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Fibroblast growth factor 21 (FGF21) plays a key role in hepatic lipid metabolism and long-acting FGF21 analogs have emerged aspromising drug candidates for the treatment of nonalcoholic steatohepatitis (NASH). It remains to characterize this drug class intranslational animal models that recapitulate the etiology and hallmarks of human disease. To this end, we evaluated the longactingFGF21 analog PF-05231023 in the GAN (Gubra Amylin NASH) diet-induced obese (DIO) and biopsy-confirmed mousemodel of NASH. Male C57BL/6J mice were fed the GAN diet high in fat, fructose, and cholesterol for 34 wk before the start ofthe study. GAN DIO-NASH mice with biopsy-confirmed NAFLD Activity Score (NAS 5) and fibrosis (stage F1) were biweeklyadministered with PF-05231023 (10 mg/kg sc) or vehicle (sc) for 12 wk. Vehicle-dosed chow-fed C57BL/6J mice served ashealthy controls. Pre-to-post liver biopsy histopathological scoring was performed for within-subject evaluation of NAFLD ActivityScore (NAS) and fibrosis stage. Terminal endpoints included quantitative liver histology and transcriptome signatures as well asblood and liver biochemistry. PF-05231023 significantly reduced body weight, hepatomegaly, plasma transaminases, andplasma/liver lipids in GAN DIO-NASH mice. Notably, PF-05231023 reduced both NAS (2-point improvement) and fibrosis stage(1-point improvement). Improvements in NASH and fibrosis severity were supported by reduced quantitative histological markersof steatosis, inflammation, and fibrogenesis as well as improvements in disease-associated liver transcriptome signatures. In conclusion,PF-05231023 reduces NASH and fibrosis severity in a translational biopsy-confirmed mouse model of NASH, supportingdevelopment of FGF21 analogs for the treatment of NASH.NEW & NOTEWORTHY It is unclear if long-acting FGF21 analogs have antifibrotic efficacy in NASH. We therefore profiled theclinically relevant FGF21 analog PF-05231023 in a translational diet-induced obese and biopsy-confirmed mouse model ofNASH. We found PF-05231023 to exert hepatoprotective effects as indicated by notable improvements in plasma markers andhistological hallmarks of NASH, including improved fibrosis stage. Collectively, the present study supports the continued explorationof long-acting FGF21 analogs for the treatment of NASH and other fibrotic diseases. Fibroblast growth factor 21 (FGF21) plays a key role in hepatic lipid metabolism and long-acting FGF21 analogs have emerged as promising drug candidates for the treatment of nonalcoholic steatohepatitis (NASH). It remains to characterize this drug class in translational animal models that recapitulate the etiology and hallmarks of human disease. To this end, we evaluated the long-acting FGF21 analog PF-05231023 in the GAN (Gubra Amylin NASH) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. Male C57BL/6J mice were fed the GAN diet high in fat, fructose, and cholesterol for 34 wk before the start of the study. GAN DIO-NASH mice with biopsy-confirmed NAFLD Activity Score (NAS ≥5) and fibrosis (stage ≥F1) were biweekly administered with PF-05231023 (10 mg/kg sc) or vehicle (sc) for 12 wk. Vehicle-dosed chow-fed C57BL/6J mice served as healthy controls. Pre-to-post liver biopsy histopathological scoring was performed for within-subject evaluation of NAFLD Activity Score (NAS) and fibrosis stage. Terminal endpoints included quantitative liver histology and transcriptome signatures as well as blood and liver biochemistry. PF-05231023 significantly reduced body weight, hepatomegaly, plasma transaminases, and plasma/liver lipids in GAN DIO-NASH mice. Notably, PF-05231023 reduced both NAS (≥2-point improvement) and fibrosis stage (1-point improvement). Improvements in NASH and fibrosis severity were supported by reduced quantitative histological markers of steatosis, inflammation, and fibrogenesis as well as improvements in disease-associated liver transcriptome signatures. In conclusion, PF-05231023 reduces NASH and fibrosis severity in a translational biopsy-confirmed mouse model of NASH, supporting development of FGF21 analogs for the treatment of NASH.NEW & NOTEWORTHY It is unclear if long-acting FGF21 analogs have antifibrotic efficacy in NASH. We therefore profiled the clinically relevant FGF21 analog PF-05231023 in a translational diet-induced obese and biopsy-confirmed mouse model of NASH. We found PF-05231023 to exert hepatoprotective effects as indicated by notable improvements in plasma markers and histological hallmarks of NASH, including improved fibrosis stage. Collectively, the present study supports the continued exploration of long-acting FGF21 analogs for the treatment of NASH and other fibrotic diseases.

AIM: Liraglutide treatment is associated with gallbladder-related disorders and has been shown to delay postprandial gallbladder refilling. The gut hormones cholecystokinin (CCK), fibroblast growth factor 19 (FGF19) and glucagon-like peptide 2 (GLP-2), are known to regulate gallbladder motility and may be implicated in gallbladder-related disorders associated with liraglutide treatment.MATERIALS AND METHODS: In a double-blind, 12-week trial, 52 participants [50% male, age 47.6 ± 10.0 years, body mass index 32.6 ± 3.4 kg/m 2 (mean ± standard deviation)] with obesity were randomized 1:1 to once-daily subcutaneous liraglutide (escalated from 0.6 mg to 3.0 mg once-daily) or placebo. During liquid meal tests performed at baseline, after the first dose and following 12 weeks of treatment, we evaluated postprandial gallbladder dynamics and plasma responses of CCK, FGF19 and GLP-2. RESULTS: Liraglutide reduced postprandial FGF19 after the first dose [area under the curve (AUC) 0-240 min 24.8 vs. 48.0 min × ng/ml, treatment ratio (TR) (95% confidence interval) 0.52 (0.39; 0.69)] and following 12 weeks of treatment [AUC 0-240 min 33.7 vs. 48.5 ng/ml × min, TR 0.69 (0.52; 0.93)]. Liraglutide also reduced postprandial GLP-2 responses (AUC 0-240 min 3650 vs. 4894 min × pmol/L, TR 0.75 (0.62; 0.90)] following the first dose as well as after 12 weeks [AUC 0-240 min 3760 vs. 4882 min × pmol/L, TR 0.77 (0.60; 0.99)]. Liraglutide increased postprandial responses of CCK after the first dose [AUC 0-240 min 762 vs. 670 min × pmol/L; TR 1.14 (0.97; 1.33)] and following 12 weeks of treatment [AUC 0-240 min 873 vs. 628 min × pmol/L; TR 1.39 (1.12; 1.73)]. CONCLUSION: Compared with placebo, treatment with liraglutide decreased postprandial FGF19 and GLP-2 concentrations and increased postprandial CCK concentrations, which may explain the delayed postprandial gallbladder refilling observed in individuals with obesity treated with liraglutide.

