Gordon Holmes Syndrome Model Mice Exhibit Alterations in Microglia, Age, and Sex-Specific Disruptions in Cognitive and Proprioceptive Function.

Gordon Holmes syndrome (GHS) is a neurological disorder associated with neuroendocrine, cognitive, and motor impairments with corresponding neurodegeneration. Mutations in the E3 ubiquitin ligase RNF216 are strongly linked to GHS. Previous studies show that deletion of Rnf216 in mice led to sex-specific neuroendocrine dysfunction due to disruptions in the hypothalamic-pituitary-gonadal axis. To address RNF216 action in cognitive and motor functions, we tested Rnf216 knock-out (KO) mice in a battery of motor and learning tasks for a duration of 1 year. Although male and female KO mice did not demonstrate prominent motor phenotypes, KO females displayed abnormal limb clasping. KO mice also showed age-dependent strategy and associative learning impairments with sex-dependent alterations of microglia in the hippocampus and cortex. Additionally, KO males but not females had more negative resting membrane potentials in the CA1 hippocampus without any changes in miniature excitatory postsynaptic current (mEPSC) frequencies or amplitudes. Our findings show that constitutive deletion of Rnf216 alters microglia and neuronal excitability, which may provide insights into the etiology of sex-specific impairments in GHS.

Effects of Dietary Methionine Restriction on Cognition in Mice.

Dietary restriction of the essential amino acid, methionine, has been shown to induce unique metabolic protection. The peripheral benefits of methionine restriction (MR) are well established and include improvements in metabolic, energy, inflammatory, and lifespan parameters in preclinical models. These benefits all occur despite MR increasing energy intake, making MR an attractive dietary intervention for the prevention or reversal of many metabolic and chronic conditions. New and emerging evidence suggests that MR also benefits the brain and promotes cognitive health. Despite widespread interest in MR over the past few decades, many findings are limited in scope, and gaps remain in our understanding of its comprehensive effects on the brain and cognition. This review details the current literature investigating the impact of MR on cognition in various mouse models, highlights some of the key mechanisms responsible for its cognitive benefits, and identifies gaps that should be addressed in MR research moving forward. Overall findings indicate that in animal models, MR is associated with protection against obesity-, age-, and Alzheimer's disease-induced impairments in learning and memory that depend on different brain regions, including the prefrontal cortex, amygdala, and hippocampus. These benefits are likely mediated by increases in fibroblast growth factor 21, alterations in methionine metabolism pathways, reductions in neuroinflammation and central oxidative stress, and potentially alterations in the gut microbiome, mitochondrial function, and synaptic plasticity.

Berry consumption mitigates the hypertensive effects of a high-fat, high-sucrose diet via attenuation of renal and aortic AT1R expression resulting in improved endothelium-derived NO bioavailability.

Dysregulation of the renin-angiotensin system (RAS) is a contributor to high-fat diet-related blood pressure (BP) increases. Deleterious effects of dysregulated RAS result in an overproduction of reactive oxygen species and a decrease in endothelial nitric oxide (NO) bioavailability due to increased NADPH oxidase (NOX) expression. Dietary polyphenols have been shown to mitigate the imbalance in the redox state and protect against endothelial dysfunction induced by a high-fat diet. Thus, we aim to determine whether polyphenol-rich blackberry and raspberry, alone and in combination, attenuate the detrimental effects of a high-fat, high-sucrose (HFHS) diet on the vascular endothelium and kidneys of mice. We show that a HFHS diet increased the expression of renal and aortic angiotensin type 1 receptor (AT1R). Further, NOX1 and NOX4 expression were increased in the kidney contributing to fibrotic damage. In human aortic endothelial cells (HAECs), palmitic acid increased the expression of NOX4, potentially driving oxidative damage in the aorta, as evidenced by increased nitrotyrosine expression. Berries reduced the expression of renal and aortic AT1R, leading to a subsequent decrease in renal NOX expression and reduced aortic oxidative stress evidenced by reduced nitrotyrosine expression. Blackberry and raspberry in combination increased the expression of NRF2 and its downstream proteins in HAECs, thereby reducing the oxidative burden to the endothelium. In combination, blackberry and raspberry also increased serum levels of NO metabolites. These findings indicate that blackberry and raspberry unique polyphenols may act synergistically to favorably modulate the abovementioned pathways and attenuate HFHS diet-induced increases in BP.

