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Previously, we have shown that Maternal Separation and Early Weaning (MSEW) exacerbates high fat diet (HF)-induced visceral obesity in female offspring compared to normally reared female mice. Stress hormones such as glucocorticoids and mineralocorticoids are critical mediators in the process of fat expansion, and both can activate the mineralocorticoid receptor (MR) in the adipocyte. Therefore, this study aimed to, comprehend the specific effects of MSEW on adipose tissue basic homeostatic function, and investigate whether female MSEW mice show an exacerbated obesogenic response mediated by MR. Gonadal white adipose tissue (gWAT), a type of visceral fat, was collected to assess lipidomics, transcriptomics, and in vitro lipolysis assay. Obese female MSEW mice showed increased adiposity, elevated 44:2/FA 18:2 + NH4 lipid class and reduced mitochondrial DNA density compared to obese control counterparts. In addition, single-cell RNA sequencing in isolated pre- and mature adipocytes showed a ~9-fold downregulation of aquaglycerolporin 3 (Aqp3), a channel responsible for glycerol efflux in adipocytes. Obese MSEW mice showed high levels of circulating aldosterone and gWAT-derived corticosterone compared to controls. Further, the MR blocker spironolactone (Spiro, 100 mg/kg/day, 2 weeks) normalized the elevated intracellular glycerol levels, the greater in vitro lipolysis response, and the number of large size adipocytes in MSEW mice compared to the controls. Our data suggests that MR plays a role promoting adipocyte hypertrophy in female MSEW mice by preventing lipolysis via glycerol release in favor of triglyceride formation and storage., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The lactate dehydrogenase isoform A (LDHA) is a key metabolic enzyme that preferentially catalyzes the conversion of pyruvate to lactate. Whereas LDHA is highly expressed in many tissues, its expression is turned off in the differentiated adult β-cell within the pancreatic islets. The repression of LDHA under normal physiological condition and its inappropriate upregulation under a diabetogenic environment is well-documented in rodent islets/β-cells but little is known about LDHA expression in human islet cells and whether its abundance is altered under diabetic conditions. Analysis of public single-cell RNA-seq (sc-RNA seq) data as well as cell type-specific immunolabeling of human pancreatic islets showed that LDHA was mainly localized in human α-cells while it is expressed at a very low level in β-cells. Furthermore, LDHA, both at mRNA and protein, as well as lactate production is upregulated in human pancreatic islets exposed to chronic high glucose treatment. Microscopic analysis of stressed human islets and autopsy pancreases from individuals with type 2 diabetes (T2D) showed LDHA upregulation mainly in human α-cells. Pharmacological inhibition of LDHA in isolated human islets enhanced insulin secretion under physiological conditions but did not significantly correct the deregulated secretion of insulin or glucagon under diabetic conditions., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity is associated with alterations in hepatic lipid metabolism. We previously identified the prorenin receptor (PRR) as a potential contributor to liver steatosis. Therefore, we aimed to determine the relative contribution of PRR and its soluble form, sPRR, to lipid homeostasis. PRR-floxed male mice were treated with an adeno-associated virus with thyroxine-binding globulin promoter-driven Cre to delete PRR in the liver [liver PRR knockout (KO) mice]. Hepatic PRR deletion did not change the body weight but increased liver weights. The deletion of PRR in the liver decreased peroxisome proliferator-activated receptor gamma (PPARγ) and triglyceride levels, but liver PRR KO mice exhibited higher plasma cholesterol levels and lower hepatic low-density lipoprotein receptor (LDLR) and Sortilin 1 (SORT1) proteins than control (CTL) mice. Surprisingly, hepatic PRR deletion elevated hepatic cholesterol, and up-regulated hepatic sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-R) genes. In addition, the plasma levels of sPRR were significantly higher in liver PRR KO mice than in controls. In vitro studies in HepG2 cells demonstrated that sPRR treatment upregulated SREBP2, suggesting that sPRR could contribute to hepatic cholesterol biosynthesis. Interestingly, PRR, total cleaved and noncleaved sPRR contents, furin, and Site-1 protease (S1P) were elevated in the adipose tissue of liver PRR KO mice, suggesting that adipose tissue could contribute to the circulating pool of sPRR. Overall, this work supports previous works and opens a new area of investigation concerning the function of sPRR in lipid metabolism and adipose tissue-liver cross talk. NEW & NOTEWORTHY Hepatic PRR and its soluble form, sPRR, contribute to triglyceride and cholesterol homeostasis and hepatic inflammation. Deletion of hepatic PRR decreased triglyceride levels through a PRR-PPARγ-dependent mechanism but increased hepatic cholesterol synthesis through sPRR-medicated upregulation of SREBP-2. Our study highlighted a new paradigm of cross talk between the liver and the adipose tissue involving cholesterol and sPRR.