The gut microbiota impacts systemic levels of multiple metabolites including NAD+ precursors through diverse pathways. Nicotinamide riboside (NR) is an NAD+ precursor capable of regulating mammalian cellular metabolism. Some bacterial families express the NR-specific transporter, PnuC. We hypothesized that dietary NR supplementation would modify the gut microbiota across intestinal sections. We determined the effects of 12 weeks of NR supplementation on the microbiota composition of intestinal segments of high-fat diet-fed (HFD) rats. We also explored the effects of 12 weeks of NR supplementation on the gut microbiota in humans and mice. In rats, NR reduced fat mass and tended to decrease body weight. Interestingly, NR increased fat and energy absorption but only in HFD-fed rats. Moreover, 16S rRNA gene sequencing analysis of intestinal and fecal samples revealed an increased abundance of species within Erysipelotrichaceae and Ruminococcaceae families in response to NR. PnuC-positive bacterial strains within these families showed an increased growth rate when supplemented with NR. The abundance of species within the Lachnospiraceae family decreased in response to HFD irrespective of NR. Alpha and beta diversity and bacterial composition of the human fecal microbiota were unaltered by NR, but in mice, the fecal abundance of species within Lachnospiraceae increased while abundances of Parasutterella and Bacteroides dorei species decreased in response to NR. In conclusion, oral NR altered the gut microbiota in rats and mice, but not in humans. In addition, NR attenuated body fat mass gain in rats, and increased fat and energy absorption in the HFD context.

Aims/hypothesis Metabolic effects of intermittent unhealthy lifestyle in young adults are poorly studied. We investigated the gluco-metabolic and hepatic effects of participation in Roskilde Festival (1 week of binge drinking and junk food consumption) in young, healthy males. Methods Fourteen festival participants (FP) were studied before, during and after 1 week’s participation in Roskilde Festival. Fourteen matched controls (CTRL) who did not participate in Roskilde Festival or change their lifestyle in other ways were investigated along a similar timeline. Results The FP group consumed more alcohol compared to their standard living conditions (2.0 ± 3.9 vs 16.3 ± 8.3 units/day, P < 0.001). CTRLs did not change their alcohol consumption. AUC for glucose during OGTT did not change in either group. C-peptide responses increased in the FP group (206 ± 24 vs 236 ± 17 min × nmol/L, P = 0.052) and the Matsuda index of insulin sensitivity decreased (6.2 ± 2.4 vs 4.7 ± 1.4, P = 0.054). AUC for glucagon during oral glucose tolerance test (OGTT) increased in the FP group (1037 ± 90 vs 1562 ± 195 min × pmol/L, P = 0.003) together with fasting fibroblast growth factor 21 (FGF21) (62 ± 30 vs 132 ± 72 pmol/L, P < 0.001), growth differentiation factor 15 (GDF5) (276 ± 78 vs 330 ± 83 pg/mL, P = 0.009) and aspartate aminotransferase (AST) levels (37.6 ± 6.8 vs 42.4 ± 11 U/L, P = 0.043). Four participants (29%) developed ultrasound-detectable steatosis and a mean strain elastography-assessed liver stiffness increased (P = 0.026) in the FP group. Conclusions/Interpretation Participation in Roskilde Festival did not affect oral glucose tolerance but was associated with a reduction in insulin sensitivity, increases in glucagon, FGF21, GDF15 and AST and lead to increased liver stiffness and, in 29% of the participants, ultrasound-detectable hepatic steatosis.

Acute and adaptive changes in systemic markers of oxidatively generated nucleic acid modifications (i.e., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)) as well as inflammatory cytokines (i.e., C-reactive protein, interleukin-6, interleukin-10, and tumour necrosis factor alpha), a liver hormone (i.e., fibroblast growth factor 21 (FGF21)), and bone metabolism markers (sclerostin, osteocalcin, C-terminal telopeptide, and N-terminal propeptide of type 1 procollagen) were investigated following a marathon in 20 study participants. Immediate changes were observed in inflammatory cytokines, FGF21, and bone metabolism markers following the marathon. In contrast, no immediate changes in urinary excretion of 8-oxodG and 8-oxoGuo were evident. Four days after the marathon, decreased urinary excretion of 8-oxodG (-2.9 (95% CI -4.8;-1.1) nmol/24 h,8-oxodG: 8-oxo-7,8-dihydro-2'-deoxyguanosine; 8-oxoGuo: 8-oxo-7,8-dihydroguanosine; CI: confidence interval; CTX: C-terminal telopeptide of type 1 collagen; DXA: dual-energy X-ray absorptiometry; ELISA: enzyme-linked immunosorbent assay; FGF21: Fibroblast growth factor 21; h: hour; hsCRP: high sensitivity C-reactive protein; IL: interleukin; IQR: interquartile range; MS: mass spectrometry: P1NP: N-terminal propeptide of type 1 procollagen; TNFα: tumour necrosis factor alpha; UPLC: ultra-performance liquid chromatography.

