ABSTRACT Insulin signaling is a critical determinant of metabolic health, and impairments in insulin action contribute to the development of type 2 diabetes. The kinase Akt is a central mediator of insulin signaling and is required for insulin’s suppression of hepatic glucose output. Although the regulation of Akt by the insulin receptor-PI3K pathway is well understood, there are instances in which signaling downstream of Akt is dissociated from proximal insulin signaling, for example in insulin resistance. Nonetheless, little is known about PI3K-independent mechanisms of Akt regulation. Here, we discovered that hepatocyte manganese concentrations are a key determinant of PI3K-independent Akt function in vivo. We further demonstrated that manganese increases Akt’s catalytic efficiency, and quantitative phosphoproteomics revealed that manganese and insulin act additively to enhance Akt activity. Moreover, we uncovered that hepatic manganese concentrations fluctuate during fasting and feeding via carbohydrate-dependent transcriptional regulation of the manganese efflux transporter Slc30a10. This dynamic metal–signaling axis provides a mechanistic link between nutrient status and Akt activation, and suggests a molecular explanation for the glucose-lowering effects of manganese observed in humans. Our findings establish manganese as a physiologically regulated cofactor for Akt and position metal bioavailability as a previously unrecognized layer of insulin signaling control.

Bile acids (BAs) are signaling molecules involved in energy expenditure, glucose homeostasis, and immune system regulation via activation of BA receptors, such as Takeda G-Protein-Coupled Receptor 5 (TGR5), Farnesoid X Receptor (FXR), and Vitamin D Receptor (VDR). The structure of BA, especially the hydroxyl group position, plays an important role in exerting its function. Previously, we reported that 16α-hydroxylated BA, also known as pythocholic acid (PCA), has beneficial effects on metabolic function and lipid metabolism in mammals. However, the molecular mechanism of PCA in mammals is yet to be explored because 16α-hydroxylated BA has not been seen in mammals. This study aims to investigate the binding interaction of PCA to human bile acid receptors, TGR5, FXR, and VDR, using a luciferase reporter assay. Luciferase reporter assay showed that PCA and tauro-conjugated-PCA (TPCA) activated TGR5, but did not activate FXR or VDR. Additionally, PCA and TPCA did not show an antagonistic effect on any of the BA receptors. TPCA treatment significantly decreased lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-α) expression in mouse peritoneal macrophages, and inhibition of TGR5 by SBI-115 canceled the anti-inflammatory effect of TPCA. Our data suggests that PCA and TPCA are ligands for mammalian TGR5 receptors.

Introduction & Objective: Metals are known to be necessary for cellular processes such as signal transduction and in protein conformation, though the characterization of which proteins bind metals is incomplete. Manganese (Mn) is known to be required for life but is one of the least understood metals. We aim to: (i) determine the mechanisms that regulate Mn and (ii) the molecular targets of Mn. Methods: We used a mouse model of Mn excess that depletes the Mn efflux transporter, Slc30a10, from the liver and intestine (Slc30a10Tbg,Vil). We conducted metabolic studies on mice kept on a standard chow diet or fed a high-fat diet for eight weeks. We used primary hepatocytes treated with Mn ex vivo to perform glucose production assays and assess the phosphorylation cascade in the insulin signaling pathway. Results: We found that Slc30a10Tbg,Vil mice have a significant improvement in glucose tolerance on a standard chow diet and are protected from impaired glucose tolerance when put on a high-fat diet. We treated hepatocytes with Mn ex vivo and found a significant reduction in glucose production. These data were associated with increased phosphorylation of Akt targets without increased phosphorylation of Akt itself. Using knockout and inhibitor approaches, we found that Mn acts downstream of both insulin receptor and PI3 kinase, suggesting that Mn acts directly on Akt. Fasting and refeeding studies on wildtype mice found Slc30a10 is low during fasting and increases rapidly after refeeding. Conclusions: These data suggest Mn acts directly on Akt to sensitize hepatocytes to insulin. Further, our finding that Slc30a10 is regulated nutritionally suggests the possibility that controlled access to Mn may be a physiological mechanism to regulate Akt activity. These results highlight a novel role of Mn in the insulin signaling pathway and shed light on how Mn homeostasis is regulated in the liver. Disclosure J. Gamarra: None. Y. Xie: None. S. Higuchi: None. R. Haeusler: None.

