Fecal microbiota transplantation from female donors restores gut permeability and reduces liver injury and inflammation in middle-aged male mice exposed to alcohol.

Background: Alcohol misuse, binge drinking pattern, and gender-specific effects in the middle-aged population has been clearly underestimated. In the present study, we focused on understanding gender-specific effects of alcohol exposure on the gut-liver axis and the role of gut microbiota in modulating gender-specific responses to alcohol consumption. Methods: Fifty-two-week-old female and male C57BL/6 mice were fasted for 12 h, and then administered a single oral dose of ethanol (EtOH) (6 g/kg). Controls were given a single dose of PBS. Animals were sacrificed 8 h later. Alternatively, fecal microbiota transplantation (FMT) was performed in 52-week-old male mice from female donors of the same age. Permeability of the large intestine (colon), gut microbiota, liver injury, and inflammation was thoroughly evaluated in all groups. Results: Middle-aged male mice exposed to EtOH showed a significant increase in gut permeability in the large intestine, evaluated by FITC-dextran assay and ZO-1, OCCLUDIN and MUCIN-2 immuno-staining, compared to PBS-treated animals, whilst female mice of the same age also increased their gut permeability, but displayed a partially maintained intestinal barrier integrity. Moreover, there was a significant up-regulation of TLRs and markers of hepatocellular injury, cell death (AST, TUNEL-positive cells) and lipid accumulation (ORO) in male mice after EtOH exposure. Interestingly, FMT from female donors to male mice reduced gut leakiness, modified gut microbiota composition, ameliorated liver injury and inflammation, TLR activation and the senescence phenotype of middle-aged mice. Conclusion: Our findings highlighted the relevance of gender in middle-aged individuals who are exposed to alcohol in the gut-liver axis. Moreover, our study revealed that gender-specific microbiota transplantation might be a plausible therapy in the management of alcohol-related disorders during aging.

Central and Peripheral Inflammation: A Common Factor Causing Addictive and Neurological Disorders and Aging-Related Pathologies.

Many diseases and degenerative processes affecting the nervous system and peripheral organs trigger the activation of inflammatory cascades. Inflammation can be triggered by different environmental conditions or risk factors, including drug and food addiction, stress, and aging, among others. Several pieces of evidence show that the modern lifestyle and, more recently, the confinement associated with the COVID-19 pandemic have contributed to increasing the incidence of addictive and neuropsychiatric disorders, plus cardiometabolic diseases. Here, we gather evidence on how some of these risk factors are implicated in activating central and peripheral inflammation contributing to some neuropathologies and behaviors associated with poor health. We discuss the current understanding of the cellular and molecular mechanisms involved in the generation of inflammation and how these processes occur in different cells and tissues to promote ill health and diseases. Concomitantly, we discuss how some pathology-associated and addictive behaviors contribute to worsening these inflammation mechanisms, leading to a vicious cycle that promotes disease progression. Finally, we list some drugs targeting inflammation-related pathways that may have beneficial effects on the pathological processes associated with addictive, mental, and cardiometabolic illnesses.

Chronic Exposure to High Fat Diet Affects the Synaptic Transmission That Regulates the Dopamine Release in the Nucleus Accumbens of Adolescent Male Rats.

Obesity is a pandemic caused by many factors, including a chronic excess in hypercaloric and high-palatable food intake. In addition, the global prevalence of obesity has increased in all age categories, such as children, adolescents, and adults. However, at the neurobiological level, how neural circuits regulate the hedonic consumption of food intake and how the reward circuit is modified under hypercaloric diet consumption are still being unraveled. We aimed to determine the molecular and functional changes of dopaminergic and glutamatergic modulation of nucleus accumbens (NAcc) in male rats exposed to chronic consumption of a high-fat diet (HFD). Male Sprague-Dawley rats were fed a chow diet or HFD from postnatal day (PND) 21 to 62, increasing obesity markers. In addition, in HFD rats, the frequency but not amplitude of the spontaneous excitatory postsynaptic current is increased in NAcc medium spiny neurons (MSNs). Moreover, only MSNs expressing dopamine (DA) receptor type 2 (D2) increase the amplitude and glutamate release in response to amphetamine, downregulating the indirect pathway. Furthermore, NAcc gene expression of inflammasome components is increased by chronic exposure to HFD. At the neurochemical level, DOPAC content and tonic dopamine (DA) release are reduced in NAcc, while phasic DA release is increased in HFD-fed rats. In conclusion, our model of childhood and adolescent obesity functionally affects the NAcc, a brain nucleus involved in the hedonic control of feeding, which might trigger addictive-like behaviors for obesogenic foods and, through positive feedback, maintain the obese phenotype.