BACKGROUND & AIMS: The sucrase-isomaltase (SI) c.273_274delAG loss-of-function variant is common in Arctic populations and causes congenital sucrase-isomaltase deficiency, which is an inability to break down and absorb sucrose and isomaltose. Children with this condition experience gastrointestinal symptoms when dietary sucrose is introduced. We aimed to describe the health of adults with sucrase-isomaltase deficiency.METHODS: The association between c.273_274delAG and phenotypes related to metabolic health was assessed in 2 cohorts of Greenlandic adults (n = 4922 and n = 1629). A sucrase-isomaltase knockout (Sis-KO) mouse model was used to further elucidate the findings.RESULTS: Homozygous carriers of the variant had a markedly healthier metabolic profile than the remaining population, including lower body mass index (β [standard error], -2.0 [0.5] kg/m2; P = 3.1 × 10-5), body weight (-4.8 [1.4] kg; P = 5.1 × 10-4), fat percentage (-3.3% [1.0%]; P = 3.7 × 10-4), fasting triglyceride (-0.27 [0.07] mmol/L; P = 2.3 × 10-6), and remnant cholesterol (-0.11 [0.03] mmol/L; P = 4.2 × 10-5). Further analyses suggested that this was likely mediated partly by higher circulating levels of acetate observed in homozygous carriers (β [standard error], 0.056 [0.002] mmol/L; P = 2.1 × 10-26), and partly by reduced sucrose uptake, but not lower caloric intake. These findings were verified in Sis-KO mice, which, compared with wild-type mice, were leaner on a sucrose-containing diet, despite similar caloric intake, had significantly higher plasma acetate levels in response to a sucrose gavage, and had lower plasma glucose level in response to a sucrose-tolerance test.CONCLUSIONS: These results suggest that sucrase-isomaltase constitutes a promising drug target for improvement of metabolic health, and that the health benefits are mediated by reduced dietary sucrose uptake and possibly also by higher levels of circulating acetate.

AIMS: To evaluate the effect of curcumin treatment on hepatic fat content in obese individuals.MATERIALS AND METHODS: In a double-blind, parallel-group trial, 37 obese, non-diabetic individuals were randomised to placebo or curcumin treatment for six weeks. Curcumin was dosed as lecithin-formulated tablet; 200 mg twice daily. Primary endpoint was hepatic fat content as assessed by magnetic resonance spectroscopy (MRS). Other endpoints included anthropometric measurements, hepatic biomarkers including FibroScan® measurements, metabolic parameters, inflammation markers, appetite measures and ad libitum food intake.RESULTS: Baseline characteristics (mean ± SD): age 46 ±14 years; hepatic fat content 12.2 ± 8.8 percent points; body mass index 38.8 ±6.1 kg/m 2 ; waist circumference 125.8 ± 12.3 cm. After six weeks treatment with curcumin, hepatic fat content was changed by -0.86 percent points (95% CI -3.65;1.94) compared to 0.71 percent points (95% CI -2.08;3.51) with placebo, thus resulting in a non-significant estimated treatment difference of -1.57 percent points (95% CI -5.36; 2.22, P = 0.412). Compared to placebo, curcumin treatment caused small reductions in fasting plasma glucose (estimated treatment difference (ETD) -0.24 mmol/L (95% CI -0.45; -0.03)), triglycerides (ETD (percentage change) -20.22% (95% CI -33.21; -6.03)), and gamma glutamyltransferase (ETD (percentage change) -15.70 (95% CI -23.32; -7.32)), but except for gamma glutamyltransferase, none of these differences remained statistically significant after adjusting for multiple testing. Treatment was well tolerated. CONCLUSIONS: Compared to placebo, curcumin treatment for six weeks had no significant effect on MRS-assessed hepatic fat content in obese individuals with primarily mild steatosis. Curcumin was well tolerated. This article is protected by copyright. All rights reserved.

The liver-derived hormone fibroblast growth factor 21 (FGF21) has recently been linked to preference for sweet-tasting food. We hypothesized, that surgery-induced changes in FGF21 could mediate the reduction in sweet food intake and preference following bariatric surgery. Forty participants (35 females) with severe obesity (BMI ≥ 35 kg/m2 ) scheduled for roux-en-y gastric bypass (n = 30) or sleeve gastrectomy (n = 10) were included. Pre-and postprandial responses of intact plasma FGF21 as well as intake of sweet-tasting food assessed at a buffet meal test, the hedonic evaluation of sweet taste assessed using an apple juice with added sucrose and visual analog scales, and sweet taste sensitivity were assessed before and 6 months after bariatric surgery. In a cross-sectional analysis pre-surgery, pre-and postprandial intact FGF21 levels were negatively associated with the hedonic evaluation of a high-sucrose juice sample (p = 0.03 and p = 0.02). However, no changes in pre- (p = 0.24) or postprandial intact FGF21 levels were found 6 months after surgery (p = 0.11), and individual pre-to postoperative changes in pre-and postprandial intact FGF21 levels were not found to be associated with changes in intake of sweet foods, the hedonic evaluation of sweet taste or sweet taste sensitivity (all p ≥ 0.10). In conclusion, we were not able to show an effect of bariatric surgery on circulating FGF21, and individual postoperative changes in FGF21 were not found to mediate an effect of surgery on sweet food intake and preference.

Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.

Background Ingestion of the calorically dense compound alcohol may cause metabolic disturbances including hypoglycaemia, hepatic steatosis and insulin resistance, but the underlying mechanisms are uncertain. The gastrointestinal tract is well recognised as a major influencer on glucose, protein and lipid metabolism, but its role in alcohol metabolism remains unclear. Objective To examine the effects of oral and intravenous alcohol, respectively, on plasma concentrations of several gluco-regulatory hormones including serum/plasma insulin, C-peptide, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1) and fibroblast growth factor 21 (FGF21). Design and methods In a double-blinded, randomised, crossover design, we subjected 12 healthy men to intragastric ethanol infusion (IGEI) and an isoethanolaemic intravenous ethanol infusion (IVEI) (0.7 g alcohol per kg body weight), respectively, on two separate experimental days. Results Isoethanolaemia during the two alcohol administration forms was obtained (P = 0.38). During both interventions, plasma glucose peaked after ~30 min and thereafter fell below baseline concentrations. GIP and GLP-1 concentrations were unaffected by the two interventions. Insulin concentrations were unaffected by IGEI but decreased during IVEI. C-peptide, insulin secretion rate and glucagon concentrations were lowered similarly during IGEI and IVEI. FGF21 concentrations increased dramatically (nine-fold) and similarly during IGEI and IVEI. Conclusions Alcohol does not seem to affect the secretion of incretin hormones but decreased insulin and glucagon secretion independently of gut-derived factors. IGEI as well as IVEI potently stimulate FGF21 secretion indicating a gut-independent effect of alcohol on FGF21 secretion in humans.