Bidirectional communication between the brain and gastrointestinal tract, called the gut-brain axis, is linked with our emotions. Intestinal lipids, hormones, and molecules, such as bile acids (BAs), impact our mood, motivation, and emotions via the gut-brain axis. BAs are synthesized from cholesterol in the liver and serve as a regulator of lipid metabolism and hormonal secretion in the intestine. Human studies have indicated that the alteration of plasma BA levels is associated with depression and anxiety. Several possible mechanisms, such as BA receptor-dependent and receptor-independent mechanisms, have been reported for emotional control. Animal studies have indicated that the deletion of BA receptors shows behavioral abnormalities. BAs regulate gut hormones, glucagon-like peptide-1 secretion, bioactive lipids, oleoylethanolamide, and the immune system function, which influences neural activities. Thus, BAs act as an emotional regulator. This review aims to summarize the following: clinical evidence of BA concentration linked to mental disorders, including depression and anxiety; and animal studies of BA-related signaling correlated with its neurobehavioral effect supporting its mechanism. We will also discuss future research required for further neurobehavioral treatment.

Modulation of bile acid (BA) structure is a potential strategy for obesity and metabolic disease treatment. BAs act not only as signaling molecules involved in energy expenditure and glucose homeostasis, but also as regulators of food intake. The structure of BAs, particularly the position of the hydroxyl groups of BAs, impacts food intake partly by intestinal effects: (1) modulating the activity of N-acyl phosphatidylethanolamine phospholipase D, which produces the anorexigenic bioactive lipid oleoylethanolamide (OEA) or (2) regulating lipid absorption and the gastric emptying-satiation pathway. We hypothesized that 16α-hydroxylated BAs uniquely regulate food intake because of the long intermeal intervals in snake species in which these BAs are abundant. However, the effects of 16α-hydroxylated BAs in mammals are completely unknown because they are not naturally found in mammals. To test the effect of 16α-hydroxylated BAs on food intake, we isolated the 16α-hydroxylated BA pythocholic acid from ball pythons (Python regius). Pythocholic acid or deoxycholic acid (DCA) was given by oral gavage in mice. DCA is known to increase N-acyl phosphatidylethanolamine phospholipase D activity better than other mammalian BAs. We evaluated food intake, OEA levels, and gastric emptying in mice. We successfully isolated pythocholic acid from ball pythons for experimental use. Pythocholic acid treatment significantly decreased food intake in comparison to DCA treatment, and this was associated with increased jejunal OEA, but resulted in no change in gastric emptying or lipid absorption. The exogenous BA pythocholic acid is a novel regulator of food intake and the satiety signal for OEA in the mouse intestine.

ABSTRACT Objective Modulation of bile acid (BA) structure is a potential strategy for obesity and metabolic disease treatment. BAs act not only as signaling molecules involved in energy expenditure and glucose homeostasis, but also as regulators of food intake. The structure of BAs, particularly the position of the hydroxyl groups of BAs impacts food intake partly by intestinal effects: (1) modulating the activity of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), which produces the anorexigenic bioactive lipid oleoylethanolamide (OEA), or (2) regulating lipid absorption and the gastric emptying-satiation pathway. We hypothesized that 16α-hydroxylated BAs uniquely regulate food intake, because of the long intermeal intervals in snake species in which these BAs are abundant. However, the effects of 16α-hydroxylated BAs in mammals are completely unknown, because 16α-hydroxylated BAs are not naturally found in mammals. To test the effect of 16α-hydroxylated BAs on food intake, we isolated the 16α-hydroxylated BA pythocholic acid from ball pythons ( Python regius ). Methods Pythocholic acid or deoxycholic acid (DCA) were given by oral gavage in mice. DCA is known to increase NAPE-PLD activity better than other mammalian BAs. We evaluated food intake, OEA levels and gastric emptying in mice. Results We successfully isolated pythocholic acid from ball pythons for experimental use. Pythocholic treatment significantly decreased food intake compared with DCA treatment, and this was associated with increased jejunal OEA, but no change in gastric emptying or lipid absorption. Conclusion The exogenous bile acid pythocholic acid is a novel regulator of food intake and the satiety signal OEA in the mouse intestine. Highlights Pythocholic acid decreases food intake. Pythocholic acid increases intestinal OEA and other fatty acid ethanolamides. The effects of pythocholic acid on OEA and hypophagia are greater than the effects of DCA. Pythocholic acid does not affect lipid absorption or gastric emptying.

