Postprandial Plasma Lipidomics Reveal Specific Alteration of Hepatic-derived Diacylglycerols in Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Hepatic energy metabolism is a dynamic process modulated by multiple stimuli. In nonalcoholic fatty liver disease (NAFLD), human studies typically focus on the static fasting state. We hypothesized that unique postprandial alterations in hepatic lipid metabolism are present in NAFLD. METHODS: In a prospective clinical study, 37 patients with NAFLD and 10 healthy control subjects ingested a standardized liquid meal with pre- and postprandial blood sampling. Postprandial plasma lipid kinetics were characterized at the molecular lipid species level by untargeted lipidomics, cluster analysis, and lipid particle isolation, then confirmed in a mouse model. RESULTS: There was a specific increase of multiple plasma diacylglycerol (DAG) species at 4 hours postprandially in patients with NAFLD but not in controls. This was replicated in a nonalcoholic steatohepatitis mouse model, where postprandial DAGs increased in plasma and concomitantly decreased in the liver. The increase in plasma DAGs appears early in the disease course, is dissociated from NAFLD severity and obesity, and correlates with postprandial insulin levels. Immunocapture isolation of very low density lipoprotein in human samples and stable isotope tracer studies in mice revealed that elevated postprandial plasma DAGs reflect hepatic secretion of endogenous, rather than meal-derived lipids. CONCLUSIONS: We identified a selective insulin-related increase in hepatic secretion of endogenously derived DAGs after a mixed meal as a unique feature of NAFLD. DAGs are known to be lipotoxic and associated with atherosclerosis. Although it is still unknown whether the increased exposure to hepatic DAGs contributes to extrahepatic manifestations and cardiovascular risk in NAFLD, our study highlights the importance of extending NAFLD research beyond the fasting state.

Non-Nutritive Sweeteners in Human Amniotic Fluid and Cord Blood: Evidence of Transplacental Fetal Exposure.

OBJECTIVE: This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood. STUDY DESIGN: Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid (n = 13) and cord blood samples (n = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health. RESULTS: Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples. CONCLUSION: Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health. KEY POINTS: · NNS consumption during pregnancy has increased in recent years.. · Maternal NNS intake during pregnancy is associated with preterm birth and higher infant weight gain in epidemiologic studies.. · In rodents, in utero NNS exposure induces metabolic abnormalities in mothers and their offspring, alters offspring gut microbiota composition, and promotes sweet taste preference in adulthood.. · It is presently unknown whether and to what degree maternal NNS ingestion in humans leads to direct in utero exposure.. · This study provides the first evidence of in utero NNS exposure in humans and highlights the urgent need to investigate clinical consequences of early life NNS exposure on metabolism, weight, taste preference, and general health..

Metformin improves blood glucose by increasing incretins independent of changes in gluconeogenesis in youth with type 2 diabetes.

