Codon-optimized FAM132b gene therapy prevents dietary obesity by blockading adrenergic response and insulin action.

BACKGROUND: FAM132b (myonectin) has been identified as a muscle-derived myokine with exercise and has hormone activity in circulation to regulate iron homeostasis and lipid metabolism via unknown receptors. Here, we aim to explore the potential of adeno-associated virus to deliver FAM132b in vivo to develop a gene therapy against obesity. METHODS: Adeno-associated virus AAV9 were engineered to induce overexpression of FAM132b with two mutations, A136T and P159A. Then, AAV9 was delivered into high-fat diet mice through tail vein, and glucose homeostasis and obesity development of mice were observed. Methods of structural biology were used to predict the action site or receptor of the FAM132b mutant. RESULTS: Treatment of high-fat diet-fed mice with AAV9 improved glucose intolerance and insulin resistance, and resulted in reductions in body weight, fat depot, and adipocyte size. Codon-optimized FAM132b (coFAM132b) reduced the glycemic response to epinephrine (EPI) in the whole body and increased the lipolytic response to EPI in adipose tissues. However, FAM132b knockdown by shRNA significantly increased the glycemic response to EPI in vivo and reduced adipocyte response to EPI and adipose tissue browning. Structural analysis predicted that the FAM132b mutant with A136T and P159A may form a weak bond with ß2 adrenergic receptor (ADRB2) and may have more affinity for insulin and insulin-receptor complexes. CONCLUSIONS: Our study underscores the potential of FAM132b gene therapy with codon optimization to treat obesity by modulating the adrenergic response and insulin action. Both structural biological analysis and in vivo experiments suggest that the adrenergic response and insulin action are most likely blockaded by FAM132b mutants.

The Role of Fgf9 in the Antidepressant Effects of Exercise and Fluoxetine in Chronic Unpredictable Mild Stress Mice.

OBJECTIVE: The neurotrophic hypothesis of depression posits that stress and depression decrease neurotrophic factor expression in brain, whereas antidepressants and exercise can contribute to the blockade of stress effects and produce antidepressant effects. Fibroblast growth factor 9 (FGF9), a member of the fibroblast growth factor (FGF) family, has been reported to be dysregulated in depression. The present study aimed to determine whether and how Fgf9 mediates the antidepressant effects of fluoxetine and exercise in chronic unpredictable mild stress (CUMS) mice. METHODS: Male C57BL/6 mice were exposed to CUMS for 7 weeks. From the fourth week, CUMS-exposed mice were subjected to fluoxetine treatment or swimming exercise for 4 weeks. Forced swim test, tail suspension test, and hole-board test were used to assess behaviors of mice. Real-time polymerase chain reaction was used to examine hippocampal messenger RNA levels of Fgf9, Fgf2, FgfR1, FgfR2, and FgfR3. Western blotting was used to examine the protein levels of Fgf9, protein kinase B (Akt), and phosphorylation of Akt at Ser473 in mouse hippocampus. RESULTS: Our results demonstrated that CUMS induced depression-like behaviors, which were reversed by fluoxetine treatment and swimming exercise. Moreover, we found that CUMS resulted in a dysregulation of Fgf9, Fgf2, and FgfR2 expression, whereas fluoxetine and swimming restored the FGF expression in CUMS-exposed mice. An analysis of the proteins suggests that the antidepressant effects of fluoxetine and exercise in CUMS-exposed mice were associated with ameliorated Fgf9/Akt signaling. CONCLUSIONS: Our findings have demonstrated that swimming exercise mimics the antidepressant effects of fluoxetine by regulating Fgf9 in CUMS-exposed mice, which may offer new mechanism-based therapeutic targets for depression.

Interactions between Endoplasmic Reticulum Stress and Autophagy: Implications for Apoptosis and Neuroplasticity-Related Proteins in Palmitic Acid-Treated Prefrontal Cells.

