Ketogenic diet administration later in life improves memory by modifying the synaptic cortical proteome via the PKA signaling pathway in aging mice.

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated in vivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.

A ketogenic diet reduces age-induced chronic neuroinflammation in mice Running title: ketogenic diet and brain inflammaging.

Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionally in vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.

Macrophage fatty acid oxidation inhibits atherosclerosis progression.

Atherosclerosis is a chronic disorder of the vessel wall. One key regulator of disease progression is lipid handling in macrophages. However, the role of macrophage mitochondrial-dependent fatty acid ß-oxidation (FAO) in atherosclerosis is not well defined. To address this, we focused on carnitine palmitoyltransferase (CPT) 1 and 2, which play an essential role in the transport of long chain fatty acids (FAs) into the mitochondria. Using conditional alleles of these mitochondrial enzymes, we have generated myeloid-specific Cpt1a and Cpt2 knockout mutants (CPT1a M-KO and CPT2 M-KO). In culture, macrophages derived from CPT1a and CPT2 M-KO mice have impaired FAO, enhanced expression of the CD36 scavenger receptor, increased uptake of oxidized low-density lipoprotein (oxLDL), and augmented transformation into cholesterol-rich foam cells. In line with these in vitro observations, in the atherosclerosis-susceptible apolipoprotein E (ApoE) KO background, CPT2 M-KO mice demonstrated augmented atherosclerosis, accompanied by increased accumulation of aortic macrophages with elevated CD36 expression. These data suggest that macrophage FAO is athero-protective and that augmenting FAO may potentially slow atherosclerotic progression.

TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue.

Remodelling of energy storing white fat into energy expending beige fat could be a promising strategy to reduce adiposity. Here, we show that the bile acid-responsive membrane receptor TGR5 mediates beiging of the subcutaneous white adipose tissue (scWAT) under multiple environmental cues including cold exposure and prolonged high-fat diet feeding. Moreover, administration of TGR5-selective bile acid mimetics to thermoneutral housed mice leads to the appearance of beige adipocyte markers and increases mitochondrial content in the scWAT of Tgr5 +/+ mice but not in their Tgr5 -/- littermates. This phenotype is recapitulated in vitro in differentiated adipocytes, in which TGR5 activation increases free fatty acid availability through lipolysis, hence fuelling ß-oxidation and thermogenic activity. TGR5 signalling also induces mitochondrial fission through the ERK/DRP1 pathway, further improving mitochondrial respiration. Taken together, these data identify TGR5 as a druggable target to promote beiging with potential applications in the management of metabolic disorders.

TGR5 reduces macrophage migration through mTOR-induced C/EBPß differential translation.

The bile acid-responsive G protein-coupled receptor TGR5 is involved in several metabolic processes, and recent studies suggest that TGR5 activation may promote pathways that are protective against diet-induced diabetes. Here, we investigated the role of macrophage-specific TGR5 signaling in protecting adipose tissue from inflammation and associated insulin resistance. Examination of adipose tissue from obese mice lacking macrophage Tgr5 revealed enhanced inflammation, increased chemokine expression, and higher macrophage numbers compared with control obese animals. Moreover, macrophage-specific deletion of Tgr5 exacerbated insulin resistance in obese animals. Conversely, pharmacological activation of TGR5 markedly decreased LPS-induced chemokine expression in primary macrophages. This reduction was mediated by AKT-dependent activation of mTOR complex 1, which in turn induced the differential translation of the dominant-negative C/EBPß isoform, liver inhibitory protein (LIP). Overall, these studies reveal a signaling pathway downstream of TGR5 that modulates chemokine expression in response to high-fat diet and suggest that targeting this pathway has the potential to be therapeutically exploited for prevention of chronic inflammatory diseases and type 2 diabetes mellitus.

Adult headform impact tests of three Japanese child bicycle helmets into a vehicle.