AIMS/HYPOTHESIS: Metformin is the only approved oral agent for youth with type 2 diabetes but its mechanism of action remains controversial. Recent data in adults suggest a primary role for the enteroinsular pathway, but there are no data in youth, in whom metformin efficacy is only ~50%. Our objectives were to compare incretin concentrations and rates of glucose production and gluconeogenesis in youth with type 2 diabetes before and after short-term metformin therapy compared with peers with normal glucose tolerance (NGT). METHODS: This is a case-control observational study in youth with type 2 diabetes who were not on metformin (n = 18) compared with youth with NGT (n = 10) who were evaluated with a 2 day protocol. A 75 g OGTT was administered to measure intact glucagon-like 1 peptide (iGLP-1), gastric inhibitory polypeptide (GIP) and peptide YY (PYY). Insulinogenic index (IGI) and whole-body insulin sensitivity were calculated using glucose and insulin levels from the OGTT. Basal rates of gluconeogenesis (2H2O), glucose production ([6,6-2H2]glucose) and whole-body lipolysis ([2H5]glycerol) were measured after an overnight fast on study day 2. Youth with type 2 diabetes (n = 9) were subsequently evaluated with an identical 2 day protocol after 3 months on the metformin study. RESULTS: Compared with individuals with NGT, those with type 2 diabetes had higher fasting (7.8 ± 2.5 vs 5.1 ± 0.3 mmol/l, mean ± SD p = 0.002) and 2 h glucose concentrations (13.8 ± 4.5 vs 5.9 ± 0.9 mmol/l, p = 0.001), higher rates of absolute gluconeogenesis (10.0 ± 1.7 vs 7.2 ± 1.1 µmol [kg fat-free mass (FFM)]-1 min-1, p < 0.001) and whole-body lipolysis (5.2 ± 0.9 vs 4.0 ± 1.4 µmol kgFFM-1 min-1, p < 0.01), but lower fasting iGLP-1 concentrations (0.5 ± 0.5 vs 1.3 ± 0.7 pmol/l, p < 0.01). Metformin decreased 2 h glucose (pre metformin 11.4 ± 2.8 vs post metformin 9.9 ± 1.9 mmol/l, p = 0.04) and was associated with ~20-50% increase in IGI (median [25th-75th percentile] pre 1.39 [0.89-1.47] vs post 1.43 [0.88-2.70], p = 0.04), fasting iGLP-1 (pre 0.3 ± 0.2 vs post 1.0 ± 0.7 pmol/l, p = 0.02), 2 h iGLP (pre 0.4 ± 0.2 vs post 1.2 ± 0.9 pmol/l, p = 0.06), fasting PYY (pre 6.3 ± 2.2 vs post 10.5 ± 4.3 pmol/l, p < 0.01) and 2 h PYY (pre 6.6 ± 2.9 vs post 9.0 ± 4.0 pmol/l, p < 0.01). There was no change in BMI, insulin sensitivity or GIP concentrations pre vs post metformin. There were no differences pre vs post metformin in rates of glucose production (15.0 ± 3.9 vs 14.9 ± 2.2 µmol kgFFM-1 min-1, p = 0.84), absolute gluconeogenesis (9.9 ± 1.8 vs 9.7 ± 1.7 µmol kgFFM-1 min-1, p = 0.76) or whole-body lipolysis (5.0 ± 0.7 vs 5.3 ± 1.3 µmol kgFFM-1 min-1, p = 0.20). Post metformin iGLP-1 and PYY concentrations in youth with type 2 diabetes were comparable to levels in youth with NGT. CONCLUSIONS/INTERPRETATION: Overall, the improved postprandial blood glucose levels and increase in incretins observed in the absence of changes in insulin sensitivity and gluconeogenesis, support an enteroinsular mechanistic pathway in youth with type 2 diabetes treated with short-term metformin.

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans.

Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available Escherichia coli plasmid, pNEB193, and cannot replicate autonomously in methanogens. The designed plasmids facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from the methanogen, M. acetivorans.

Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus.

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.

Increased 24-hour ad libitum food intake is associated with lower plasma irisin concentrations the following morning in adult humans.

BACKGROUND: The relationship between food intake and irisin concentrations in humans is unclear. OBJECTIVES: To determine whether the previous day's intake impacts fasting plasma irisin concentrations, or whether fasting irisin concentrations associate with subsequent ad libitum food intake. METHODS: Sixty-six nondiabetic adults (42 men) were admitted for a study of the determinants of energy intake. After 6 days of a weight maintaining diet, ad libitum energy intake over 3 days was assessed using a vending machine paradigm. Fasting plasma irisin concentrations were measured on the morning of the second day of the vending period. RESULTS: There were no correlations between irisin and demographic or anthropometric parameters. On day 1, subjects consumed 144 ± 52% of weight maintaining energy needs. Every additional 500 kcal consumed on day 1 associated with a 3.4% lower irisin concentration the following morning (95% CI -6.2, -0.4%, p = 0.01; adjusted for age, sex and race). If energy intake was expressed as a percentage of weight maintaining energy needs, every 10% increase associated with a 1.9% lower irisin concentration (95% CI -3.7, -0.1%; adjusted p = 0.02). A 100 kcal increase in carbohydrate or fat consumption associated with a 1.3% (95% CI -2.5, -0.1%, p = 0.01) and a 0.6% (95% CI -1.1, -0.0%, p = 0.02) lower irisin concentration, respectively. There was no association between fasting irisin concentrations and subsequent energy intake on day 2 (r = 0.19, p = 0.1). CONCLUSIONS: Higher ad libitum 24 h energy intake was associated with lower fasting irisin concentrations the following morning, but fasting irisin concentrations did not predict subsequent energy intake. The decrease in irisin concentrations with increased energy intake is consistent with the detrimental metabolic effects of overeating.