Fibroblast growth factor 21 (FGF21) is a liver-derived hormone with pleiotropic metabolic effects. Its production is induced by various dietary imbalances in mice (including low-protein and ketogenic diets, fructose feeding and ethanol), hinting that it might influence food preference given the role of the liver in maintaining homeostatic levels of circulating nutrients. In 2016, it was shown that FGF21 selectively inhibits consumption of sugars and the primary product of their fermentation, ethanol, but not intake of fat, protein or complex carbohydrates. Since then, studies have sought to unravel this selectivity, its physiological purpose and translational relevance, as well as delineate the neural mechanisms involved. Initially found to impact ingestive behaviours in mice and non-human primates, FGF21 is also induced in humans by sugars and, far more dramatically, by acute alcohol intake. Genetic studies have revealed that patterns of weekly candy and alcohol consumption are associated with genetic variants in FGF21 and its co-receptor β-klotho (KLB), suggesting that liking for sugar, and fermented sugar, may be influenced by natural variation in FGF21 signal strength in humans. Herein, we discuss our nascent understanding of FGF21 as a selective negative regulator of sugar and alcohol appetite as well as reasons why such a peculiar system may have evolved in mammals. Uncovering the regulatory network governing sugar, and fermented sugar, intake could provide new opportunities to improve dietary choices in a population suffering from Western diet-induced diseases fuelled in part by a runaway sweet - and alcohol - tooth.

Aim/hypothesis Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved. Methods We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis. Results In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3’s cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion. Conclusions/interpretation Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions. Graphical abstract

Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid–derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid–containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet–induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid–based therapeutics.

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD+. As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet–fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression.

Ethanol intake increases plasma concentrations of triglycerides and chronic ethanol use impairs lipid metabolism and causes chronic inflammation. The gut plays an important role in metabolic handling of nutrients, including lipids, and a leaky gut associated with alcohol intake, allowing inflammatory signals to the portal vein, has been proposed to constitute a mechanism by which ethanol induces hepatic inflammation. We compared the effects of enteral and parenteral administration of ethanol on a range of circulating inflammation markers (including soluble CD163, a marker of liver macrophage activation), lipids, cholecystokinin (CCK) and fibroblast growth factor 19 (FGF19) as well as gallbladder volume. On two separate and randomized study days, we subjected healthy men (n = 12) to double-blinded intragastric ethanol infusion (IGEI) and isoethanolemic intravenous ethanol infusion (IVEI). Blood was sampled and ultrasonographic evaluation of gallbladder volume was performed at frequent intervals for 4 h after initiation of ethanol administration on both days. Little or no effects were observed on plasma levels of inflammation markers during IGEI and IVEI, respectively. Circulating levels of total, low-density lipoprotein and high-density lipoprotein cholesterol decreased after ethanol administration independently of the administration form. Triglyceride and very low-density lipoprotein (VLDL) cholesterol concentrations increased more after IGEI compared to IVEI. IVEI had no effect on plasma CCK and caused an increased gallbladder volume whereas IGEI elicited a CCK response (P < 0.0001) without affecting gallbladder volume. Circulating FGF19 concentrations decreased equally in response to both ethanol administration forms. In conclusion, by evaluating a range of circulating inflammation markers during IGEI and IVEI we were not able to detect signs of systemic low-grade inflammation originating from the presence of ethanol in the gut. IVEI increased gallbladder volume whereas IGEI increased plasma CCK (with neutral effect on gallbladder volume), increased plasma VLDL cholesterol and triglyceride concentrations; indicating that the enteral route of administration may influence ethanol's effects on lipid metabolism.

Fibroblast growth factor (FGF) 21, a unique, largely liver-derived endocrine member of the FGF superfamily, is often thought of as a fasting factor owing to its induction in rodents during starvation. However, FGF21 is not increased by fasting for periods of

Excessive alcohol consumption is a major health and social issue in our society. Pharmacologic administration of the endocrine hormone fibroblast growth factor 21 (FGF21) suppresses alcohol consumption through actions in the brain in rodents, and genome wide association studies have identified single nucleotide polymorphisms in genes involved with FGF21 signaling as being associated with increased alcohol consumption in humans. However, the neural circuit(s) through which FGF21 signals to suppress alcohol consumption are unknown, as are its effects on alcohol consumption in higher organisms. Here, we demonstrate that administration of an FGF21 analogue to alcohol-preferring non-human primates reduces alcohol intake by 50%. Further, we reveal that FGF21 suppresses alcohol consumption through a projection-specific sub-population of KLB-expressing neurons in the basolateral amygdala. Our results illustrate how FGF21 suppresses alcohol consumption through a specific population of neurons in the brain and demonstrate its therapeutic potential in non-human primate models of excessive alcohol consumption.

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to nicotinamide adenine dinucleotide (NAD+). As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease (NAFLD), we hypothetized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine, choline-free 60% high-fat diet (MCD), hepatocyte-specific Nampt knockout mice (HNKO) accumulated less triglyceride than wild-type littermates, but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet (PD). This HNKO phenotype was also associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in PD-fed HNKO liver. In addition, fibrotic area in HNKO liver sections negatively correlated with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside (NR) and nicotinic acid. Mass spectrometry (MS)-based proteomic analysis revealed that NR supplementation rescued hepatic levels of oxidoreductase- and OXPHOS proteins. Finally, single nucleus RNAseq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis due to low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and NAFLD progression.