Objective: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches., Methods: We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and β-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression., Results: SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets., Conclusions: The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Obesity-related hypertension is a major public health concern. We recently demonstrated that plasma levels of the soluble form of the prorenin receptor (sPRR) were elevated in obesity-associated hypertension. Therefore, in the present study, we investigated the contribution of sPRR to blood pressure (BP) elevation in the context of obesity. High fat-fed C57BL/6 male mice were infused with vehicle or sPRR (30 µg/kg per day) via subcutaneously implanted osmotic minipump for 4 weeks. BP parameters were recorded using radiotelemetry devices. Male mice infused with sPRR exhibited higher systolic BP and mean arterial pressure and lower spontaneous baroreflex sensitivity than mice infused with vehicle. To define mechanisms involved in systolic BP elevation, mice were injected with an AT1R (Ang II [angiotensin II] type 1 receptor) antagonist (losartan), a muscarinic receptor antagonist (atropine), a β-adrenergic antagonist (propranolol), and a ganglionic blocker (chlorisondamine). Losartan did not blunt sPRR-induced elevation in systolic BP. Chlorisondamine treatment exacerbated the decrease in mean arterial pressure in male mice infused with sPRR. These results demonstrated that sPRR induced autonomic nervous dysfunction. Interestingly, plasma leptin levels were increased in high fat-fed C57BL/6 male mice infused with sPRR. Overall, our results indicated that sPRR increased systolic BP through an impairment of the baroreflex sensitivity and an increase in the sympathetic tone potentially mediated by leptin in high fat-fed C57BL/6 male mice.

Deletion of the prorenin receptor (PRR) in adipose tissue elevates systolic blood pressure (SBP) and the circulating soluble form of PRR (sPRR) in male mice fed a high-fat (HF) diet. However, sex differences in the contribution of adipose-PRR and sPRR to the regulation of the renin-angiotensin system (RAS) in key organs for blood pressure control are undefined. Therefore, we assessed blood pressure and the systemic and intrarenal RAS status in adipose-PRR knockout (KO) female mice. Blockade of RAS with losartan blunted SBP elevation in HF diet-fed adipose-PRR KO mice. ANG II levels were significantly increased in the renal cortex of HF diet-fed adipose-PRR KO female mice, but not systemically. HF diet-fed adipose-PRR KO mice exhibited higher vasopressin levels, water retention, and lower urine output than wild-type (WT) mice. The results also showed that deletion of adipose-PRR increased circulating sPRR and total hepatic sPRR contents, suggesting the liver as a major source of elevated plasma sPRR in adipose-PRR KO mice. To mimic the elevation of circulating sPRR and define the direct contribution of systemic sPRR to the regulation of the RAS and vasopressin, C57BL/6 female mice fed a standard diet were infused with recombinant sPRR. sPRR infusion increased plasma renin levels, renal and hepatic angiotensinogen expression, and vasopressin. Together, these results demonstrate that the deletion of adipose-PRR induced an elevation of SBP likely mediated by an intrarenal ANG II-dependent mechanism and that sPRR participates in RAS regulation and body fluid homeostasis via its capacity to activate the RAS and increase vasopressin levels. NEW & NOTEWORTHY The elevation of systolic blood pressure appears to be primarily mediated by cortical ANG II in high-fat diet-fed adipose-prorenin receptor knockout female mice. In addition, our data support a role for soluble prorenin receptor in renin-angiotensin system activation and vasopressin regulation.