Ultrasound assessment of cervical length at 18-21 weeks' gestation in an Australian obstetric population: comparison of transabdominal and transvaginal approaches.

BACKGROUND: Using a fixed cut-off of ≤25 mm, ultrasound assessment of cervical length during the 18-23 week anomaly scan has been shown to identify approximately 50% of pregnancies that would deliver prior to 34 weeks. AIM: To determine whether a policy of reverting to transvaginal cervical assessment only if the cervix appears short (≤25 mm) on transabdominal assessment affects the efficiency of screening. METHODS: Women with a singleton pregnancy that presented for a routine anomaly scan had their cervical length assessed transabdominally, initially with the maternal bladder full (TABF) and then empty (TABE). Cervical length was then assessed transvaginally (TV). RESULTS: One hundred and ninety-eight women agreed to participate in the study. Identification of the internal and external cervical os was possible during TABF, TABE and TV sonography in 97.0, 82.8 and 100%, respectively. Compared with TV sonography, TABF overestimates cervical length (6.1 mm difference in median values; P < 0.01). There was no significant difference between TV and TABE. However, TABE assessment was not possible in one in six women. If TABF sonography was to be used as a screening tool and using ≤25 mm as the critical cut-off, the sensitivity and specificity was 15.4 and 93.2%, respectively. CONCLUSION: This study has shown that assessment of cervical length using a TA approach is only routinely possible when the bladder is full. However, measurements are significantly overestimated. Therefore, we feel that TV assessment of cervical length is the preferred method of reliable cervical assessment. As such, all women should be offered a TV assessment of cervical length at the time of the fetal anomaly ultrasound as a screening test for preterm birth.

The effects of an exercise program on inflammation in adults who differ according to obstructive sleep apnea severity.

Exercise improves chronic inflammation and is recommended as a first-line medical or behavioral treatment for OSA with obesity. We examined whether the effects of an exercise program on inflammatory blood markers differed according to severity of OSA among obese adults. Overweight (BMI > 27 kg/m2) adults were evaluated for OSA using overnight polysomnography and subsequently classified as exhibiting no-to-mild OSA (AHI < 15 events/hour) or moderate-to-severe OSA (AHI ≥ 15 events/hour). Cardiorespiratory fitness, body composition assessed by DXA, fasting metabolic parameters and adipokines (i.e., glucose, insulin, leptin and adioponectin), and multiple markers of inflammation (i.e., CRP, IL-4, IL-8 and TNF-α) were measured at baseline (Pre) and following a 6-week (3 days per week) comprehensive exercise program (Post). Ten adults (Age: 48 ± 8 years; W:6; M:4) with no/mild OSA and 12 adults (Age: 54 ± 8 years; W:5; M:7) with moderate/severe OSA completed all aspects of the trial. No significant differences in age, cardiorespiratory fitness, body composition, fasting metabolic parameters and most inflammatory markers were observed between groups at baseline. Exercise training decreased total fat mass (Pre: 41,167 ± 13,315 g; Post: 40,311 ± 12,657 g; p = 0.008), leptin (Pre: 26.7 ± 29.6 pg/ml; Post: 22.7 ± 19.4 pg/ml; p = 0.028) and adiponectin (Pre: 16.6 ± 10.9 µg/ml; Post: 11.0 ± 10.6 µg/ml; p = 0.004) in those with moderate/severe OSA. Among those with no/mild OSA, exercise training resulted in a decrease in total fat mass (Pre = 37,332 ± 20,258 g; Post: 37,068 ± 18,268 g, p = 0.037). These data suggest that while 6 weeks of exercise reduced adipokines in those with moderate-to-severe OSA, it was not sufficient to improve common markers of inflammation among overweight adults with OSA.

Impaired metabolic flexibility to fasting is associated with increased ad libitum energy intake in healthy adults.