The head is the body region that most frequently incurs fatal and serious injuries of cyclists in collisions against vehicles. Many research studies investigated helmet effectiveness in preventing head injuries using accident data. In this study, the impact attenuation characteristics of three Japanese child bicycle helmets were examined experimentally in impact tests into a concrete surface and a vehicle. A pedestrian adult headform with and without a Japanese child bicycle helmet was dropped onto a concrete surface and then propelled into a vehicle at 35 km/h in various locations such as the bonnet, roof header, windshield and A-pillar. Accelerations were measured and head injury criterion (HIC) calculated. In the drop tests using the adult headform onto a concrete surface from the height of 1.5m, the HIC for a headform without a child helmet was 6325, and was reduced by around 80% when a child helmet was fitted to the headform. In the impact tests, where the headform was fired into the vehicle at 35 km/h at various locations on a car, the computed acceleration based HIC varied depending on the vehicle impact locations. The HIC was reduced by 10-38% for impacts headforms with a child helmet when the impact was onto a bonnet-top and roof header although the HIC was already less than 1000 in impacts with the headform without a child helmet. Similarly, for impacts into the windshield (where a cyclist's head is most frequently impacted), the HIC using the adult headform without a child helmet was 122; whereas when the adult headform was used with a child helmet, a higher HIC value of more than 850 was recorded. But again, the HIC values are below 1000. In impacts into the A-pillar, the HIC was 4816 for a headform without a child helmet and was reduced by 18-38% for a headform with a child helmet depending on the type of Japanese child helmet used. The tests demonstrated that Japanese child helmets are effective in reducing accelerations and HIC in a drop test using an adult headform onto a relatively rigid hard surface, i.e., simulating a road surface or concrete path. However, when the impact tests are into softer surfaces, the child helmet's capacity to decrease accelerations is accordingly reduced. Impacts into the windshield, while below the critical HIC value of 1000, indicated higher HIC values for a headform with a child helmet compared to an adult headform without a child helmet. The unpredictable nature of the results indicates further research work is required to assess how representative the stiffness of an adult headform is when compared to an actual head.

TGR5 potentiates GLP-1 secretion in response to anionic exchange resins.

Anionic exchange resins are bona fide cholesterol-lowering agents with glycemia lowering actions in diabetic patients. Potentiation of intestinal GLP-1 secretion has been proposed to contribute to the glycemia lowering effect of these non-systemic drugs. Here, we show that resin exposure enhances GLP-1 secretion and improves glycemic control in diet-induced animal models of "diabesity", effects which are critically dependent on TGR5, a G protein-coupled receptor that is activated by bile acids. We identified the colon as a major source of GLP-1 secretion after resin treatment. Furthermore, we demonstrate that the boost in GLP-1 release by resins is due to both enhanced TGR5-dependent production of the precursor transcript of GLP-1 as well as to the local enrichment of TGR5 agonists in the colon. Thus, TGR5 represents an essential component in the pathway mediating the enhanced GLP-1 release in response to anionic exchange resins.

TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.

The G protein-coupled receptor TGR5 has been identified as an important component of the bile acid signaling network, and its activation has been linked to enhanced energy expenditure and improved glycemic control. Here, we demonstrate that activation of TGR5 in macrophages by 6α-ethyl-23(S)-methylcholic acid (6-EMCA, INT-777), a semisynthetic BA, inhibits proinflammatory cytokine production, an effect mediated by TGR5-induced cAMP signaling and subsequent NF-κB inhibition. TGR5 activation attenuated atherosclerosis in Ldlr(-/-)Tgr5(+/+) mice but not in Ldlr(-/-)Tgr5(-/-) double-knockout mice. The inhibition of lesion formation was associated with decreased intraplaque inflammation and less plaque macrophage content. Furthermore, Ldlr(-/-) animals transplanted with Tgr5(-/-) bone marrow did not show an inhibition of atherosclerosis by INT-777, further establishing an important role of leukocytes in INT-777-mediated inhibition of vascular lesion formation. Taken together, these data attribute a significant immune modulating function to TGR5 activation in the prevention of atherosclerosis, an important facet of the metabolic syndrome.

The bile acid membrane receptor TGR5: a valuable metabolic target.

Bile acids (BAs) are amphipathic molecules that facilitate the uptake of lipids, and their levels fluctuate in the intestines as well as in the circulation depending on food intake. Besides their role in dietary lipid absorption, BAs function as signaling molecules that activate specific BA receptors and trigger downstream signaling cascades. The BA receptors and the signaling pathways they control are not only important in the regulation of BA synthesis and their metabolism, but they also regulate glucose homeostasis, lipid metabolism and energy expenditure - processes relevant in the context of the metabolic syndrome. In addition to the function of the nuclear receptor FXRα in regulating local effects of BAs in the organs of the enterohepatic axis, increasing evidence points to a crucial role of the G-protein-coupled receptor TGR5 in mediating systemic actions of BAs. Here we review the current knowledge on BA receptors, with a strong focus on the cell membrane receptor TGR5, which has emerged as a promising target for intervention in metabolic diseases.