AIM To evaluate the pharmacodynamics of compounds in clinical development for nonalcoholic steatohepatitis (NASH) in obese mouse models of biopsy-confirmed NASH. METHODS Male wild-type C57BL/6J mice (DIO-NASH) and Lepob/ob (ob/ob-NASH) mice were fed a diet high in trans-fat (40%), fructose (20%) and cholesterol (2%) for 30 and 21 wk, respectively. Prior to treatment, all mice underwent liver biopsy for confirmation and stratification of liver steatosis and fibrosis, using the nonalcoholic fatty liver disease activity score (NAS) and fibrosis staging system. The mice were kept on the diet and received vehicle, liraglutide (0.2 mg/kg, SC, BID), obeticholic acid (OCA, 30 mg/kg PO, QD), or elafibranor (30 mg/kg PO, QD) for eight weeks. Within-subject comparisons were performed on changes in steatosis, inflammation, ballooning degeneration, and fibrosis scores. In addition, compound effects were evaluated by quantitative liver histology, including percent fractional area of liver fat, galectin-3, and collagen 1a1. RESULTS Liraglutide and elafibranor, but not OCA, reduced body weight in both models. Liraglutide improved steatosis scores in DIO-NASH mice only. Elafibranor and OCA reduced histopathological scores of hepatic steatosis and inflammation in both models, but only elafibranor reduced fibrosis severity. Liraglutide and OCA reduced total liver fat, collagen 1a1, and galectin-3 content, driven by significant reductions in liver weight. The individual drug effects on NASH histological endpoints were supported by global gene expression (RNA sequencing) and liver lipid biochemistry. CONCLUSION DIO-NASH and ob/ob-NASH mouse models show distinct treatment effects of liraglutide, OCA, and elafibranor, being in general agreement with corresponding findings in clinical trials for NASH. The present data therefore further supports the clinical translatability and utility of DIO-NASH and ob/ob-NASH mouse models of NASH for probing the therapeutic efficacy of compounds in preclinical drug development for NASH.

Objective Augmenting nicotinamide adenine dinucleotide (NAD+) metabolism through dietary provision of NAD+ precursor vitamins translates to improved glucose handling in rodent models of obesity and diabetes. Preclinical evidence suggests that the NAD+/SIRT1 axis may be implicated in modulating important gut-related aspects of glucose regulation. We sought to test whether NAD+ precursor supplementation with nicotinamide riboside (NR) affects β-cell function, α-cell function, and incretin hormone secretion as well as circulating bile acid levels in humans. Design A 12-week randomized, double-blind, placebo-controlled, parallel-group trial in 40 males with obesity and insulin resistance allocated to NR at 1000 mg twice daily (n = 20) or placebo (n = 20). Two-hour 75-g oral glucose tolerance tests were performed before and after the intervention, and plasma concentrations of glucose, insulin, C-peptide, glucagon, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were determined. β-Cell function indices were calculated based on glucose, insulin, and C-peptide measurements. Fasting plasma concentrations of bile acids were determined. Results NR supplementation during 12 weeks did not affect fasting or postglucose challenge concentrations of glucose, insulin, C-peptide, glucagon, GLP-1, or GIP, and β-cell function did not respond to the intervention. Additionally, no changes in circulating adipsin or bile acids were observed following NR supplementation. Conclusion The current study does not provide evidence to support that dietary supplementation with the NAD+ precursor NR serves to impact glucose tolerance, β-cell secretory capacity, α-cell function, and incretin hormone secretion in nondiabetic males with obesity. Moreover, bile acid levels in plasma did not change in response to NR supplementation.

Background There is a marked need for improved animal models of nonalcoholic steatohepatitis (NASH) to facilitate the development of more efficacious drug therapies for the disease. Methods Here, we investigated the development of fibrotic NASH in male Wistar rats fed a choline-deficient l-amino acid-defined (CDAA) diet with or without cholesterol supplementation for subsequent assessment of drug treatment efficacy in NASH biopsy-confirmed rats. The metabolic profile and liver histopathology were evaluated after 4, 8, and 12 weeks of dieting. Subsequently, rats with biopsy-confirmed NASH were selected for pharmacological intervention with vehicle, elafibranor (30 mg/kg/day) or obeticholic acid (OCA, 30 mg/kg/day) for 5 weeks. Results The CDAA diet led to marked hepatomegaly and fibrosis already after 4 weeks of feeding, with further progression of collagen deposition and fibrogenesis-associated gene expression during the 12-week feeding period. Cholesterol supplementation enhanced the stimulatory effect of CDAA on gene transcripts associated with fibrogenesis without significantly increasing collagen deposition. Pharmacological intervention with elafibranor, but not OCA, significantly reduced steatohepatitis scores, and fibrosis-associated gene expression, however, was unable to prevent progression in fibrosis scores. Conclusion CDAA-fed rats develop early-onset progressive NASH, which offers the opportunity to probe anti-NASH compounds with potential disease-modifying properties.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone produced by the liver that regulates nutrient and metabolic homeostasis. FGF21 production is increased in response to macronutrient imbalance and signals to the brain to suppress sugar intake and sweet-taste preference. However, the central targets mediating these effects have been unclear. Here, we identify FGF21 target cells in the hypothalamus and reveal that FGF21 signaling to glutamatergic neurons is both necessary and sufficient to mediate FGF21-induced sugar suppression and sweet-taste preference. Moreover, we show that FGF21 acts directly in the ventromedial hypothalamus (VMH) to specifically regulate sucrose intake, but not non-nutritive sweet-taste preference, body weight, or energy expenditure. Finally, our data demonstrate that FGF21 affects neuronal activity by increasing activation and excitability of neurons in the VMH. Thus, FGF21 signaling to glutamatergic neurons in the VMH is an important component of the neurocircuitry that functions to regulate sucrose intake.

Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD(+)-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD(+) levels, but the metabolic phenotype of Nampt-deficient hepatocytes in mice is unknown. Here, we assessed Nampt's role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific Nampt knockout (HNKO) mice, having a 50% reduction of liver NAD(+) levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD(+) levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD(+), suggesting that the mitochondrial NAD(+) pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [(15)N]glutamine, HNKO hepatocytes had a higher [(15)N]NAD(+) enrichment than WT hepatocytes, indicating that HNKO mice compensate through de novo NAD(+) synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD(+) levels and that the Nampt knockout has only minor consequences for mitochondrial function in the mouse liver.