OBJECTIVE: We investigated how changes in 24-h respiratory exchange ratio (RER) and substrate oxidation during fasting versus an energy balance condition influence subsequent ad libitum food intake. METHODS: Forty-four healthy, weight-stable volunteers (30 male and 14 female; mean [SD], age 39.3 [11.0] years; BMI 31.7 [8.3] kg/m2) underwent 24-h energy expenditure measurements in a respiratory chamber during energy balance (50% carbohydrate, 30% fat, and 20% protein) and 24-h fasting. Immediately after each chamber stay, participants were allowed 24-h ad libitum food intake from computerized vending machines. RESULTS: Twenty-four-hour RER decreased by 9.4% (95% CI: -10.4% to -8.5%; p < 0.0001) during fasting compared to energy balance, reflecting a decrease in carbohydrate oxidation (mean [SD], -2.6 [0.8] MJ/day; p < 0.0001) and an increase in lipid oxidation (2.3 [0.9] MJ/day; p < 0.0001). Changes in 24-h RER and carbohydrate oxidation in response to fasting were correlated with the subsequent energy intake such that smaller decreases in fasting 24-h RER and carbohydrate oxidation, but not lipid oxidation, were associated with greater energy intake after fasting (r = 0.31, p = 0.04; r = 0.40, p = 0.007; and r = -0.27, p = 0.07, respectively). CONCLUSIONS: Impaired metabolic flexibility to fasting, reflected by an inability to transition away from carbohydrate oxidation, is linked with increased energy intake.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Glycemia and Gluconeogenesis With Metformin and Liraglutide: A Randomized Trial in Youth-onset Type 2 Diabetes.

OBJECTIVE: Elevated rates of gluconeogenesis are an early pathogenic feature of youth-onset type 2 diabetes (Y-T2D), but targeted first-line therapies are suboptimal, especially in African American (AA) youth. We evaluated glucose-lowering mechanisms of metformin and liraglutide by measuring rates of gluconeogenesis and ß-cell function after therapy in AA Y-T2D. METHODS: In this parallel randomized clinical trial, 22 youth with Y-T2D-age 15.3 ± 2.1 years (mean ± SD), 68% female, body mass index (BMI) 40.1 ± 7.9 kg/m2, duration of diagnosis 1.8 ± 1.3 years-were randomized to metformin alone (Met) or metformin + liraglutide (Lira) (Met + Lira) and evaluated before and after 12 weeks. Stable isotope tracers were used to measure gluconeogenesis [2H2O] and glucose production [6,6-2H2]glucose after an overnight fast and during a continuous meal. ß-cell function (sigma) and whole-body insulin sensitivity (mSI) were assessed during a frequently sampled 2-hour oral glucose tolerance test. RESULTS: At baseline, gluconeogenesis, glucose production, and fasting and 2-hour glucose were comparable in both groups, though Met + Lira had higher hemoglobin A1C. Met + Lira had a greater decrease from baseline in fasting glucose (-2.0 ± 1.3 vs -0.6 ± 0.9 mmol/L, P = .008) and a greater increase in sigma (0.72 ± 0.68 vs -0.05 ± 0.71, P = .03). The change in fractional gluconeogenesis was similar between groups (Met + Lira: -0.36 ± 9.4 vs Met: 0.04 ± 12.3%, P = .9), and there were no changes in prandial gluconeogenesis or mSI. Increased glucose clearance in both groups was related to sigma (r = 0.63, P = .003) but not gluconeogenesis or mSI. CONCLUSION: Among Y-T2D, metformin with or without liraglutide improved glycemia but did not suppress high rates of gluconeogenesis. Novel therapies that will enhance ß-cell function and target the elevated rates of gluconeogenesis in Y-T2D are needed.

Atorvastatin active metabolite inhibits oxidative modification of small dense low-density lipoprotein.