Lipotoxicity of palmitic acid (PA) or high-fat diets has been reported to increase endoplasmic reticulum (ER) stress and autophagy in peripheral tissue as well as apoptotic cell death. It also can lead to an AD-like pathological pattern. However, it has been unknown that PA-induced ER stress and autophagy are involved in the regulation of neuroplastic abnormalities. Here, we investigated the roles of ER stress and autophagy in apoptosis and neuroplasticity-related protein expression in PA-treated prefrontal cells. Prefrontal cells dissected from newborn Sprague-Dawley rats were treated with PA compound with ER stress inhibitor 4-phenylbutyric acid (4-PBA) and autophagy inhibitor 3-methyladenine (3-MA) or PA alone. PA promoted ER stress and autophagy and also cause apoptosis as well as a decline in the expression of neuroplasticity-related proteins. Inhibition of ER stress decreased the expressions of neuroplasticity-related proteins and reduced autophagy activation and apoptosis in PA-treated prefrontal cells. Inhibition of autophagy exacerbated apoptosis and enhanced ER stress in PA-treated prefrontal cells. The present study illustrated that both ER stress and autophagy could be involved in apoptosis and decreased neuroplasticity-related proteins, and the interaction between ER stress and autophagy may play a critical role in apoptosis in PA-treated prefrontal cells. Our results provide new insights into the molecular mechanisms in vitro of lipotoxicity in obesity-related cognitive dysfunction.

The roles of GRP81 as a metabolic sensor and inflammatory mediator.

GPR81 (also named as HCA1) is a member of a subfamily of orphan G-protein coupled receptors (GPCRs), coupled to Gi -type G proteins. GPR81 was discovered in 2001 and identified as the only known endogenous receptor of lactate under physiological conditions in 2008, which opened a new field of research on how lactate may act as a signal molecule along with the GPR81 expression in the roles of metabolic process and inflammatory response. Recent studies showed that the physiological functions of GPR81 include lipid metabolism in adipose tissues, metabolic excitability in the brain, cellular development, and inflammatory response modulation. These findings may reveal a novel therapeutic strategy to treat clinical, metabolic, and inflammatory diseases. This article will summarize past research on GPR81, including its characteristics of distribution and expression, functional residues, pharmacological, and physiological agonists, involvement in signal transduction, and pharmacological applications.

[Aerobic exercise reduces the expression of pyroptosis-related proteins and inflammatory factors in hippocampus of mice with insulin resistance].

The aim of the present study was to observe the expression of pyroptosis- and inflammation-related proteins in the hippocampus of mice with insulin resistance (IR) after aerobic exercise, and to explore the possible mechanism of exercise to improve IR. C57BL/6J male mice of 6 weeks old were randomly fed with normal diet (n = 12) and high-fat diet (HFD) (n = 26) for 12 weeks respectively. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to determine whether IR occurred in HFD mice. Then the mice were randomly divided into control group (n = 12), IR group (n = 10) and IR + aerobic exercise group (AE, n = 10). Mice in AE group performed a 12-week progressive speed treadmill training after being adapted to the treadmill for one week. After the intervention, the expression of pyroptosis- and inflammation-related proteins in hippocampus was detected by Western blot. The results showed that compared with control group, NFκB, Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), pyroptosis-related proteins like pro-Caspase-1, gasdermin D (GSDMD), GSDMD-N, and inflammatory factors IL-1ß, IL-18 were significantly increased. The inflammasome-related protein NIMA-related kinase 7 (NEK7) and pyroptosis-related protein Caspase-1 showed an increasing trend, but there was no significant difference. Compared with the IR group, progressive speed treadmill training significantly reduced the expression of NFκB, NLRP3, NEK7, ASC, pro-Caspase-1, GSDMD, GSDMD-N, IL-1ß, and IL-18 in the hippocampus of mice with IR. These results suggested 12-week progressive speed treadmill training can significantly reduce the expression of pyroptosis-related proteins and inflammatory factors in the hippocampus of mice with IR, and inhibit pyroptosis.

Leptin receptor knockout-induced depression-like behaviors and attenuated antidepressant effects of exercise are associated with STAT3/SOCS3 signaling.