Despite the high prevalence of obesity among middle-aged subjects, it is unclear if sex differences in middle age affect the metabolic outcomes of obesity therapies. Accordingly, in this study, middle-aged obese female and male mice were randomized to one of three groups: sleeve gastrectomy (SG), sham surgery ad libitum (SH-AL), or sham surgery with weight matching to SG through intermittent fasting with calorie restriction (SH-IF). Comprehensive measures of energy and glucose homeostasis, including energy intake, body weight, energy expenditure, glucose and insulin tolerance, and interscapular brown adipose tissue (iBAT) sympathetic innervation density were obtained. At the end of 8 wk, SG and SH-IF females had better metabolic outcomes than their male counterparts. SG females had improved weight loss maintenance, preservation of fat-free mass (FFM), higher total energy expenditure (TEE), normal locomotor activity, and reduced plasma insulin and white adipose tissue (WAT) inflammatory markers. SH-IF females also had lower plasma insulin and WAT inflammatory markers, and higher TEE than SH-IF males, despite their lower FFM. In addition, SH-IF females had higher iBAT sympathetic nerve density than SG and SH-AL females, whereas there were no differences among males. Notably, SH-IF mice of both sexes had the most improved glucose tolerance, highlighting the benefits of fasting, irrespective of weight loss. Results from this study demonstrate that in middle-aged obese mice, female sex is associated with better metabolic outcomes after SG or IF with calorie restriction. Clinical studies are needed to determine if sex differences should guide the choice of obesity therapies.NEW & NOTEWORTHY SG or IF with calorie restriction produces better metabolic outcomes in females than in males. IF with calorie restriction prevents metabolic adaptation, even in the face of fat-free mass loss. IF with calorie restriction in females only, is associated with increased iBAT sympathetic innervation, which possibly mitigates reductions in energy expenditure secondary to fat-free mass loss. Lastly, IF leads to better glucose homeostasis than SG, irrespective of sex.

BACKGROUND AND AIMS:Stomach cells can be converted to insulin-producing cells by Neurog3, MafA, and Pdxl over-expression. Enteroendocrine cells can be similarly made to produce insulin by the deletion of FOXO1. Characteristics and functional properties of FOXO1-expressing stomach cells are not known. METHODS:Using mice bearing a FOXO1-GFP knock-in allele and primary cell cultures, we examined the identity of FOXO1-expressing stomach cells and analyzed their features through loss-of-function studies with red-to-green fluorescent reporters. RESULTS:FOXO1 localizes to a subset of Neurog3 and parietal cells. FOXO1 deletion ex vivo or in vivo using Neurog3-cre or Atp4b-cre increased numbers of parietal cells, generated insulin- and C-peptide-immunoreactive cells, and raised Neurog3 messenger RNA. Gene expression and ChIP- seq experiments identified the cell cycle regulator cyclin E1 (CCNE1) as a FOXO1 target. CONCLUSION:FOXO1 is expressed in a subset of stomach cells. Its ablation increases parietal cells and yields insulin-immunoreactive cells, consistent with a role in lineage determination.

ABSTRACT Objective Murine-specific muricholic acids (MCAs) are reported to protect against obesity and associated metabolic disorders. However, the response of mice with genetic depletion of MCA to an obesogenic diet has not been evaluated. We used Cyp2c-deficient (Cyp2c −/− ) mice, which lack MCAs and thus have a human-like bile acid (BA) profile, to directly investigate the potential role of MCAs in diet-induced obesity. Methods Male and female Cyp2c −/− mice and wild-type controls were fed a standard chow diet or a high fat diet (HFD) for 18 weeks. We measured BA composition from a pool of liver, gallbladder, and intestine, as well as weekly body weight, food intake, lean and fat mass, systemic glucose homeostasis, energy expenditure, intestinal lipid absorption, fecal lipid, and energy content. Results Cyp2c deficiency depleted MCAs and caused other changes in BA composition, namely a decrease in the ratio of 12α-hydroxylated (12α-OH) BAs to non-12α-OH BAs, without altering the total BA levels. While wild-type male mice became obese after HFD-feeding, Cyp2c −/− male mice were protected from obesity and associated metabolic dysfunctions. Cyp2c −/− male mice also showed reduced intestinal lipid absorption and increased lipid excretion, which was reversed by oral gavage with the 12α-OH BA, taurocholic acid. Cyp2c −/− mice also showed increased liver damage, which appeared stronger in females. Conclusion MCA does not protect against diet-induced obesity but may protect against liver injury. Reduced lipid absorption in Cyp2c-deficient male mice is potentially due to a reduced ratio of 12α-OH/non-12α-OH BAs.