Erratum: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis.

[This corrects the article DOI: 10.1016/j.isci.2022.104386.].

The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis.

Recessive mutations in RNF216/TRIAD3 cause Gordon Holmes syndrome (GHS), in which dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis and neurodegeneration are thought to be core phenotypes. We knocked out Rnf216/Triad3 in a gonadotropin-releasing hormone (GnRH) hypothalamic cell line. Rnf216/Triad3 knockout (KO) cells had decreased steady-state GnRH and calcium transients. Rnf216/Triad3 KO adult mice had reductions in GnRH neuron soma size and GnRH production without changes in neuron densities. In addition, KO male mice had smaller testicular volumes that were accompanied by an abnormal release of inhibin B and follicle-stimulating hormone, whereas KO females exhibited irregular estrous cycling. KO males, but not females, had reactive microglia in the hypothalamus. Conditional deletion of Rnf216/Triad3 in neural stem cells caused abnormal microglia expression in males, but reproductive function remained unaffected. Our findings show that dysfunction of RNF216/TRIAD3 affects the HPG axis and microglia in a region- and sex-dependent manner, implicating sex-specific therapeutic interventions for GHS.

The Origins, Evolution, and Future of Dietary Methionine Restriction.

The original description of dietary methionine restriction (MR) used semipurified diets to limit methionine intake to 20% of normal levels, and this reduction in dietary methionine increased longevity by ∼30% in rats. The MR diet also produces paradoxical increases in energy intake and expenditure and limits fat deposition while reducing tissue and circulating lipids and enhancing overall insulin sensitivity. In the years following the original 1993 report, a comprehensive effort has been made to understand the nutrient sensing and signaling systems linking reduced dietary methionine to the behavioral, physiological, biochemical, and transcriptional components of the response. Recent work has shown that transcriptional activation of hepatic fibroblast growth factor 21 (FGF21) is a key event linking the MR diet to many but not all components of its metabolic phenotype. These findings raise the interesting possibility of developing therapeutic, MR-based diets that produce the beneficial effects of FGF21 by nutritionally modulating its transcription and release.

Raspberry and blackberry act in a synergistic manner to improve cardiac redox proteins and reduce NF-κB and SAPK/JNK in mice fed a high-fat, high-sucrose diet.

BACKGROUND AND AIMS: Increased cardiac inflammation and oxidative stress are common features in obesity, and toll-like receptor (TLR)4 signaling is a key inflammatory pathway in this deleterious process. This study aimed to investigate whether berries could attenuate the detrimental effects of a high-fat, high-sucrose (HFHS) diet on the myocardium at the molecular level. METHODS AND RESULTS: Eight-week-old male C57BL/6 mice consumed a low-fat, low-sucrose (LFLS) diet alone or supplemented with 10% blackberry (BL), 10% raspberry (RB) or 10% blackberry + raspberry (BL + RB) for four weeks. Animals were then switched to a HFHS diet for 24 weeks with or without berry supplementation or maintained on a LFLS control diet without berry supplementation. Left ventricles of the heart were isolated for protein and mRNA analysis. Berry consumption, particularly BL + RB reduced NADPH-oxidase (NOX)1 and NOX2 and increased catalase (CAT) and superoxide dismutase (SOD)2, expression while BL and RB supplementation alone was less efficacious. Downstream TLR4 signaling was attenuated mostly by both RB and BL + RB supplementation, while NF-κB pathway was attenuated by BL + RB supplementation. Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) was also attenuated by BL + RB supplementation, and reduced TNF-α transcription and protein expression was observed only with BL + RB supplementation. CONCLUSION: The synergistic effects of BL + RB may reduce obesity-induced cardiac inflammation and oxidative stress to a greater extent than BL or RB alone.

Nutritional Regulation of Hepatic FGF21 by Dietary Restriction of Methionine.