High-sugar intake and senescence share common deleterious effects, in particular in liver, but combination of these two factors was little studied. Our aims were to examine the effect of a high-sucrose diet in liver of old rats and also the potential benefices of a polyphenol/micronutrient supplementation. Four groups of 22-month-old male rats fed during 5 months with a diet containing either 13 or 62% sucrose, supplemented or not with rutin, vitamin E, A, D, selenium, and zinc were compared. We measured liver macronutrient composition, glycation/oxidative stress, enzyme activities (lipogenesis, β-oxidation, fructokinase), gene expression (enzymes and transcription factors), in vivo protein synthesis rates and plasma parameters. Sucrose induced an increase in plasma and liver lipid content, and a stimulation of liver protein synthesis rates. Gene expression was little changed by sucrose, with lower levels for LXR-α and LXR-β. Polyphenol/micronutrient supplementation tended to limit liver triglyceride infiltration through variations in fatty acid synthase, acyl coA oxidase, and possibly ATP-citrate lyase activities. In conclusion, despite differences in enzymatic regulations, and blunted responses of gene expression, high-sucrose diet was still able to induce a marked increase in liver lipid content in old animals. However, it probably attenuated the positive impact of polyphenol/micronutrients.

Little is still known about brain protein synthesis. In order to increase our knowledge of it, we aimed to modulate brain protein synthesis rates through aging, variations in nutritional state (fed state vs. fasted state), high sucrose diet and micronutrient supplementation. Four groups of 16 month-old male rats were fed for five months with a diet containing either 13% or 62% sucrose (wheat starch was replaced with sucrose), supplemented or not with rutin (5 g kg-1 diet), vitamin E (4×), A (2×), D (5×), selenium (10×) and zinc (+44%) and compared with an adult control group. We measured cerebellum protein synthesis and hippocampus gene expression of antioxidant enzymes, inflammatory cytokines and transcription factors. We showed that cerebellum protein synthesis was unchanged by the nutritional state, decreased during aging (-8%), and restored to the adult level by micronutrient supplementation. Sucrose diet did not change protein synthesis but reduced the protein content. Micronutrient supplementation had no effect in sucrose fed rats. Hippocampus gene expressions were affected by age (an increase of TNF-α), sucrose treatment (an increase of IL-1β and IL-6), and micronutrient supplementation (a decrease of heme oxygenase, catalase, glutathione peroxidase, TNF-α, and Nrf2). We noted that cerebellum protein synthesis and hippocampus TNF-α gene expression were modulated by the same factors: they were affected by aging and micronutrient supplementation and unchanged by feeding and by high sucrose diet.

In our societies, the proportions of elderly people and of obese individuals are increasing. Both factors are associated with high health-related costs. During obesity, many authors suggest that it is a high chronic intake of added sugars (HCIAS) that triggers the shift towards pathology. However, the majority of studies were performed in young subjects and only a few were interested in the interaction with the ageing process. Our purpose was to discuss the metabolic effects of HCIAS, compare with the effects of ageing, and evaluate how deleterious the combined action of HCIAS and ageing could be. This effect of HCIAS seems mediated by fructose, targeting the liver first, which may lead to all subsequent metabolic alterations. The first basic alterations induced by fructose are increased oxidative stress, protein glycation, inflammation, dyslipidaemia and insulin resistance. These alterations are also present during the ageing process, and are closely related to each other, one leading to the other. These basic alterations are also involved in more complex syndromes, which are also favoured by HCIAS, and present during ageing. These include non-alcoholic fatty liver disease, hypertension, neurodegenerative diseases, sarcopenia and osteoporosis. Cumulative effects of ageing and HCIAS have been seldom tested and may not always be strictly additive. Data also suggest that some of the metabolic alterations that are more prevalent during ageing could be related more with nutritional habits than to intrinsic ageing. In conclusion, it is clear that HCIAS interacts with the ageing process, accelerates the accumulation of metabolic alterations, and that it should be avoided.

We aimed to determine the time-course of metabolic changes related to the early onset of insulin resistance (IR), trying to evidence breaking points preceding the appearance of the clinical IR phenotype. The model chosen was the fructose (FRU)-fed rat compared to controls fed with starch. We focused on the hepatic metabolism after 0, 5, 12, 30, or 45 days of FRU intake. The hepatic molecular metabolic changes followed indeed a multistep trajectory rather than a continuous progression. After 5 d of FRU feeding, we observed deep modifications in the hepatic metabolism, driven by the induction of lipogenic genes and important glycogen depletion. Thereafter, a steady-state period between days 12 and 30 was observed, characterized by a switch from carbohydrate to lipid utilization at the hepatic level and increased insulin levels aiming at alleviating lipid accumulation and hyperglycemia, respectively. The FRU-fed animals were only clinically IR at day 45 (altered homeostasis model assessment-estimated insulin resistance and muscle glucose transport). Furthermore, the urine metabolome revealed even earlier metabolic trajectory changes that precede the hepatic alterations. We identified several candidate metabolites linked to the tryptophan-nicotinamide metabolism and the installation of fasting hyperglycemia that suggest a role of this metabolic pathway on the development of the IR phenotype in the FRU-fed rats.