Relatively little has been known about pathophysiological mechanisms contributing to the development of neuropsychiatric symptoms in the context of metabolic syndrome. Impaired leptin signaling activation in db/db mice has been proposed as a potential link between behavioral and metabolic disorders. Our previous studies have shown that exercise has the beneficial effects on a depression-like and insulin-resistant state in mice. The present study aimed to determine whether and how leptin receptor knockout (db/db) induces depression-like behaviors, and to identify the antidepressant effects of swimming exercise in db/db mice. Our results support the validity of db/db mice as an animal model to study depression with metabolic abnormalities, but fail to confirm the improvement of exercise on depression. LepRb knockout-induced depression-like behaviors are associated with STAT3/SOCS3 signaling but independent of IKKß/NFκB signaling. Our findings suggest the potential importance of LepRb as an exercise-regulated target for depression, also representing a new target underlying treatment-resistant depression.

The potential mechanisms of lactate in mediating exercise-enhanced cognitive function: a dual role as an energy supply substrate and a signaling molecule.

Lactate has previously been considered a metabolic waste and is mainly involved in exercise-induced fatigue. However, recent studies have found that lactate may be a mediator of the beneficial effects of exercise on brain health. Lactate plays a dual role as an energy supply substrate and a signaling molecule in this process. On the one hand, astrocytes can uptake circulating glucose or degrade glycogen for glycolysis to produce lactate, which is released into the extracellular space. Neurons can uptake extracellular lactate as an important supplement to their energy metabolism substrates, to meet the demand for large amounts of energy when synaptic activity is enhanced. Thus, synaptic activity and energy transfer show tight metabolic coupling. On the other hand, lactate acts as a signaling molecule to activate downstream signaling transduction pathways by specific receptors, inducing the expression of immediate early genes and cerebral angiogenesis. Moderate to high-intensity exercise not only increases lactate production and accumulation in muscle and blood but also promotes the uptake of skeletal muscle-derived lactate by the brain and enhances aerobic glycolysis to increase brain-derived lactate production. Furthermore, exercise regulates the expression or activity of transporters and enzymes involved in the astrocyte-neuron lactate shuttle to maintain the efficiency of this process; exercise also activates lactate receptor HCAR1, thus affecting brain plasticity. Rethinking the role of lactate in cognitive function and the regulatory effect of exercise is the main focus and highlights of the review. This may enrich the theoretical basis of lactate-related to promote brain health during exercise, and provide new perspectives for promoting a healthy aging strategy.

Lactate Is Answerable for Brain Function and Treating Brain Diseases: Energy Substrates and Signal Molecule.

Research to date has provided novel insights into lactate's positive role in multiple brain functions and several brain diseases. Although notable controversies and discrepancies remain, the neurobiological role and the metabolic mechanisms of brain lactate have now been described. A theoretical framework on the relevance between lactate and brain function and brain diseases is presented. This review begins with the source and route of lactate formation in the brain and food; goes on to uncover the regulatory effect of lactate on brain function; and progresses to gathering the application and concentration variation of lactate in several brain diseases (diabetic encephalopathy, Alzheimer's disease, stroke, traumatic brain injury, and epilepsy) treatment. Finally, the dual role of lactate in the brain is discussed. This review highlights the biological effect of lactate, especially L-lactate, in brain function and disease studies and amplifies our understanding of past research.

Elevated Lactate by High-Intensity Interval Training Regulates the Hippocampal BDNF Expression and the Mitochondrial Quality Control System.

High-intensity interval training (HIIT) is reported to be beneficial to brain-derived neurotrophic factor (BDNF) biosynthesis. A key element in this may be the existence of lactate, the most obvious metabolic product of exercise. In vivo, this study investigated the effects of a 6-week HIIT on the peripheral and central lactate changes, mitochondrial quality control system, mitochondrial function and BDNF expression in mouse hippocampus. In vitro, primary cultured mice hippocampal cells were used to investigate the role and the underlying mechanisms of lactate in promoting mitochondrial function during HIIT. In vivo studies, we firstly reported that HIIT can potentiate mitochondrial function [boost some of the mitochondrial oxidative phosphorylation (OXPHOS) genes expression and ATP production], stimulate BDNF expression in mouse hippocampus along with regulating the mitochondrial quality control system in terms of promoting mitochondrial fusion and biogenesis, and suppressing mitochondrial fission. In parallel to this, the peripheral and central lactate levels elevated immediately after the training. In vitro study, our results revealed that lactate was in charge of regulating mitochondrial quality control system for mitochondrial function and thus may contribute to BDNF expression. In conclusion, our study provided the mitochondrial mechanisms of HIIT enhancing brain function, and that lactate itself can mediate the HIIT effect on mitochondrial quality control system in the hippocampus.