The work reported here is an extension of our previous findings in which supercritical composite particles (SCP) of alpha lipoic acid (ALA) masked with hydrogenated colza oil (HCO) named as ALA/HCO/SCP were obtained by the modified particles from gas-saturated solutions (PGSS) process in supercritical carbon dioxide in order to obscure the unpleasant taste and odor of ALA. The masking effect on ALA/HCO/SCP was compared with the widely used mechano-chemically masked formulation of ALA and HCO named as MC-50F. In the present study, ALA/HCO/SCP particles were found to have a significant improvement in regard to bitterness, numbness, and smell compared to ALA bulk powders suggesting they were well coated. The pharmacokinetic parameters for ALA/HCO/SCP and ALA bulk powder gave similar values but were significantly different from those of MC-50F. The amount of ALA absorbed into the body, in the administered ALA/HCO/SCP, was comparable to that absorbed by ALA bulk powder, whereas about half portion of ALA of the MC-50F was not absorbed, because the ALA/HCO/SCP particles were small enough and the particles of MC-50F were relatively large and had smaller specific surface area. Therefore, this study suggested a newly masked candidate may offer functional particles with maintained efficacy.

Background Some degree of weight regain is typically observed in human patients who undergo Sleeve Gastrectomy (SG), even if the majority of them do not return to their presurgical body weight. Although the majority of bariatric surgery patients are middle aged, most preclinical models of bariatric surgery utilize juvenile male mice. A long-term characterization of the response of mature, wild type, obese male mice to SG has not been performed. Methods Eight-month old C57bl/6J obese male mice were randomized to undergo SG, sham surgery without caloric restriction (SH) or sham surgery with caloric restriction to match body weight to the SG group (SWM). Body weight, body composition and glucose tolerance were matched at baseline. Mice were followed for 60 days following their respective surgeries. Results SG mice had a more pronounced percent weight loss than the SH group in the first post-operative month (p<0.05), along with fat mass loss (p<0.01). By the second post-operative month, the SG group started to regain fat mass, although it continued to be statistically lower than the SH group (p<0.05). Cumulative food intake was significantly lower in the SG group compared to SH group only in the first post-operative week (p<0.05), with both groups having similar cumulative food intake thereafter (p>0.05). SWM group had a significantly lower cumulative food intake throughout the study, except for week 1 (p<0.01). Glucose tolerance was only demonstrably better in the SG group compared to SH group at 8 weeks post-operatively (p<0.01). Plasma leptin was significantly lower in the SG group compared to both SWM and SH groups group by the second post-operative month (p<0.01), in spite of SG’s increasing fat mass accumulation. In the second post-operative month, both FGF-21 and GDF-15 were increased in the SH group compared to the SG and SWM groups (p<0.05), while there was no difference in plasma insulin among the three groups. Heat production was surprisingly higher in the SH group compared to the other two groups (p<0.05), even though brown adipose tissue Peroxisome Proliferator-Activated Receptor Gamma (PPARg) and Cidea mRNA expression were significantly higher in SG and SWM compared to SH (p<0.01). There was no change in BAT UCP-1 mRNA expression among the groups (p>0.05). There was also no change in fecal lipid content among the groups (p>0.05). Conclusions SG in obese, middle aged male mice leads is accompanied by fat mass regain in the second post-operative month, while plasma leptin levels continue to be significantly lower. This raises the question of whether the observed fat mass regain consists mostly of visceral adipose tissue. ### Competing Interest Statement The authors have declared no competing interest.