Background: Today, high chronic intake of added sugars is frequent, which leads to inflammation, oxidative stress, and insulin resistance. These 3 factors could reduce meal-induced stimulation of muscle protein synthesis and thus aggravate the age-related loss of muscle mass (sarcopenia)., Objectives: Our aims were to determine if added sugars could accelerate sarcopenia and to assess the capacity of antioxidants and anti-inflammatory agents to prevent this., Methods: For 5 mo, 16-mo-old male rats were starch fed (13% sucrose and 49% wheat starch diet) or sucrose fed (62% sucrose and 0% wheat starch diet) with or without rutin (5 g/kg diet), vitamin E (4 times), vitamin A (2 times), vitamin D (5 times), selenium (10 times), and zinc (+44%) (R) supplementation. We measured the evolution of body composition and inflammation, plasma insulin-like growth factor 1 (IGF-I) concentration and total antioxidant status, insulin sensitivity (oral-glucose-tolerance test), muscle weight, superoxide dismutase activity, glutathione concentration, and in vivo protein synthesis rates., Results: Sucrose-fed rats lost significantly more lean body mass (-8.1% vs. -5.4%, respectively) and retained more fat mass (+0.2% vs. -33%, respectively) than starch-fed rats. Final muscle mass was 11% higher in starch-fed rats than in sucrose-fed rats. Sucrose had little effect on inflammation, oxidative stress, and plasma IGF-I concentration but reduced the insulin sensitivity index (divided by 2). Meal-induced stimulation of muscle protein synthesis was significantly lower in sucrose-fed rats (+7.3%) than in starch-fed rats (+22%). R supplementation slightly but significantly reduced oxidative stress and increased muscle protein concentration (+4%) but did not restore postprandial stimulation of muscle protein synthesis., Conclusions: High chronic sucrose intake accelerates sarcopenia in older male rats through an alteration of postprandial stimulation of muscle protein synthesis. This effect could be explained by a decrease of insulin sensitivity rather than by changes in plasma IGF-I, inflammation, and/or oxidative stress., (© 2015 American Society for Nutrition.)

Our aim was to compare and combine 3 nutritional strategies to slow down the age-related loss of muscle mass in healthy old rats: 1) increase protein intake, which is likely to stimulate muscle protein anabolism; 2) use leucine rich, rapidly digested whey proteins as protein source (whey proteins are recognized as the most effective proteins to stimulate muscle protein anabolism). 3) Supplement animals with a mixture of chamomile extract, vitamin E, vitamin D (reducing inflammation and oxidative stress is also effective to improve muscle anabolism). Such comparisons and combinations were never tested before. Nutritional groups were: casein 12% protein, whey 12% protein, whey 18% protein and each of these groups were supplemented or not with polyphenols/antioxidants. During 6 months, we followed changes of weight, food intake, inflammation (plasma fibrinogen and alpha-2-macroglobulin) and body composition (DXA). After 6 months, we measured muscle mass, in vivo and ex-vivo fed and post-absorptive muscle protein synthesis, ex-vivo muscle proteolysis, and oxidative stress parameters (liver and muscle glutathione, SOD and total antioxidant activities, muscle carbonyls and TBARS). We showed that although micronutrient supplementation reduced inflammation and oxidative stress, the only factor that significantly reduced the loss of lean body mass was the increase in whey protein intake, with no detectable effect on muscle protein synthesis, and a tendency to reduce muscle proteolysis. We conclude that in healthy rats, increasing protein intake is an effective way to delay sarcopenia.