Association of Polyphenols Consumption with Risk for Gestational Diabetes Mellitus and Preeclampsia: A Systematic Review and Meta-Analysis.

Gestational Diabetes Mellitus (GDM) and preeclampsia (PE) affects 6-25% of pregnancies and are characterized by an imbalance in natural prooxidant/antioxidant mechanisms. Due to their antioxidant and anti-inflammatory properties, polyphenols consumption during the pregnancy might exert positive effects by preventing GDM and PE development. However, this association remains inconclusive. This systematic review and metanalysis is aimed to analyze the association between polyphenol-rich food consumption during pregnancy and the risk of GDM and PE. A systematic search in MEDLINE, EMBASE, and Web of Science (Clarivate Analytics, London, United Kingdom) for articles dated between 1 January 1980 and July 2022 was undertaken to identify randomized controlled trials and observational studies evaluating polyphenol-rich food consumption and the risk of GDM and PE. The Newcastle-Ottawa Scale was used to evaluate the quality of these included studies. Twelve studies were included, of which eight articles evaluated GDM and four studied PE. A total of 3785 women presented with GDM (2.33%). No association between polyphenol consumption and GDM was found (ES = 0.85, 95% CI 0.71-1.01). When total polyphenol intake was considered, a lower likelihood to develop GDM was noted (ES = 0.78, 95% CI 0.69-0.89). Furthermore, polyphenol consumption was not associated with PE development (ES = 0.90, 95% CI 0.57-1.41). In conclusion, for both outcomes, pooled analyses showed no association with polyphenol-rich food consumption during pregnancy. Therefore, association of polyphenol intake with a decreased risk of GDM and PE remains inconclusive.

Activation of the NLRP3 Inflammasome Increases the IL-1ß Level and Decreases GLUT4 Translocation in Skeletal Muscle during Insulin Resistance.

Low-grade chronic inflammation plays a pivotal role in the pathogenesis of insulin resistance (IR), and skeletal muscle has a central role in this condition. NLRP3 inflammasome activation pathways promote low-grade chronic inflammation in several tissues. However, a direct link between IR and NLRP3 inflammasome activation in skeletal muscle has not been reported. Here, we evaluated the NLRP3 inflammasome components and their role in GLUT4 translocation impairment in skeletal muscle during IR. Male C57BL/6J mice were fed with a normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. The protein levels of NLRP3, ASC, caspase-1, gasdermin-D (GSDMD), and interleukin (IL)-1ß were measured in both homogenized and isolated fibers from the flexor digitorum brevis (FDB) or soleus muscle. GLUT4 translocation was determined through GLUT4myc-eGFP electroporation of the FBD muscle. Our results, obtained using immunofluorescence, showed that adult skeletal muscle expresses the inflammasome components. In the FDB and soleus muscles, homogenates from HFD-fed mice, we found increased protein levels of NLRP3 and ASC, higher activation of caspase-1, and elevated IL-1ß in its mature form, compared to NCD-fed mice. Moreover, GSDMD, a protein that mediates IL-1ß secretion, was found to be increased in HFD-fed-mice muscles. Interestingly, MCC950, a specific pharmacological NLRP3 inflammasome inhibitor, promoted GLUT4 translocation in fibers isolated from the FDB muscle of NCD- and HFD-fed mice. In conclusion, we found increased NLRP3 inflammasome components in adult skeletal muscle of obese insulin-resistant animals, which might contribute to the low-grade chronic metabolic inflammation of skeletal muscle and IR development.

Pannexin-1 and CaV1.1 show reciprocal interaction during excitation-contraction and excitation-transcription coupling in skeletal muscle.

One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltage-sensing protein CaV1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processes. Specifically, knockdown of CaV1.1 produces chronically elevated extracellular ATP concentrations at rest, consistent with disruption of the normal control of Panx1 activity. Conversely, knockdown of Panx1 affects not only activation of transcription but also CaV1.1 function on the control of muscle fiber contraction. Altogether, our results establish the presence of bidirectional functional regulations between the molecular machineries involved in the control of contraction and transcription induced by membrane depolarization of adult muscle fibers. Our results are important for an integrative understanding of skeletal muscle function and may impact our understanding of several neuromuscular diseases.

NLRP3 Inflammasome: Potential Role in Obesity Related Low-Grade Inflammation and Insulin Resistance in Skeletal Muscle.