Introduction: The mechanisms behind the detrimental effects of ethanol on the liver remain uncertain. It has been suggested that the presence of ethanol in the gut lumen promotes a ’leaky gut’ allowing bacterial products to translocate to the bloodstream causing an inflammatory response. Furthermore, the ethanol-induced hormone fibroblast growth factor 21 (FGF-21) has emerged as a potential safeguard from ethanol-induced liver injury. Here, we aimed to investigate how ethanol administered intragastrically and intravenously, respectively, influences circulating markers of inflammation and FGF-21. Materials and Method: In a double-blinded, randomized cross-over design, we subjected 12 fasted healthy men (age 25.3±3.9 years; body mass index (BMI) 22.6±2.6 kg/m2 (mean±SD)) to intragastrically instilled ethanol (0.70 g ethanol per kg body weight in a 20% (v/w) solution infused over 5 minutes) and isoethanolaemic iv ethanol infusion, respectively, on two separate experimental days, and evaluated a range of circulating markers of inflammation and liver function as well as FGF-21. Results: No increment in plasma sCD163, or any other inflammation markers (lipopolysaccharide binding protein, tumor necrosis factor-α, interferon-γ, interleukin-10 (IL), IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, and IL-8), was observed. Plasma FGF-21 levels increased significantly (nine times baseline level), similarly and throughout both experimental days. Conclusion: Neither ’oral’ nor iv ethanol elicited significant changes in circulating markers of macrophage activation or inflammation, but increased plasma FGF-21 significantly regardless of the administration form suggesting a direct ethanol effect on FGF-21 secretion. Disclosure A.R. Lanng: None. L.S. Gasbjerg: Stock/Shareholder; Self; Antag Therapeutics. N.C. Bergmann: Employee; Self; Zealand Pharma A/S. H. Møller: Other Relationship; Self; IQ-products. M. Gillum: None. H. Gronbaek: Board Member; Self; Ipsen Biopharmaceuticals, Inc., Novartis AG. Research Support; Self; Intercept Pharmaceuticals, Inc., AbbVie Inc., Novo Nordisk A/S. T. Vilsbøll: Advisory Panel; Self; Amgen Inc., Boehringer Ingelheim GmbH, Eli Lilly and Company, AstraZeneca, MSD K.K., Sanofi, Novo Nordisk A/S. Consultant; Self; Amgen Inc., Boehringer Ingelheim GmbH, Eli Lilly and Company, AstraZeneca, MSD K.K., Sanofi, Novo Nordisk A/S. Speaker's Bureau; Self; Amgen Inc., Boehringer Ingelheim GmbH, Eli Lilly and Company, AstraZeneca, MSD K.K., Sanofi, Novo Nordisk A/S. Research Support; Self; Eli Lilly and Company, Novo Nordisk A/S. F.K. Knop: Advisory Panel; Self; AstraZeneca. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca. Speaker's Bureau; Self; AstraZeneca. Advisory Panel; Self; Eli Lilly and Company. Speaker's Bureau; Self; Eli Lilly and Company. Advisory Panel; Self; Amgen Inc.. Speaker's Bureau; Self; Merck Sharp & Dohme Corp.. Advisory Panel; Self; Novo Nordisk A/S. Consultant; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Consultant; Self; Amgen Inc.. Advisory Panel; Self; MedImmune, Sanofi. Consultant; Self; Sanofi. Research Support; Self; Sanofi. Speaker's Bureau; Self; Sanofi.

BACKGROUND. Exercise has profound pleiotropic health benefits, yet the underlying mechanisms remain incompletely understood. Endocrine FGF21, bile acids (BAs), and BA-induced FGF19 have emerged as metabolic signaling molecules. Here, we investigated if dissimilar modes of exercise, resistance exercise (RE) and endurance exercise (EE), regulate plasma BAs, FGF19, and FGF21 in humans. METHODS. Ten healthy, moderately trained males were enrolled in a randomized crossover study of 1 hour of bicycling at 70% of VO2peak (EE) and 1 hour of high-volume RE. Hormones and metabolites were measured in venous blood and sampled before and after exercise and at 15, 30, 60, 90, 120, and 180 minutes after exercise. RESULTS. We observed exercise mode–specific changes in plasma concentrations of FGF19 and FGF21. Whereas FGF19 decreased following RE (P < 0.001), FGF21 increased in response to EE (P < 0.001). Total plasma BAs decreased exclusively following RE (P < 0.05), but the composition of BAs changed in response to both types of exercise. Notably, circulating levels of the potent TGR5 receptor agonist, lithocholic acid, increased with both types of exercise (P < 0.001). CONCLUSION. This study reveals divergent effects of EE and RE on circulating concentrations of the BA species, FGF19, and FGF21. We identify temporal relationships between decreased BA and FGF19 following RE and a sharp disparity in FGF21 concentrations, with EE eliciting a clear increase parallel to that of glucagon. FUNDING. The Novo Nordisk Foundation (NNF17OC0026114) and the Lundbeck Foundation (R238-2016-2859).

Summary The liver is an important integrator of nutrient metabolism, yet no liver-derived factors regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine fibroblast growth factor 21 (FGF21), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF21 in mice increases sucrose consumption, whereas acute administration or overexpression of FGF21 suppresses the intake of both sugar and non-caloric sweeteners. FGF21 does not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF21 production is elevated by sucrose ingestion. We conclude that the liver functions to regulate macronutrient-specific intake by producing an endocrine satiety signal that acts centrally to suppress the intake of "sweets."