We tested the hypothesis that atorvastatin active metabolite (ATM), on the basis of its distinct structural features and potent antioxidant activity, preferentially inhibits lipid oxidation in human small dense low-density lipoprotein (sdLDL) and other small lipid vesicles. LDL, sdLDL, and various subfractions were isolated from human plasma by sequential ultracentrifugation, treated with ATM, atorvastatin, pravastatin, rosuvastatin, or simvastatin and were subjected to copper-induced oxidation. Lipid oxidation was measured spectrophotometrically as a function of thiobarbituric acid reactive substances formation. Similar analyses were performed in reconstituted lipid vesicles enriched in polyunsaturated fatty acids and prepared at various sizes. ATM was found to inhibit sdLDL oxidation in a dose-dependent manner. The antioxidant effects of ATM in sdLDL were 1.5 and 4.7 times greater (P < 0.001) than those observed in large buoyant LDL and very low-density lipoprotein subfractions, respectively. ATM had similar dose- and size-dependent effects in reconstituted lipid vesicles. None of these effects were reproduced by atorvastatin (parent) or any of the other statins examined in this study. These data suggest that ATM interacts with sdLDL in a specific manner that also confers preferential resistance to oxidative stress. Such interactions may reduce sdLDL atherogenicity and improve clinical outcomes in patients with cardiovascular disease.

Discordance between gut-derived appetite hormones and energy intake in humans.

Gut-derived hormones affect appetite and are thought to play an important role in body weight regulation. Dietary macronutrient composition can influence gut-derived appetite hormone concentrations, thereby providing theoretical basis for why some diets might facilitate weight loss better than others. We investigated postprandial gut-derived appetite hormones in 20 inpatient adults after 2 weeks of eating either a low carbohydrate (LC) or a low fat (LF) diet followed by the alternate diet in random order. A LC meal resulted in significantly greater postprandial GLP-1, GIP, and PYY but lower ghrelin compared to an isocaloric LF meal (all p≤0.02). However, differences in gut-derived appetite hormones were incommensurate with subsequent ad libitum energy intake over the rest of the day, which was 551±103 kcal (p<0.0001) greater with the LC as compared to the LF diet. The effects of gut-derived appetite hormones on ad libitum energy intake can be dominated by other diet-related factors, at least in the short-term.

Insulin Signaling Through the Insulin Receptor Increases Linear Growth Through Effects on Bone and the GH-IGF-1 Axis.

CONTEXT: Childhood overnutrition is associated with increased growth and bone mineral density (BMD) vs the opposite for undernutrition. The role of insulin receptor (InsR) signaling in these phenotypes is unclear. Rare disease patients with hyperinsulinemia and impaired InsR function (homozygous [-/-] or heterozygous [+/-] INSR pathogenic variants, type B insulin resistance [TBIR]) model increased InsR signaling, while patients with intact InsR function (congenital generalized lipodystrophy, CGL) model decreased InsR signaling. OBJECTIVE: This work aimed to understand mechanisms whereby InsR signaling influences growth. METHODS: A cross-sectional comparison was conducted of CGL (N = 23), INSR-/- (N = 13), INSR+/- (N = 17), and TBIR (N = 8) at the National Institutes of Health. Main outcome measures included SD scores (SDS) for height, body mass index, insulin-like growth factor (IGF)-1, and BMD, and IGF binding proteins (IGFBP)-1 and -3. RESULTS: INSR-/- vs CGL had higher insulin (median 266 [222-457] vs 33 [15-55] mcU/mL), higher IGFBP-1 (72 350 [55 571-103 107] vs 6453 [1634-26 674] pg/mL), lower BMI SDS (-0.7 ± 1.1 vs 0.5 ± 0.9), lower height SDS (-1.9[-4.3 to -1.3] vs 1.1 [0.5-2.5]), lower BMD SDS (-1.9 ± 1.4 vs 1.9 ± 0.7), and lower IGFBP-3 (0.37 [0.19-1.05] vs 2.00 [1.45-2.67] µg/mL) (P < .05 for all). INSR +/- were variable. Remission of TBIR lowered insulin and IGFBP-1, and increased IGF-1 and IGFBP-3 (P < .05). CONCLUSION: Patients with hyperinsulinemia and impaired InsR function exhibit impaired growth and lower BMD, whereas elevated InsR signaling (CGL) causes accelerated growth and higher BMD. These patients demonstrate that insulin action through the InsR stimulates direct anabolic effects in bone and indirect actions through the growth hormone (GH)-IGF-1 axis. TBIR patients exhibit abnormalities in the GH axis that resolve when InsR signaling is restored, supporting a causal relationship between InsR and GH axis signaling.