Among multiple mechanisms, low-grade inflammation is critical for the development of insulin resistance as a feature of type 2 diabetes. The nucleotide-binding oligomerization domain-like receptor family (NOD-like) pyrin domain containing 3 (NLRP3) inflammasome has been linked to the development of insulin resistance in various tissues; however, its role in the development of insulin resistance in the skeletal muscle has not been explored in depth. Currently, there is limited evidence that supports the pathological role of NLRP3 inflammasome activation in glucose handling in the skeletal muscle of obese individuals. Here, we have centered our focus on insulin signaling in skeletal muscle, which is the main site of postprandial glucose disposal in humans. We discuss the current evidence showing that the NLRP3 inflammasome disturbs glucose homeostasis. We also review how NLRP3-associated interleukin and its gasdermin D-mediated efflux could affect insulin-dependent intracellular pathways. Finally, we address pharmacological NLRP3 inhibitors that may have a therapeutical use in obesity-related metabolic alterations.

High extracellular ATP levels released through pannexin-1 channels mediate inflammation and insulin resistance in skeletal muscle fibres of diet-induced obese mice.

AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1ß and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/µg RNA) compared with controls (32 ± 10 pmol ATP/µg RNA). ATP release in obese mice fibres was reduced by application of 100 µmol/l oleamide (OLE) and 5 µmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/µg RNA vs 252 ± 37 pmol ATP/µg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 µmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/µg RNA vs 222 ± 28 pmol ATP/µg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

Metformin during Pregnancy: Effects on Offspring Development and Metabolic Function.

Maternal obesity during pregnancy and gestational diabetes mellitus (GDM) are both associated with of several postnatal diseases in the offspring, including obesity, early onset hypertension, diabetes mellitus, and reproductive alterations. Metformin is an oral drug that is being evaluated to treat GDM, obesity-associated insulin resistance, and polycystic ovary syndrome (PCOS) during pregnancy. The beneficial effects of metformin on glycemia and pregnancy outcomes place it as a good alternative for its use during pregnancy. In this line of thought, improving the metabolic status of the pregnant mother by using metformin should avoid the consequences of insulin resistance on the offspring's fetal and postnatal development. However, some human and animal studies have shown that metformin during pregnancy could amplify these alterations and be associated with excessive postnatal weight gain and obesity. In this minireview, we discuss not only the clinical and experimental evidence that supports the benefits of using metformin during pregnancy but also the evidence showing a possible negative impact of this drug on the offspring's development.

Physalis peruviana L. Pulp Prevents Liver Inflammation and Insulin Resistance in Skeletal Muscles of Diet-Induced Obese Mice.

A chronic high-fat diet (HFD) produces obesity, leading to pathological consequences in the liver and skeletal muscle. The fat in the liver leads to accumulation of a large number of intrahepatic lipid droplets (LD), which are susceptible to oxidation. Obesity also affects skeletal muscle, increasing LD and producing insulin signaling impairment. Physalis peruviana L. (PP) (Solanaceae) is rich in peruvioses and has high antioxidant activity. We assessed the ability of PP to enhance insulin-dependent glucose uptake in skeletal muscle and the capacity to prevent both inflammation and lipoperoxidation in the liver of diet-induced obese mice. Male C57BL/6J mice were divided into groups and fed for eight weeks: control diet (C; 10% fat, 20% protein, 70% carbohydrates); C + PP (300 mg/kg/day); HFD (60% fat, 20% protein, 20% carbohydrates); and HFD + PP. Results suggest that PP reduces the intracellular lipoperoxidation level and the size of LD in both isolated hepatocytes and skeletal muscle fibers. PP also promotes insulin-dependent skeletal muscle glucose uptake. In conclusion, daily consumption of 300 mg/kg of fresh pulp of PP could be a novel strategy to prevent the hepatic lipoperoxidation and insulin resistance induced by obesity.

Testosterone signals through mTOR and androgen receptor to induce muscle hypertrophy.

