys of db/db mice. In particular, the pathways for glycine, serine and threonine metabolism, as well as the metabolite of glycine, were significantly upregulated in CANA-treated kidneys. Glycine supplementation ameliorated renal lesions in db/db mice by inhibiting food intake, improving insulin sensitivity and reducing blood glucose levels. Glycine supplementation improved apoptosis of human proximal tubule cells via the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. CONCLUSIONS/INTERPRETATION: In conclusion, our study shows that CANA ameliorates DKD by inducing fasting-like and aestivation-like metabolic patterns. Furthermore, DKD was ameliorated by glycine supplementation, and the beneficial effects of glycine were probably due to the activation of the AMPK/mTOR pathway.

Organ protection by SGLT2 inhibitors: role of metabolic energy and water conservation.

Therapeutic inhibition of the sodium-glucose co-transporter 2 (SGLT2) leads to substantial loss of energy (in the form of glucose) and additional solutes (in the form of Na+ and its accompanying anions) in urine. However, despite the continuously elevated solute excretion, long-term osmotic diuresis does not occur in humans with SGLT2 inhibition. Rather, patients on SGLT2 inhibitor therapy adjust to the reduction in energy availability and conserve water. The metabolic adaptations that are induced by SGLT2 inhibition are similar to those observed in aestivation - an evolutionarily conserved survival strategy that enables physiological adaptation to energy and water shortage. Aestivators exploit amino acids from muscle to produce glucose and fatty acid fuels. This endogenous energy supply chain is coupled with nitrogen transfer for organic osmolyte production, which allows parallel water conservation. Moreover, this process is often accompanied by a reduction in metabolic rate. By comparing aestivation metabolism with the fuel switches that occur during therapeutic SGLT2 inhibition, we suggest that SGLT2 inhibitors induce aestivation-like metabolic patterns, which may contribute to the improvements in cardiac and renal function observed with this class of therapeutics.

Differential gene expression in the liver of the African lungfish, Protopterus annectens, after 6 months of aestivation in air or 1 day of arousal from 6 months of aestivation.

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase) of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of aestivation in the liver of P. annectens.

Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens, and their mRNA expression levels in the liver, kidney, brain and skeletal muscle during aestivation.

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.

Seasonal changes in the hypothalamic vasopressinergic system of a wild Sahelian rodent, Taterillus petteri.

Seasonal variations in the immunoreactivity of vasopressinergic perikarya in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic nuclei (SCN), and in the labelling of vasopressinergic fibres in the internal zone of the median eminence were studied in Taterillus petteri, a rodent that is found in the north Burkina Faso (formerly Upper Volta). In this region, there are four seasonal climatic combinations: the humid and hot, humid and cold, dry and cold, and dry and hot seasons. In the dry hot season, the rodents experience phases of torpor (adaptation to dryness). Immunoreactivity of the PVN and SON is highest during the dry cold season. Labelling is intense during the dry hot and humid hot seasons, and is at its lowest during the humid cold season. In the SCN, labelling of the perikarya is only dense during the dry hot season, whereas for the rest of the year, the immunoreactivity is weak or undetectable. The pattern of immunoreactive variations of vasopressin-positive fibres located in the internal zone of the median eminence is similar to those of vasopressinergic perikarya in the PVN and SON. These results suggest that there is an association between: (1) seasonal modifications in the immunoreactivity of PVN and SON vasopressinergic perikarya and vasopressinergic fibres of the internal median eminence, and (2) climatic conditions, water metabolism, behavioural activity and diet. It is not possible to establish a correlation between seasonal variations in water availability and fluctuations in the labelling of vasopressinergic perikarya in the SCN. However, labelling is intense when the animals are in torpor during the dry hot season.

The reproductive cycle of male uinta ground squirrels: some anatomical and biochemical correlates.

1. Maximum testicular development and plasma testosterone levels occurred at spring emergence from aestivation-hibernation (A-H), followed by a regression to basal state in June. 2. A slight testicular recrudescense occurred just prior to immergence into A-H, but this development was not accompanied by a definitive increase in plasma testosterone levels or seminal vesicle weights. 3. Four lines of evidence were consistent with the hypothesis that FSH stimulates testicular MAO activity. 4. Changes in pituitary and hypothalamic MAO activity suggested a functional role for this enzyme in controlling releasing factor and gonadotropin production by these organs.

[Morphometric data on pineal gland of loir (Glis glis) and lerot (Elioyms quercinus) during the annual cycle]. / Données morphométriques sur la glande pinéale du loir (Clis glis) et du lérot (Eliomys quercinus) au cours du cycle anuel

The important involution of the pineal gland of Glis glis and Eliomys quercinus during the months of july and august belongs to a polyglandular involution (anterior lobe of the hypophysis, male and female genital glands) characteristic of estivation.