ObjectivesLipid mediators in the GI tract regulate satiation and satiety. Bile acids (BAs) regulate the absorption and metabolism of dietary lipid in the intestine, but their effects on lipid-regulated satiation and satiety are completely unknown. Investigating this is challenging because introducing excessive BAs or eliminating BAs strongly impacts GI functions. We used a mouse model (Cyp8b1–/–mice) with normal total BA levels, but alterations in the composition of the BA pool that impact multiple aspects of intestinal lipid metabolism. We tested two hypotheses: BAs affect food intake by (1) regulating production of the bioactive lipid oleoylethanolamide (OEA), which enhances satiety; or (2) regulating the quantity and localisation of hydrolysed fat in small intestine, which controls gastric emptying and satiation.DesignWe evaluated OEA levels, gastric emptying and food intake in wild-type and Cyp8b1–/–mice. We assessed the role of the fat receptor GPR119 in these effects using Gpr119–/–mice.ResultsCyp8b1–/–mice on a chow diet showed mild hypophagia. Jejunal OEA production was blunted in Cyp8b1–/–mice, thus these data do not support a role for this pathway in the hypophagia of Cyp8b1–/–mice. On the other hand, Cyp8b1 deficiency decreased gastric emptying, and this was dependent on dietary fat. GPR119 deficiency normalised the gastric emptying, gut hormone levels, food intake and body weight of Cyp8b1–/–mice.ConclusionBAs regulate gastric emptying and satiation by determining fat-dependent GPR119 activity in distal intestine.

Background Sleeve gastrectomy (SG) is currently the most frequently performed bariatric surgery in the United States. The majority of patients undergoing SG are middle aged women. Most preclinical models of bariatric surgery, however, utilize juvenile male mice. A long-term characterization of the response of mature wild type, obese female mice to SG has not been performed. Thus, we set out to characterize the response of middle aged obese female mice to SG. Methods Ten-month old C57bl/6J obese female mice were randomized to undergo SG, sham surgery without caloric restriction (SH) or sham surgery with caloric restriction to match body weight to the SG group (SWM). Body weight, body composition and glucose tolerance were matched at baseline. Mice were followed for 60 days following their respective surgeries. Results The SG group had a more pronounced percent weight loss than the SH and SWM control groups (p<0.05), while consuming more calories than the SWM group (p<0.05). The SG group had a significant improvement in glucose tolerance compared to the SH control group (p<0.05). Plasma leptin was significantly decreased in the SG and SWM group, compared to the SH group (p<0.01). Unexpectedly, FGF-21 was increased in the SH group compared to the SG and SWM groups (p<0.01), while there was no difference in plasma insulin among the three groups. Heat production was increased in the SG group compared to SWM and SH groups (p<0.001). SG also had a significantly increased mRNA expression of Uncoupling Protein 1 (UCP-1), Adiponectin and Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha (PGC1-alpha) in brown adipose tissue (BAT), compared to SWM and SH groups. Both SG and SWM groups had increased fecal lipid excretion (p<0.05), compared to the SH group. Conclusions SG in obese, middle aged female mice leads to sustained weight loss and blood glucose improvement. It appears that increased metabolism in BAT may be linked to these effects. ### Competing Interest Statement The authors have declared no competing interest.