FGF21 is a potent metabolic regulator of energy balance, body composition, lipid metabolism, and glucose homeostasis. Initial studies reported that it was increased by fasting and the associated increase in ketones, but more recent work points to the importance of dietary protein and sensing of essential amino acids in FGF21 regulation. For example, dietary restriction of methionine produces a rapid transcriptional activation of hepatic FGF21 that results in a persistent 5- to 10-fold increase in serum FGF21. Although FGF21 is a component of a complex transcriptional program activated by methionine restriction (MR), loss-of-function studies show that FGF21 is an essential mediator of the resulting effects of the MR diet on energy balance, remodeling of adipose tissue, and enhancement of insulin sensitivity. These studies also show that FGF21 signaling in the brain is required for the MR diet-induced increase in energy expenditure (EE) and reduction of adiposity. Collectively, the evidence supports the view that the liver functions as a sentinel to detect and respond to changes in dietary amino acid composition, and that the resulting mobilization of hepatic FGF21 is a key element of the homeostatic response. These findings raise the interesting possibility that therapeutic diets could be developed that produce sustained, biologically effective increases in FGF21 by nutritionally modulating its transcription and release.

The acute transcriptional responses to dietary methionine restriction are triggered by inhibition of ternary complex formation and linked to Erk1/2, mTOR, and ATF4.

The initial sensing of dietary methionine restriction (MR) occurs in the liver where it activates an integrated stress response (ISR) that quickly reduces methionine utilization. The ISR program is regulated in part by ATF4, but ATF4's prototypical upstream regulator, eIF2α, is not acutely activated by MR. Bioinformatic analysis of RNAseq and metabolomics data from liver samples harvested 3 h and 6 h after initiating MR shows that general translation is inhibited at the level of ternary complex formation by an acute 50% reduction of hepatic methionine that limits formation of initiator methionine tRNA. The resulting ISR is induced by selective expression of ATF4 target genes that mediate adaptation to reduced methionine intake and return hepatic methionine to control levels within 4 days of starting the diet. Complementary in vitro experiments in HepG2 cells after knockdown of ATF4, or inhibition of mTOR or Erk1/2 support the conclusion that the early induction of genes by MR is partially dependent on ATF4 and regulated by both mTOR and Erk1/2. Taken together, these data show that initiation of dietary MR induces an mTOR- and Erk1/2-dependent stress response that is linked to ATF4 by the sharp, initial drop in hepatic methionine and resulting repression of translation pre-initiation.

Berries as a Treatment for Obesity-Induced Inflammation: Evidence from Preclinical Models.

Inflammation that accompanies obesity is associated with the infiltration of metabolically active tissues by inflammatory immune cells. This propagates a chronic low-grade inflammation associated with increased signaling of common inflammatory pathways such as NF-κB and Toll-like receptor 4 (TLR4). Obesity-associated inflammation is linked to an increased risk of chronic diseases, including type 2 diabetes, cardiovascular disease, and cancer. Preclinical rodent and cell culture studies provide robust evidence that berries and their bioactive components have beneficial effects not only on inflammation, but also on biomarkers of many of these chronic diseases. Berries contain an abundance of bioactive compounds that have been shown to inhibit inflammation and to reduce reactive oxygen species. Therefore, berries represent an intriguing possibility for the treatment of obesity-induced inflammation and associated comorbidities. This review summarizes the anti-inflammatory properties of blackberries, blueberries, strawberries, and raspberries. This review highlights the anti-inflammatory mechanisms of berries and their bioactive components that have been elucidated through the use of preclinical models. The primary mechanisms mediating the anti-inflammatory effects of berries include a reduction in NF-κB signaling that may be secondary to reduced oxidative stress, a down-regulation of TLR4 signaling, and an increase in Nrf2.

Synergistic Impact of Xanthorrhizol and d-δ-Tocotrienol on the Proliferation of Murine B16 Melanoma Cells and Human DU145 Prostate Carcinoma Cells.