[Effects of resistance exercise on pyroptosis-related proteins in hippocampus of insulin resistant mice].

Objective: To investigate the changes of pyroptosis-related proteins in the hippocampus of insulin-resistant mice and the regulation of resistance training on pyroptosis-related proteins. Methods: Six-week-old male C57BL/6J mice were randomly divided into control group (C, n=12) and high-fat diet group (HFD, n=26) for normal or high-fat diet for 12 weeks. Subsequently, according to the results of glucose tolerance test (GTT) and insulin tolerance test (ITT), the rats fed with high-fat diet were divided into insulin resistance group (IR, n=10) and resistance exercise group (RT, n=10) as well as to maintain high-fat diet. At the same time, mice in the RT group were subjected to resistance training. After 12 weeks, all mice were sacrificed after anesthesia, brain was removed and hippocampus was exfoliated, and the expressions of pyroptosis-related proteins were detected by Western blot. Results: Compared with the C group, NF-κB, the NLRP3 inflammasome proteins, their downstream pyroptosis-related proteins GSDMD-N and GSDMD as well as inflammation factors IL-1ß and IL-18 in hippocampus of IR group were significantly increased (P＜0.05), and the expression levels of SIRT1 and p-AMPK protein were significantly decreased (P＜0.05). Compared with the IR group, NF-κB, the NLRP3 inflammasome proteins, their downstream pyroptosis-related proteins GSDMD-N and GSDMD as well as inflammation factors IL-1ß and IL-18 in hippocampus of RT group were significantly decreased (P＜0.05), and the expression levels of SIRT1 and p-AMPK protein were significantly increased (P＜0.01). Conclusion: NLRP3 inflammasome in the hippocampus of insulin-resistant mice is activated, which mediates pyroptosis in the hippocampus. Twelve weeks of resistance training can effectively inhibit the activation of NLRP3 inflammasome and decrease pyroptosis and improve inflammation in the hippocampus.

Swimming exercise reverses CUMS-induced changes in depression-like behaviors and hippocampal plasticity-related proteins.

BACKGROUND: Stress-induced failed resilience of brain plasticity can contribute to the onset and recurrence of depression. Chronic stress has been reported to open windows of epigenetic plasticity in hippocampus. However, how hippocampal plasticity underlies depression-like behaviors and how it adapts in response to stress has not been addressed. The present study aimed to investigate the signaling mechanisms of CUMS affecting hippocampal plasticity-related proteins expression and the regulation of swimming exercise in mice. METHODS: Male C57BL/6 mice were subjected to chronic unpredictable mild stress (CUMS) for 7 weeks. From the 4th week, CUMS mice were trained in a moderate swimming program for a total of 4 weeks. A videocomputerized tracking system was used to record behaviors of animals for a 5-min session. Real-time PCR and Western Blotting were used to examine gene expression in mouse hippocampus. RESULTS: Our results demonstrated that CUMS induced depression-like behaviors, which were reversed by swimming exercise. Moreover, the behavioral changes induced by CUMS and exercise were correlated with hippocampal plasticity-related proteins expression of growth-associated protein-43 (GAP-43) and synaptophysin (SYN). The molecular mechanisms regulating this plasticity may include SIRT1/mircoRNA, CREB/BDNF, and AKT/GSK-3ß signaling pathways. LIMITATIONS: We did not establish a correlation between depression-like behaviors induced by chronic stress and epigenetic changes of hippocampal plasticity, either a causal molecular signaling underling this plasticity. CONCLUSIONS: Our findings have identified swimming exercise effects on CUMS-induced changes in depression-like behaviors and hippocampal plasticity-related proteins, which provide a framework for developing new strategies to treat stress-induced depression.

OGT-related mitochondrial motility is associated with sex differences and exercise effects in depression induced by prenatal exposure to glucocorticoids.