Abstract A promising new therapy for type 1 diabetes is the reprogramming of gut enteroendocrine cells into cells that produce insulin. The mechanism by which gut epithelial cells are converted into cells that make insulin remains unknown. We have previously found that elimination of Foxo1 in neurogenin3 (Ngn3)-expressing cells of the intestine generates glucose-sensing, insulin-producing cells that are capable of reversing streptozotocin-induced diabetes. Others have reported that stomach cells have a similar property when made to express β-cell factors Ngn3, Pdx1, and MafA. Using mice bearing a Foxo1-GFP knock-in allele, we traced Foxo1-expressing cells in the gut to subpopulations of Ngn3+, as well as acid-secreting parietal stomach cells. To study these cells, we established a 2D co-culture method in which primary stomach cells are isolated from mice and cultured with embryonic fibroblasts. Deletion of Foxo1 in this system generated cells immunoreactive for insulin and C-peptide. Interestingly, Foxo1 ablation also altered the abundance of other gastric cell populations, including more parietal cells and decreased expression of stem cell marker, Lgr5. Tissue-specific elimination of Foxo1 in vivo in either Ngn3+ or parietal cells also resulted in the appearance of insulin+ cells, increased parietal cells, and reduced Lgr5 mRNA. To determine how Foxo1 regulated these changes, we used cells isolated from reporter mice that change from red to green after genetic recombination to collect Foxo1-deleted primary stomach cells using FACS. While the mRNA levels of many known Foxo1 targets did not change, cyclin E1 (CCNE1), which regulates G1 to S-phase progression of the cell cycle, was significantly decreased. Conversely, primary stomach cells overexpressing Foxo1 had increased levels of CCNE1. Finally, using ChIP-seq, we found that Foxo1 binds directly to the CCNE promoter in a nutrient-dependent manner. In summary, we show that Foxo1 is expressed in a subpopulation of stomach parietal cells and that it regulates their function through the cell cycle regulator, CCNE1.

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis.

Purpose of review Type 2 diabetes is associated with a characteristic dyslipidemia that may exacerbate cardiovascular risk. The causes of, and the effects of new antihyperglycemia medications on, this dyslipidemia, are under investigation. In an unexpected reciprocal manner, lowering LDL-cholesterol with statins slightly increases the risk of diabetes. Here we review the latest findings. Recent findings The inverse relationship between LDL-cholesterol and diabetes has now been confirmed by multiple lines of evidence. This includes clinical trials, genetic instruments using aggregate single nucleotide polymorphisms, as well as at least eight individual genes - HMGCR, NPC1L1, HNF4A, GCKR, APOE, PCKS9, TM6SF2, and PNPLA3 - support this inverse association. Genetic and pharmacologic evidence suggest that HDL-cholesterol may also be inversely associated with diabetes risk. Regarding the effects of diabetes on lipoproteins, new evidence suggests that insulin resistance but not diabetes per se may explain impaired secretion and clearance of VLDL-triglycerides. Weight loss, bariatric surgery, and incretin-based therapies all lower triglycerides, whereas SGLT2 inhibitors may slightly increase HDL-cholesterol and LDL-cholesterol. Summary Diabetes and lipoproteins are highly interregulated. Further research is expected to uncover new mechanisms governing the metabolism of glucose, fat, and cholesterol. This topic has important implications for treating type 2 diabetes and cardiovascular disease.

Prostaglandin E2 receptor 4–associated protein (EPRAP) is a key molecule in suppressing inflammatory responses in macrophages. EPRAP is expressed not only in macrophages but also in hepatocytes; however, the role of EPRAP in hepatocytes has not yet been defined. To examine the physiological role of hepatic EPRAP in mice, we performed the glucose tolerance test and the hyperinsulinemic-euglycemic clamp in high-fat sucrose diet (HFSD)-fed wild-type (WT) and Eprap null mice. We evaluated the contribution of EPRAP to gluconeogenesis by pyruvate tolerance test and primary hepatocyte experiments. Furthermore, lentivirus-expressing Eprap-specific small-hairpin RNA was injected in db/ db mice. HFSD-fed Eprap null mice had significantly lower blood glucose levels than HFSD-fed WT mice. Eprap null mice also had low glucose levels after fasting or pyruvic acid injection. Moreover, primary hepatocytes from Eprap-deficient mice showed decreased glucose production and lower expression of the Phosphoenol pyruvate carboxykinase and Glucose 6-phosphatase genes. Lentivirus-mediated hepatic Eprap suppression decreased glucose levels and the expression of gluconeogenic genes in db/ db mice. We conclude that EPRAP regulates gluconeogenesis in hepatocytes and is associated with hyperglycemia in diabetic mice. Our data suggest that suppression of EPRAP could be a novel strategy for the treatment of diabetes.