Two monoclonal antibodies have been raised against the native form of the potent cardiotoxin isolated from the venom of Naja nigricollis. The toxic action to mice as well as the depolarizing effect on muscle fibres in culture of the cardiotoxin are neutralized by the two immunoglobulins. Binding studies revealed that the radiolabelled toxin has a high affinity for both antibodies, the equilibrium dissociation constant values being equal to 0.2 and 0.4 nM. The epitopes that are recognized by the antibodies have been localized on the basis of competition experiments between the labelled toxin and a series of variants or a Trp-11 modified derivative, toward both antibodies. The data obtained indicate that the antibodies bind at topographically different antigenic sites. Knowing that the toxin is a single polypeptide chain folded in a structure that contains three adjacent loops emerging from a small globular region, it appears that one of the two antibodies binds on loop I, at a site which involves Trp-11 whereas the other binds at a site which involves one or both of loops II and III. Possible mechanisms of neutralization of the toxin by the antibodies are discussed.

Examination of 76 homologous neurotoxin sequences suggested that the "toxic" domain of these compounds consists of twelve highly conserved residues. Five of these, namely Lys-27, Trp-29, Asp-31, Arg-33 and Glu-38, together with a variant residue at position 36 are organized into a pattern which resembles that of d-tubocurarine. Two lines of experimental evidence are in agreement with the proposed topology of the "toxic" site in Naja nigricollis toxin alpha--Three highly conserved residues (Lys-27, Trp-29 and Lys-47) have been modified individually in toxin alpha. These modifications induce a decrease in binding affinity of toxin alpha for its target, the nicotinic acetylcholine receptor. In contrast, modifications of three residues (Leu-1, Lys-15 and Lys-51) excluded from the "toxic" domain, do not alter the binding properties of toxin alpha.--Five toxin derivatives carrying a nitroxide group at residues 1, 15, 27, 47 or 51 have been prepared. ESR spectra have been recorded for each derivative in both the free state and bound to the receptor. Mobility of the probes of the residues excluded from the "toxic" site is not altered upon receptor binding. In contrast mobility of the nitroxide of the presumed "toxic" Lys-47 becomes markedly reduced after toxin receptor complex formation. Lys-27 nitroxide is immobilized in both the free and bound state. The antigenic structure of N. nigricollis toxin alpha has been partially clarified using two different approaches. --Fifteen antigenically important residues of toxin alpha have been identified by analyzing cross-reactions between toxin alpha and eleven homologous neurotoxins, using polyclonal antibodies.--- One monoclonal antibody (M alpha 1) specific for toxin alpha has been prepared. Competition experiments, made with (3H) toxin alpha, six mono modified toxin derivatives or alpha three homologous neurotoxins, showed that the binding site of (M alpha 1) comprises the N-terminal group, Lys-15, Pro-18 and probably Thr-16. This site is topographically different from the "toxic" domain. (M alpha 1) inhibits the toxicity of toxin alpha under both in vivo and in vitro conditions. In addition, (M alpha 1) is capable of "removing" toxin molecules bound to the receptor, allowing a rapid recovery of the functional properties of the receptor.

Snake curaremimetic toxins are known to bind to the nicotinic acetylcholine receptor (AcChoR) [Changeux et al. (1970) Proc. Natl Acad. Sci. USA, 67, 1241-1247], thus blocking neuromuscular transmission, and producing respiratory failure in mammals. In the present paper we show that the toxic effects of Naja nigricollis toxin alpha to mammals can be efficiently reversed by toxin-alpha-specific antibodies. In vivo we observed that return to normal breathing in toxin-alpha-intoxicated and ventilated rats was 12 times faster after injection of specific antiserum or monoclonal antibody (M-alpha 1) as compared with control animals. Ex vivo we observed that return to normal contraction of a toxin-alpha-blocked phrenic nerve-hemidiaphragm preparation was 14 times more rapid after treatment with specific antiserum than after washings. In vitro we observed that antibodies accelerated the reversal of binding of [3H]toxin alpha to AcChoR prepared from rat diaphragm. The observation made in vitro furthermore indicates that antibodies are capable of destabilizing the [3H]toxin-AcChoR complex. A similar destabilization phenomenon occurs also in vivo, as inferred from measurements of receptor occupancy by [3H]toxin alpha in diaphragm of anaesthetized rats in the presence or absence of antibodies. The property of antibodies to reverse neurotoxin binding to AcChoR may be considered as a critical test for evaluation of the quality of a neurotoxin-specific antisera.