Summary Activation of energy expenditure in thermogenic fat is a promising strategy to improve metabolic health, yet the dynamic processes that evoke this response are poorly understood. Here we show that synthesis of the mitochondrial phospholipid cardiolipin is indispensable for stimulating and sustaining thermogenic fat function. Cardiolipin biosynthesis is robustly induced in brown and beige adipose upon cold exposure. Mimicking this response through overexpression of cardiolipin synthase (Crls1) enhances energy consumption in mouse and human adipocytes. Crls1 deficiency in thermogenic adipocytes diminishes inducible mitochondrial uncoupling and elicits a nuclear transcriptional response through endoplasmic reticulum stress-mediated retrograde communication. Cardiolipin depletion in brown and beige fat abolishes adipose thermogenesis and glucose uptake, which renders animals insulin resistant. We further identify a rare human CRLS1 variant associated with insulin resistance and show that adipose CRLS1 levels positively correlate with insulin sensitivity. Thus, adipose cardiolipin has a powerful impact on organismal energy homeostasis through thermogenic fat bioenergetics., Graphical Abstract, Highlights • Cardiolipin (CL) is a key effector of brown and beige fat thermogenic programs • CL mediates mitochondria-to-nucleus crosstalk through the ER stress factor CHOP-10 • Disruption of brown and beige fat mitochondria causes insulin resistance • CL synthesis in adipose tissue is linked to insulin sensitivity in humans, Sustarsic et al. reveal that synthesis of the mitochondrial phospholipid cardiolipin is a hallmark of brown and beige fat activation by cold temperature. This single lipid species in thermogenic fat not only shapes adipose mitochondrial bioenergetics but also exerts profound control over whole-body insulin sensitivity and metabolic flexibility.

cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) regulates transcription of gluconeogenic genes by specifying targets for the transcription factor CREB in response to glucagon. We used an antisense oligonucleotide directed against CRTC2 in both normal rodents and in rodent models of increased gluconeogenesis to better understand the role of CRTC2 in metabolic disease. In the context of severe hyperglycemia and elevated hepatic glucose production, CTRC2 knockdown (KD) improved glucose homeostasis by reducing endogenous glucose production. Interestingly, despite the known role of CRTC2 in coordinating gluconeogenic gene expression, CRTC2 KD in a rodent model of type 2 diabetes resulted in surprisingly little alteration of glucose production. However, CRTC2 KD animals had elevated circulating concentrations of glucagon and a ∼80% reduction in glucagon clearance. When this phenomenon was prevented with somatostatin or a glucagon-neutralizing antibody, endogenous glucose production was reduced by CRTC2 KD. Additionally, CRTC2 inhibition resulted in reduced expression of several glucagon-induced pyridoxal 5′-phosphate-dependent enzymes that convert amino acids to gluconeogenic intermediates, suggesting that it may control substrate availability as well as gluconeogenic gene expression. CRTC2 is an important regulator of gluconeogenesis with tremendous impact in models of elevated hepatic glucose production. Surprisingly, it is also part of a previously unidentified negative feedback loop that degrades glucagon and regulates amino acid metabolism to coordinately control glucose homeostasis in vivo. Background: CRTC2 translocates to the nucleus upon glucagon stimulation, yet its role in regulating blood glucose remains controversial. Results: CRTC2 promotes expression of enzymes that direct amino acids toward gluconeogenesis and is a key regulator of glucagon clearance. Conclusion: CRTC2 has antagonistic cell-autonomous and endocrine effects on glucose homeostasis. Significance: We present new insights into glucagon/CRTC2 physiology.

Inflammation accompanies obesity and its comorbidities—type 2 diabetes, non-alcoholic fatty liver disease and atherosclerosis, among others—and may contribute to their pathogenesis. Yet the cellular machinery that links nutrient sensing to inflammation remains incompletely characterized. The protein deacetylase sirtuin-1 (SirT1) is activated by energy depletion and plays a critical role in the mammalian response to fasting. More recently it has been implicated in the repression of inflammation. SirT1 mRNA and protein expression are suppressed in obese rodent and human white adipose tissue, while experimental reduction of SirT1 in adipocytes and macrophages causes low-grade inflammation that mimics that observed in obesity. Thus suppression of SirT1 during overnutrition may be critical to the development of obesity-associated inflammation. This effect is attributable to multiple actions of SirT1, including direct deacetylation of NFκB and chromatin remodeling at inflammatory gene promoters. In this work, we report that SirT1 is also suppressed by diet-induced obesity in macrophages, which are key contributors to the ontogeny of metabolic inflammation. Thus, SirT1 may be a common mechanism by which cells sense nutrient status and modulate inflammatory signaling networks in accordance with organismal energy availability.

Caspase-1 is a cysteine protease that can be activated by both endogenous and exogenous inflammatory stimuli and has been shown to have important functions in processes as diverse as proteolytic activation of cytokines, cell death, and membrane repair. Caspase-1–dependent production of the inflammatory cytokines IL-1 and IL-18 has also been implicated in the regulation of appetite, body weight, glucose homeostasis, and lipid metabolism. Consistent with the emerging views of caspase-1 in metabolic regulation, we find that caspase-1–deficient mice have dramatically accelerated triglyceride clearance, without alteration in lipid production or absorption, and resultant decrease in steady-state circulating triglyceride and fatty acid levels. Surprisingly, this effect is independent of IL-1-family signaling, supporting the concept that caspase-1 influences lipid metabolism through multiple mechanisms, not limited to cytokines.

Upon activation, brown and beige adipocytes help to fight excessive fat in rodents, and their oxidative properties can be induced by cooling, capsinoids, and fish oil. New research now suggests that synergistic anti-obesity effects can be achieved by combining such strategies, and that a novel pathway mediates part of the effect.