BackgroundChronic inflammation plays a key role in the pathogenesis of intracranial aneurysms (IAs). DPP‐4 (dipeptidyl peptidase‐4) inhibitors have anti‐inflammatory effects, including suppressing macrophage infiltration, in various inflammatory models. We examined whether a DPP‐4 inhibitor, anagliptin, could suppress the growth of IAs in a rodent aneurysm model. Methods and ResultsIAs were surgically induced in 7‐week‐old male Sprague Dawley rats, followed by oral administration of 300 mg/kg anagliptin. We measured the morphologic parameters of aneurysms over time and their local inflammatory responses. To investigate the molecular mechanisms, we used lipopolysaccharide‐treated RAW264.7 macrophages. In the anagliptin‐treated group, aneurysms were significantly smaller 2 to 4 weeks after IA induction. Anagliptin inhibited the accumulation of macrophages in IAs, reduced the expression of MCP‐1 (monocyte chemotactic protein 1), and suppressed the phosphorylation of p65. In lipopolysaccharide‐stimulated RAW264.7 cells, anagliptin treatment significantly reduced the production of tumor necrosis factor α, MCP‐1, and IL‐6 (interleukin 6) independent of GLP‐1 (glucagon‐like peptide 1), the key mediator in the antidiabetic effects of DPP‐4 inhibitors. Notably, anagliptin activated ERK5 (extracellular signal–regulated kinase 5), which mediates the anti‐inflammatory effects of statins, in RAW264.7 macrophages. Preadministration with an ERK5 inhibitor blocked the inhibitory effect of anagliptin on MCP‐1 and IL‐6 expression. Accordingly, the ERK5 inhibitor also counteracted the suppression of p65 phosphorylation in vitro. ConclusionsA DPP‐4 inhibitor, anagliptin, prevents the growth of IAs via its anti‐inflammatory effects on macrophages.

Microglia are thought to play key roles in the progression of Alzheimer disease (AD). Overactivated microglia produce proinflammatory cytokines, such as tumor necrosis factor-alpha, which appear to contribute to disease progression. Previously, we reported that prostaglandin E-2 type 4 receptor-associated protein (EPRAP) promotes microglial activation. We crossed human amyloid precursor protein transgenic mice from strain J20(+/-) onto an EPRAP-deficient background to determine the role of EPRAP in AD. Behavioral tests were performed in 5-month-old male J20(+/-)EPRAP(+/+) and J20(+/-)EPRAP(-/-) mice. EPRAP deficiency reversed the reduced anxiety of J20(+/-) mice but did not affect hyperactivity. No differences in spatial memory were observed between J20(+/-)EPRAP(+/+) and J20(+/-)EPRAP(-/-) mice. In comparison with J20(+/-)EPRAP(+/+), J20(+/-)EPRAP(-/-) mice exhibited less microglial accumulation and reductions in the Cd68 and tumor necrosis factor-alpha mRNAs in the prefrontal cortex and hippocampus. No significant differences were found between the two types of mice in the amount of amyloid-beta 40 or 42 in the cortex and hippocampus. J20(+/-)EPRAP(-/-) mice reversed the reduced anxiety-like behavior and had reduced microglial activation compared with J20(+/-)EPRAP(+/+) mice. Further research is required to identify the role of EPRAP in AD, but our results indicate that EPRAP may be related to behavioral and psychological symptoms of dementia and inflammation in patients with AD.

EP4 receptor-associated protein (EPRAP) is a newly identified molecule that regulates macrophage activation. We recently demonstrated the presence of EPRAP in the mice brain; however, little is known about the function of EPRAP in this tissue. Therefore, we investigated the role of EPRAP in behavior and emotion using behavioral analysis in mice. In this study, we subjected EPRAP-deficient (KO) mice and wild-type C57BL/6 (WT) mice to a battery of behavioral tests. EPRAP-KO mice tended to have shorter latencies to fall in the wire hang test, but had normal neuromuscular strength. EPRAP-KO mice exhibited elevated startle responses and reduced pre-pulse inhibition. Compared with WT mice, EPRAP-KO mice increased depression-like behavior in the forced swim test. These abnormal behaviors partially mimic symptoms of depression, attention deficit hyperactivity disorder (ADHD) and schizophrenia. Methylphenidate administration increased locomotor activity less in EPRAP-KO mice than in WT mice. Finally, levels of norepinephrine were reduced in the EPRAP-KO mouse brain. These behavioral abnormalities in EPRAP-KO mice may result from the dysfunction of monoamines, in particular, norepinephrine. Our results suggest that EPRAP participates in the pathogenesis of various behavioral disorders.