Isoprenoids suppress the mevalonate pathway that provides prenyl groups for the posttranslational modification of growth-regulating proteins. We hypothesize that xanthorrhizol and d-δ-tocotrienol synergistically suppress the growth of murine B16 melanoma and human DU145 prostate carcinoma cells. Xanthorrhizol (0-200 µmol/L; half maximal inhibitory concentration [IC50] = 65 µmol/L) and d-δ-tocotrienol (0-40 µmol/L; IC50 = 20 µmol/L) each induced a concentration-dependent suppression of the proliferation of B16 cells and concurrent cell cycle arrest at the G1 phase. A blend of 16.25 µmol/L xanthorrhizol and 10 µmol/L d-δ-tocotrienol suppressed B16 cell proliferation by 69%, an impact greater than the sum of those induced by xanthorrhizol (15%) and d-δ-tocotrienol (12%) individually. The blend cumulatively reduced the levels of cyclin-dependent kinase four and cyclin D1, key regulators of cell cycle progression at the G1 phase. The expression of RAS and extracellular signal-regulated kinase (ERK1/2) in the proliferation-stimulating RAS-RAF-MEK-ERK pathway was downregulated by the blend. Xanthorrhizol also induced a concentration-dependent suppression of the proliferation of DU145 cells with concomitant morphological changes. Isobologram confirmed the synergistic effect of xanthorrhizol and d-δ-tocotrienol on DU145 cell proliferation with combination index values ranging 0.61-0.94. Novel combinations of isoprenoids with synergistic actions may offer effective approaches in cancer prevention and therapy.

The Absence of Adiponectin Alters Niacin's Effects on Adipose Tissue Inflammation in Mice.

Obesity is an immunometabolic disease associated with chronic inflammation and the dysregulation of pro- and anti-inflammatory cytokines. One hallmark of obesity is reduced concentrations of the anti-inflammatory adipokine, adiponectin. Pharmacologic doses of niacin produce multiple metabolic benefits, including attenuating high-fat diet (HFD)-induced adipose tissue inflammation and increasing adiponectin concentrations. To determine if adiponectin mediates the anti-inflammatory effects of niacin, male C57BL/6J (WT) and adiponectin null (Adipoq-/-) mice were maintained on a low-fat diet (LFD) or HFD for 6 weeks, before being administered either vehicle or niacin (360 mg/kg/day) for 5 weeks. HFD-fed mice had increased expression of genes associated with macrophage recruitment (Ccl2) and number (Cd68), and increased crown-like structure (CLS) number in adipose tissue. While niacin attenuated Ccl2 expression, there were no effects on Cd68 or CLS number. The absence of adiponectin did not hinder the ability of niacin to reduce Ccl2 expression. HFD feeding increased gene expression of inflammatory markers in the adipose tissue of WT and Adipoq-/- mice. While niacin tended to decrease the expression of inflammatory markers in WT mice, niacin increased their expression in HFD-fed Adipoq-/- mice. Therefore, our results indicate that the absence of adiponectin alters the effects of niacin on markers of adipose tissue inflammation in HFD-fed mice, suggesting that the effects of niacin on tissue cytokines may involve adiponectin.

Methionine Restriction and Cancer Biology.

The essential amino acid, methionine, is important for cancer cell growth and metabolism. A growing body of evidence indicates that methionine restriction inhibits cancer cell growth and may enhance the efficacy of chemotherapeutic agents. This review summarizes the efficacy and mechanism of action of methionine restriction on hallmarks of cancer in vitro and in vivo. The review highlights the role of glutathione formation, polyamine synthesis, and methyl group donation as mediators of the effects of methionine restriction on cancer biology. The translational potential of the use of methionine restriction as a personalized nutritional approach for the treatment of patients with cancer is also discussed.

FGF21 Signals Protein Status to the Brain and Adaptively Regulates Food Choice and Metabolism.

Reduced dietary protein intake induces adaptive physiological changes in macronutrient preference, energy expenditure, growth, and glucose homeostasis. We demonstrate that deletion of the FGF21 co-receptor ßKlotho (Klb) from the brain produces mice that are unable to mount a physiological response to protein restriction, an effect that is replicated by whole-body deletion of FGF21. Mice forced to consume a low-protein diet exhibit reduced growth, increased energy expenditure, and a resistance to diet-induced obesity, but the loss of FGF21 signaling in the brain completely abrogates that response. When given access to a higher protein alternative, protein-restricted mice exhibit a shift toward protein-containing foods, and central FGF21 signaling is essential for that response. FGF21 is an endocrine signal linking the liver and brain, which regulates adaptive, homeostatic changes in metabolism and feeding behavior during protein restriction.