Hepatic gluconeogenesis is a major contributing factor to hyperglycemia in the fasting and postprandial states in type 2 diabetes mellitus (T2DM). Because Sirtuin 1 (SirT1) induces hepatic gluconeogenesis during fasting through the induction of phosphoenolpyruvate carboxylase kinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase) gene transcription, we hypothesized that reducing SirT1, by using an antisense oligonucleotide (ASO), would decrease fasting hyperglycemia in a rat model of T2DM. SirT1 ASO lowered both fasting glucose concentration and hepatic glucose production in the T2DM rat model. Whole body insulin sensitivity was also increased in the SirT1 ASO treated rats as reflected by a 25% increase in the glucose infusion rate required to maintain euglycemia during the hyperinsulinemic-euglycemic clamp and could entirely be attributed to increased suppression of hepatic glucose production by insulin. The reduction in basal and clamped rates of glucose production could in turn be attributed to decreased expression of PEPCK, FBPase, and G6Pase due to increased acetylation of signal transducer and activator of transcription 3 (STAT3), forkhead box O1 (FOXO1), and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), known substrates of SirT1. In addition to the effects on glucose metabolism, SirT1 ASO decreased plasma total cholesterol, which was attributed to increased cholesterol uptake and export from the liver. These results indicate that inhibition of hepatic SirT1 may be an attractive approach for treatment of T2DM.

Summary N-acylphosphatidylethanolamines (NAPEs) are a relatively abundant group of plasma lipids of unknown physiological significance. Here, we show that NAPEs are secreted into circulation from the small intestine in response to ingested fat and that systemic administration of the most abundant circulating NAPE, at physiologic doses, decreases food intake in rats without causing conditioned taste aversion. Furthermore, 14 C-radiolabeled NAPE enters the brain and is particularly concentrated in the hypothalamus, and intracerebroventricular infusions of nanomolar amounts of NAPE reduce food intake, collectively suggesting that its effects may be mediated through direct interactions with the central nervous system. Finally, chronic NAPE infusion results in a reduction of both food intake and body weight, suggesting that NAPE and long-acting NAPE analogs may be novel therapeutic targets for the treatment of obesity.

Proteolytic cleavage of FGF21, evaluated in human plasma, confirmed three known proline hydrolysis sites. Although DPP-IV participates in consecutive cleavages from the N-terminus, fibroblast activation protein was implicated as the enzyme responsible for the C-terminal cleavage that inactivates FGF21., Fibroblast growth factor 21 (FGF21), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF21 is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF21 protein levels are regulated. We systematically investigated human FGF21 protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF21 was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF21 processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF21 proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, fibroblast activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF21 proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF21 protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF21 inactivation extends the FGF21 signalling pathway and may enable novel approaches to augment FGF21 actions for therapeutic applications.

Dietary modulation of carcinogenesis-related pathwaysDietary item or component studied: fish oilPathways studied: adiponectin gene expressionStudy type (in vitro, animals, humans): 129Sv miceTissue/biological material/sample size: total plasmaMode of exposure (if in vivo) (acute, chronic, root of exposure): chronic (15 days) and acute dietImpact on pathway (including dose-response): 31+-2.8, n=4 vs 25.6+-3.5, n=4μgr/ml plasma adiponectin in 14.5%fish oil-fed mice. KEYWORDS CLASSIFICATION: Adiponectin;Adipose Tissue;Animals;Antigens,CD36;blood;deficiency;dietary modulation of carcinogenesis-related pathways;drug effects;DNA Primers;Epididymis;Epoxy Compounds;Fish Oils;genetics;Gene Expression Regulation;Male;Mice;Mice,Inbred Strains;Mice,Knockout;Oils;pharmacology;physiology;PPAR gamma;Research;Reverse Transcriptase Polymerase Chain Reaction;secretion;Transcription,Genetic; Adiponectin has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties, but little is known about factors that regulate its secretion. To examine the effect of fish oil on adiponectin secretion, mice were fed either a control diet or isocaloric diets containing 27% safflower oil or 27, 13.5, and 8% menhaden fish oil. Within 15 days, fish oil feeding raised plasma adiponectin concentrations two- to threefold in a dose-dependent manner, and the concentrations remained approximately twofold higher for 7 days when the fish oil diet was replaced by the safflower oil diet. Within 24 h, fish oil markedly induced transcription of the adiponectin gene in epididymal adipose tissue but not in subcutaneous fat. The increase of plasma adiponectin by fish oil was completely blocked by administration of the peroxisome proliferator-activated receptor (PPAR)gamma inhibitor bisphenol-A-diglycidyl ether. In contrast, there was no effect of fish oil feeding on adiponectin secretion in PPARalpha-null mice. These data suggest that fish oil is a naturally occurring potent regulator of adiponectin secretion in vivo and that it does so through a PPARgamma-dependent and PPARalpha-independent manner in epididymal fat.

Summary The liking and selective ingestion of palatable foods—including sweets—is biologically controlled, and dysfunction of this regulation may promote unhealthy eating, obesity, and disease. The hepatokine fibroblast growth factor 21 (FGF21) reduces sweet consumption in rodents and primates, whereas knockout of Fgf21 increases sugar consumption in mice. To investigate the relevance of these findings in humans, we genotyped variants in the FGF21 locus in participants from the Danish Inter99 cohort (n = 6,514) and examined their relationship with a detailed range of food and ingestive behaviors. This revealed statistically significant associations between FGF21 rs838133 and increased consumption of candy, as well as nominal associations with increased alcohol intake and daily smoking. Moreover, in a separate clinical study, plasma FGF21 levels increased acutely after oral sucrose ingestion and were elevated in fasted sweet-disliking individuals. These data suggest the liver may secrete hormones that influence eating behavior.

SummaryThermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids. Serotonin (5-HT) neurons in the CNS are essential for thermoregulation and accordingly may control metabolic activity of thermogenic fat. To test this, we generated mice in which the human diphtheria toxin receptor (DTR) was selectively expressed in central 5-HT neurons. Treatment with diphtheria toxin (DT) eliminated 5-HT neurons and caused loss of thermoregulation, brown adipose tissue (BAT) steatosis, and a >50% decrease in uncoupling protein 1 (Ucp1) expression in BAT and inguinal white adipose tissue (WAT). In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold, and triglycerides 6.5-fold. Similar BAT and beige fat defects occurred in Lmx1bf/fePet1Cre mice in which 5-HT neurons fail to develop in utero. We conclude 5-HT neurons play a major role in regulating glucose and lipid homeostasis, in part through recruitment and metabolic activation of brown and beige adipocytes.