PURPOSE: The anabolic hormone testosterone induces muscle hypertrophy, but the intracellular mechanisms involved are poorly known. We addressed the question whether signal transduction pathways other than the androgen receptor (AR) are necessary to elicit hypertrophy in skeletal muscle myotubes. METHODS: Cultured rat skeletal muscle myotubes were preincubated with inhibitors for ERK1/2 (PD98059), PI3K/Akt (LY294002 and Akt inhibitor VIII) or mTOR/S6K1 (rapamycin), and then stimulated with 100 nM testosterone. The expression of α-actin and the phosphorylation levels of ERK1/2, Akt and S6K1 (a downstream target for mTOR) were measured by Western blot. mRNA levels were evaluated by real time RT-PCR. Myotube size and sarcomerization were determined by confocal microscopy. Inhibition of AR was assessed by bicalutamide. RESULTS: Testosterone-induced myotube hypertrophy was assessed as increased myotube cross-sectional area (CSA) and increased α-actin mRNA and α-actin protein levels, with no changes in mRNA expression of atrogenes (MAFbx and MuRF-1). Morphological development of myotube sarcomeres was evident in testosterone-stimulated myotubes. Known hypertrophy signaling pathways were studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after testosterone stimulus at 5 and 15 min, respectively. S6K1 was phosphorylated at 60 min. This response was abolished by PI3K/Akt and mTOR inhibition but not by ERK1/2 inhibition. Similarly, the CSA increase at 12 h was abolished by inhibitors of the PI3K/Akt pathway as well as by AR inhibition. CONCLUSIONS: These results suggest a crosstalk between pathways involving fast intracellular signaling and the AR to explain testosterone-induced skeletal muscle hypertrophy.

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity.

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P3 production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P3 was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.

Electrical stimulation induces calcium-dependent up-regulation of neuregulin-1ß in dystrophic skeletal muscle cell lines.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by reduced or no expression of dystrophin, a cytoskeletal protein that provides structural integrity to muscle fibres. A promising pharmacological treatment for DMD aims to increase the level of a structural dystrophin homolog called utrophin. Neuregulin-1 (NRG-1), a growth factor that potentiates myogenesis, induces utrophin expression in skeletal muscle cells. Microarray analysis of total gene expression allowed us to determine that neuregulin-1ß (NRG-1ß) is one of 150 differentially expressed genes in electrically stimulated (400 pulses, 1 ms, 45 Hz) dystrophic human skeletal muscle cells (RCDMD). We investigated the effect of depolarization, and the involvement of intracellular Ca(2+) and PKC isoforms on NRG-1ß expression in dystrophic myotubes. Electrical stimulation of RCDMD increased NRG-1ß mRNA and protein levels, and mRNA enhancement was abolished by actinomycin D. NRG-1ß transcription was inhibited by BAPTA-AM, an intracellular Ca(2+) chelator, and by inhibitors of IP(3)-dependent slow Ca(2+) transients, like 2-APB, Ly 294002 and Xestospongin B. Ryanodine, a fast Ca(2+) signal inhibitor, had no effect on electrical stimulation-induced expression. BIM VI (general inhibitor of PKC isoforms) and Gö 6976 (specific inhibitor of Ca(2+)-dependent PKC isoforms) abolished NRG-1ß mRNA induction. Our results suggest that depolarization induced slow Ca(2+) signals stimulate NRG-1ß transcription in RCDMD cells, and that Ca(2+)-dependent PKC isoforms are involved in this process. Based on utrophin's ability to partially compensate dystrophin disfunction, knowledge on the mechanism involved on NRG-1 up-regulation could be important for new therapeutic strategies design.

IP(3)-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers.

Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP(3)Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5-7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP(3)R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10-20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP(3)R isoforms were present in adult muscle. IP(3)R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP(3)R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.

Membrane depolarization induces calcium-dependent upregulation of Hsp70 and Hmox-1 in skeletal muscle cells.

Heat shock proteins (HSPs) are a conserved family of cytoprotective polypeptides, synthesized by cells in response to stress. Hsp70 and heme oxygenase 1 (Hmox-1) are induced by a variety of cellular stressors in skeletal muscle, playing a role in long-term adaptations and muscle fibers regeneration. Though HSPs expression after exercise has been intensely investigated, the molecular mechanisms concerning Hsp70 and Hmox-1 induction are poorly understood. The aim of this work was to investigate the involvement of calcium in Hsp70 and Hmox-1 expression upon depolarization of skeletal muscle cells. We observed that depolarization of myotubes increased both mRNA levels and protein expression for Hsp70 and Hmox-1. Stimulation in the presence of intracellular calcium chelator BAPTA-AM resulted in a complete inhibition of Hsp70-induced expression. It is known that inositol-1,4,5-trisphophate (IP(3))-mediated slow Ca(2+) transients, evoked by membrane depolarization, are involved in the regulation of gene expression. Here we demonstrated that inhibition of IP(3)-dependent calcium signals decreased both Hsp70 mRNA induction and Hsp70 and Hmox-1 protein expression. Inhibitors of calcium-dependent protein kinase C also abolished Hsp70 mRNA induction. Our results provide evidence that membrane depolarization increases Hsp70 and Hmox-1 expression in cultured skeletal muscle cells, which the effect is critically dependent on Ca(2+) released from IP(3)-sensitive intracellular stores and that it involves PKC as an upstream effector in Hsp70 mRNA-induced expression.