Dietary Methionine Restriction Reduces Inflammation Independent of FGF21 Action.

OBJECTIVE: Methionine restriction (MR) decreases inflammation and improves markers of metabolic disease in rodents. MR also increases hepatic and circulating concentrations of fibroblast growth factor 21 (FGF21). Emerging evidence has suggested that FGF21 exerts anti-inflammatory effects. The purpose of this study was to determine the role of FGF21 in mediating the MR-induced reduction in inflammation. METHODS: Wild-type and Fgf21-/- mice were fed a high-fat (HF) control or HF-MR diet for 8 weeks. In a separate experiment, mice were fed a HF diet (HFD) for 10 weeks. Vehicle or recombinant FGF21 (13.6 µg/d) was administered via osmotic minipump for an additional 2 weeks. Inflammation and metabolic parameters were measured. RESULTS: Fgf21-/- mice were more susceptible to HFD-induced inflammation, and MR reduced inflammation in white adipose tissue (WAT) and liver of Fgf21-/- mice. MR downregulated activity of signal transducer and activator of transcription 3 in WAT of both genotypes. FGF21 administration reduced hepatic lipids and blood glucose concentrations. However, there was little effect of FGF21 on inflammatory gene expression in liver or adipose tissue or circulating cytokines. CONCLUSIONS: MR reduces inflammation independent of FGF21 action. Endogenous FGF21 is important to protect against the development of HFD-induced inflammation in liver and WAT, yet administration of low-dose FGF21 has little effect on markers of inflammation.

Annatto-extracted tocotrienols improve glucose homeostasis and bone properties in high-fat diet-induced type 2 diabetic mice by decreasing the inflammatory response.

Diabetes is a risk factor for osteoporosis. Annatto-extracted tocotrienols (TT) have proven benefits in preserving bone matrix. Here, we evaluated the effects of dietary TT on glucose homeostasis, bone properties, and liver pro-inflammatory mRNA expression in high-fat diet (HFD)-induced type 2 diabetic (T2DM) mice. 58 male C57BL/6 J mice were divided into 5 groups: low-fat diet (LFD), HFD, HFD + 400 mgTT/kg diet (T400), HFD + 1600 mgTT/kg diet (T1600), and HFD + 200 mg metformin/kg (Met) for 14 weeks. Relative to the HFD group, both TT-supplemented groups (1) improved glucose homeostasis by lowering the area under the curve for both glucose tolerance and insulin tolerance tests, (2) increased serum procollagen I intact N-terminal propeptide (bone formation) level, trabecular bone volume/total volume, trabecular number, connectivity density, and cortical thickness, (3) decreased collagen type 1 cross-linked C-telopeptide (bone resorption) levels, trabecular separation, and structure model index, and (4) suppressed liver mRNA levels of inflammation markers including IL-2, IL-23, IFN-γ, MCP-1, TNF-α, ITGAX and F4/80. There were no differences in glucose homeostasis and liver mRNA expression among T400, T1600, and Met. The order of osteo-protective effects was LFD ≥T1600 ≥T400 = Met >HFD. Collectively, these data suggest that TT exerts osteo-protective effects in T2DM mice by regulating glucose homeostasis and suppressing inflammation.

The Components of Age-Dependent Effects of Dietary Methionine Restriction on Energy Balance in Rats.

OBJECTIVE: Dietary methionine restriction (MR) improves biomarkers of metabolic health, in part through coordinated increases in energy intake and energy expenditure (EE). Some metabolic benefits of dietary MR are secondary to its effects on energy balance, so this study's purpose was to examine how age at initiation of MR influences its effects on energy balance and body composition. METHODS: Energy balance was examined in rats provided control or MR diets for 9 months after weaning or in rats between 6 and 12 months of age. RESULTS: Rats provided the control diet for 9 months after weaning increased their body weight (BW) and fat mass by five- and eightfold, respectively, while BW and fat accumulation in the MR group were reduced to 50% of that of controls. In adult rats fed the respective diets between 6 and 12 months of age, dietary MR increased energy intake by ∼23%, but the 15% increase in EE was sufficient to prevent increases in BW or fat mass. CONCLUSIONS: Dietary MR produces comparable increases in EE in young, growing animals and in mature animals, but young animals continue to deposit new tissue because of the proportionately larger effect of MR on energy intake relative to maintenance requirements.