BACKGROUND: Prenatal exposure to glucocorticoids (GCs) has been found to trigger abnormal behaviors and deleterious neurological effects on offspring both in animals and in humans. The sex differences in depression have been replicated in numerous studies across cultures, persisting throughout the reproductive years. As an X-linked gene in rodents and in humans, O-GlcNAc transferase (OGT) may provide a novel perspective for the sex differences in depression. METHODS: In the last third of pregnancy (gestational day 14-21), rats were subcutaneously administered either 0.13mg/kg dexamethasone-21-phosphate disodium salt (0.1mg/kg DEX) or vehicle (0.9% saline) once a day for 7 days. Adolescent (4 weeks) offspring were then trained in a swimming program or not. RESULTS: Here we found that adult offspring rats exposed to DEX prenatally exhibited sex-specific depression-like behaviors, males being more vulnerable than females. Swimming exercise ameliorated the above-mentioned depressive syndromes, which may be a compensatory effect for male disadvantage suffering from prenatal stress. Furthermore, the effects of prenatal DEX exposure and swimming exercise on depression were associated with OGT-related mitochondrial motility, including PINK1/Parkin pathway and AKT/GSK3ß pathway. LIMITATIONS: Representative kymographs of mitochondrial motility were not detected and no causal effects were obtained by OGT gene overexpression or gene knockout in this study. CONCLUSIONS: Our results provide a new perspective for better understanding sex differences and exercise effects in depression and may offer new mechanism-based therapeutic targets for depression.

Targeting viperin improves diet-induced glucose intolerance but not adipose tissue inflammation.

Viperin is an interferon-inducible antiviral protein, responsible for antiviral response to a variety of viral infections. Here, we show that silencing viperin by antisense oligonucleotides (ASO) protects against diet-induced glucose intolerance, and yet exacerbates adipose tissue inflammation. In high-fat diet-fed mice, viperin ASO improves glucose homeostasis, reduces plasma triglyceride concentrations and ameliorates diet-induced hepatic steatosis. Peripheral delivery of viperin by adeno-associated virus elevates fasting plasma glucose and insulin concentrations and reduces insulin-stimulated glucose uptake in skeletal muscle. Viperin overexpression reduces epinephrine- stimulated lipolysis in white adipose tissue, whereas viperin ASO increases expression of lipolytic genes. Targeting viperin by antisense oligonucleotides promotes reciprocal regulation of hepatic and adipose lipogenesis by reducing hepatic lipid content and increasing triacylglycerol content in adipose tissue. These findings reveal viperin as an important target to improve glucose metabolism, and suggest that suppressing antiviral potential may improve the metabolic adaptability to high-fat diet.

Long-term treatment with nicotinamide induces glucose intolerance and skeletal muscle lipotoxicity in normal chow-fed mice: compared to diet-induced obesity.

Nicotinamide (NAM), or vitamin B3, is an essential coenzyme for ATP synthesis and an inhibitor of sirtuin 1. Recently, conflicting results were reported regarding the treatment of NAM in type 2 diabetes and obesity. The aim of this study was to determine whether and how long-term treatment with NAM at lower dose would affect insulin sensitivity in mice fed chow diet. We treated mice with NAM (100 mg/kg/day) and normal chow for 8 weeks. Strikingly, NAM induced glucose intolerance and skeletal muscle lipid accumulation in nonobese mice. NAM impaired mitochondrial respiration capacity and energy production in skeletal muscle, in combination with increased expression of the mediators for mitophagy (p62, PINK1, PARK2 and NIX) and autophagy (FOXO3, Bnip3, CTSL, Beclin1 and LC-3b). Next, we treated mice with high-fat diet (HFD) and resveratrol (RSV; 100 mg/kg/day) for 8 weeks. RSV protected against HFD-induced insulin resistance and obesity. HFD increased skeletal muscle lipid content as well as NAM, but this increase was attenuated by RSV. In contrast to NAM, HFD enhanced fatty acid oxidative capacity. Muscle transcript levels of genes for mitophagy and autophagy were largely suppressed by HFD, whereas RSV did not rescue these effects. These differences suggest that skeletal muscle autophagy may represent adaptive response to NAM-induced lipotoxicity, whereas reduced autophagy in skeletal muscle may promote HFD-induced lipotoxicity. Our results demonstrate that chronic NAM supplementation in healthy individuals, although at lower dose than previously reported, is still detrimental to glucose homeostasis and skeletal muscle lipid metabolism.