The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation.

Bile acids (BAs) are amphipathic molecules that facilitate the uptake of lipids, and their levels fluctuate in the intestine as well as in the blood circulation depending on food intake. Besides their role in dietary lipid absorption, bile acids function as signaling molecules capable to activate specific receptors. These BA receptors are not only important in the regulation of bile acid synthesis and their metabolism, but also regulate glucose homeostasis, lipid metabolism, and energy expenditure. These processes are important in diabetes and other facets of the metabolic syndrome, which represents a considerable increasing health burden. In addition to the function of the nuclear receptor FXRα in regulating local effects in the organs of the enterohepatic axis, increasing evidence points to a crucial role of the G-protein coupled receptor (GPCR) TGR5 in mediating systemic actions of BAs. Here we discuss the current knowledge on BA receptors, with a strong focus on the cell membrane receptor TGR5, which emerges as a valuable target for intervention in metabolic diseases.

Head impact mechanisms of a child occupant seated in a child restraint system as determined by impact testing.

In side collision accidents, the head is the most frequently injured body region for child occupants seated in a child restraint system (CRS). Accident analyses show that a child's head can move out of the CRS shell, make hard contact with the vehicle interior, and thus sustain serious injuries. In order to improve child head protection in side collisions, it is necessary to understand the injury mechanism of a child in the CRS whose head makes contact with the vehicle interior. In this research, an SUV-to-car oblique side crash test was conducted to reconstruct such head contacts. A Q3s child dummy was seated in a CRS in the rear seat of the target car. The Q3s child dummy's head moved out beyond the CRS side wing, moved laterally, and made contact with the side window glass and the doorsill. It was demonstrated that the hard head contact, which produced a high HIC value, could occur in side collisions. A series of sled tests was carried out to reproduce the dummy kinematic behavior observed in the SUV-to-car crash test, and the sled test conditions such as sled angle, ECE seat slant angle and velocity-time history that duplicated the kinematic behavior were determined. A parametric study also was conducted with the sled tests; and it was found that the impact angle, harness slack, chest clip, and the CRS side wing shape affected the torso motion and head contact with the vehicle interior.

Inhibition of hepatic Niemann-Pick C1-like 1 improves hepatic insulin resistance.

The present study attempted to define the role of hepatic Niemann-Pick C1-like 1 (NPC1L1), a cholesterol transporter, in hepatic insulin resistance as well as hepatic steatosis. The inhibition of NPC1L1 and its molecular consequences were examined in Zucker obese fatty (ZOF) rats and cultured steatotic hepatocytes using ezetimibe, a pharmacoloigcal inhibitor of NPC1L1, and short hairpin RNA (shRNA) of NPC1L1. Ezetimibe improved hepatic insulin signaling as well as hepatic steatosis in ZOF rats. It also restored insulin sensitivity in steatotic hepatocytes in vitro through a reduction in hepatic reactive oxygen species (ROS) generation, JNK activation, and ER stress. In addition, ezetimibe recovered insulin-induced Akt activation and reduced gluconeogenic genes in the liver of ZOF rats and cultured steatotic hepatocytes. Transfection of NPC1L1 shRNA into hepatocytes also reduced ROS generation and ER stress. These results indicate that NPC1L1 contributes to hepatic insulin resistance through cholesterol accumulation, and its inhibition could be a potential therapeutic target of hepatic insulin resistance.

Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome.

Non-alcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome characterized by dislipidemia and insulin resistance. We hypothesized that ezetimibe, an inhibitor of NPC1L1, improves these metabolic disorders in Zucker obese fatty rats (ZOF). Ezetimibe significantly lowered total cholesterol and triglycerides in ZOF with prominent reduction in the remnant lipoprotein fraction and small dense low density lipoprotein fraction. Moreover, lipid deposition and fibrosis of liver were decreased by ezetimibe. Interestingly, ezetimibe improved insulin and plasma glucose response after intraperitoneal glucose injection. Further, ezetimibe enhanced insulin signaling in cultured hepatocytes. Our results indicate the potential of ezetimibe in treating the metabolic syndrome and NAFLD.