Changes in hepatic phase I and phase II biotransformation enzyme expression and glutathione levels following atrazine exposure in female rats.

1. To determine the effects of repeated atrazine (ATR) treatment on hepatic phase I and II enzymes, adult female rats were treated with vehicle or 100 mg/kg of ATR for 1, 2, 3 or 4 days. Glutathione-s-transferases (GST) mRNA expression, protein levels (mu, pi, alpha, omega), and activity (cytosolic and microsomal), along with bioavailable glutathione (GSH) were assayed. 2. GST expression, concentrations and activity were increased, along with GSH levels, in animals treated with ATR for 3 and 4 days. 3. A subsequent study was performed with animals treated with vehicle, 6.5, 50 or 100 mg/kg/day for 4, 8 or 14 days. Expression of hepatic phase I CYP 450 enzymes was evaluated in conjugation with GST expression, protein and activity. Nineteen of the 45 CYP enzymes assayed displayed increased mRNA levels after eight days of treatment in animals treated with 50 or 100 mg/kg/day. After 14 days of treatment, all CYP expression levels returned to control levels except for CYP2B2, CYP2B3, CYP2C7, CYP2C23, CYP2E1, CYP3A9, CYP4A3 and CYP27A1, which remained elevated. 4. Results indicate that there may be a habituation or adaptation of liver phase I and phase II expression following repeated ATR treatment.

Sensing and signaling mechanisms linking dietary methionine restriction to the behavioral and physiological components of the response.

Dietary methionine restriction (MR) is implemented using a semi-purified diet that reduces methionine by ∼80% and eliminates dietary cysteine. Within hours of its introduction, dietary MR initiates coordinated series of transcriptional programs and physiological responses that include increased energy intake and expenditure, decreased adiposity, enhanced insulin sensitivity, and reduction in circulating and tissue lipids. Significant progress has been made in cataloguing the physiological responses to MR in males but not females, but identities of the sensing and communication networks that orchestrate these responses remain poorly understood. Recent work has implicated hepatic FGF21 as an important mediator of MR, but it is clear that other mechanisms are also involved. The goal of this review is to explore the temporal and spatial organization of the responses to dietary MR as a model for understanding how nutrient sensing systems function to integrate complex transcriptional, physiological, and behavioral responses to changes in dietary composition.

The role of suppression of hepatic SCD1 expression in the metabolic effects of dietary methionine restriction.

Dietary methionine restriction (MR) produces concurrent increases in energy intake and expenditure, but the proportionately larger increase in energy expenditure (EE) effectively limits weight gain and adipose tissue accretion over time. Increased hepatic fibroblast growth factor-21 (FGF21) is essential to MR-dependent increases in EE, but it is unknown whether the downregulation of hepatic stearoyl-coenzyme A desaturase-1 (SCD1) by MR could also be a contributing factor. Global deletion of SCD1 mimics cold exposure in mice housed at 23 °C by compromising the insular properties of the skin. The resulting cold stress increases EE, limits fat deposition, reduces hepatic lipids, and increases insulin sensitivity by activating thermoregulatory thermogenesis. To examine the efficacy of MR in the absence of SCD1 and without cold stress, the biological efficacy of MR in Scd1-/- mice housed near thermoneutrality (28 °C) was evaluated. Compared with wild-type mice on the control diet, Scd1-/- mice were leaner, had higher EE, lower hepatic and serum triglycerides, and lower serum leptin and insulin. Although dietary MR increased adipose tissue UCP1 expression, hepatic Fgf21 messenger RNA, 24 h EE, and reduced serum triglycerides in Scd1-/- mice, it failed to reduce adiposity or produce any further reduction in hepatic triglycerides, serum insulin, or serum leptin. These findings indicate that even when thermal stress is minimized, global deletion of SCD1 mimics and effectively masks many of the metabolic responses to dietary MR. However, the retention of several key effects of dietary MR in this model indicates that SCD1 is not a mediator of the biological effects of the diet.