The association between gut hormones and diet-induced metabolic flexibility in metabolically healthy adults.

OBJECTIVE: This study investigated whether interindividual variance in diet-induced metabolic flexibility is explained by differences in gut hormone concentrations. METHODS: A total of 69 healthy volunteers with normal glucose regulation underwent 24-hour assessments of respiratory quotient (RQ) in a whole-room indirect calorimeter during eucaloric feeding (EBL; 50% carbohydrate, 30% fat) and then, in a crossover design, during 24-hour fasting and three normal-protein (20%) overfeeding diets (200% energy requirements). Metabolic flexibility was defined as the change in 24-hour RQ from EBL during standard (50% carbohydrate), high-fat (60%), and high-carbohydrate (75%) overfeeding diets. Plasma concentrations of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) after an overnight fast were measured prior to and after each diet. RESULTS: Compared with EBL, on average, 24-hour RQ decreased by ~4% during high-fat overfeeding, whereas it increased by ~4% during standard overfeeding and by ~9% during high-carbohydrate overfeeding. During high-carbohydrate overfeeding, but not during any other overfeeding diet or fasting, increased GLP-1 concentration was associated with increased RQ (r = 0.44, p < 0.001), higher/lower carbohydrate/lipid oxidation rates (r = 0.34 and r = -0.51, both p < 0.01), respectively, and increased plasma insulin concentration (r = 0.38, p = 0.02). CONCLUSIONS: Increased GLP-1 concentration following high-carbohydrate overfeeding associated with a greater shift to carbohydrate oxidation, suggesting that GLP-1 may be implicated in diet-induced metabolic flexibility to carbohydrate overload.

Dysregulated Cyclic Nucleotide Metabolism in Alcohol-Associated Steatohepatitis: Implications for Novel Targeted Therapies.

BACKGROUND: Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking. METHODS: Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH). RESULTS: Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis. CONCLUSIONS: These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.

Blood-based Proteomic Signatures Associated With MEN1-related Duodenopancreatic Neuroendocrine Tumor Progression.

PURPOSE: Patients with multiple endocrine neoplasia type 1 (MEN1) are predisposed to develop duodenopancreatic neuroendocrine tumors (dpNETs), and metastatic dpNET is the primary cause of disease-related mortality. Presently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. In the current study, we aimed to establish novel circulating molecular protein signatures associated with disease progression. EXPERIMENTAL DESIGN: Mass spectrometry-based proteomic profiling was conducted on plasmas procured through an international collaboration between MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht from a cohort of 56 patients with MEN1 [14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)]. Findings were compared to proteomic profiles generated from serially collected plasmas from a mouse model of Men1-pancreatic neuroendocrine tumors (Men1fl/flPdx1-CreTg) and control mice (Men1fl/fl). RESULTS: A total of 187 proteins were found to be elevated in MEN1 patients with distant metastasis compared to controls, including 9 proteins previously associated with pancreatic cancer and other neuronal proteins. Analyses of mouse plasmas revealed 196 proteins enriched for transcriptional targets of oncogenic MYCN, YAP1, POU5F1, and SMAD that were associated with disease progression in Men1fl/flPdx1-CreTg mice. Cross-species intersection revealed 19 proteins positively associated with disease progression in both human patients and in Men1fl/flPdx1-CreTg mice. CONCLUSIONS: Our integrated analyses identified novel circulating protein markers associated with disease progression in MEN1-related dpNET.

Structure-based redesign of an edema toxin inhibitor.

Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3',5'-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15µg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.

Effects of Short-term Fasting on Ghrelin/GH/IGF-1 Axis in Healthy Humans: The Role of Ghrelin in the Thrifty Phenotype.

CONTEXT: A greater decrease in 24-hour energy expenditure (24hEE) during short-term fasting is indicative of a thrifty phenotype. OBJECTIVE: As ghrelin and the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis are implicated in the regulation of energy intake and metabolism, we investigated whether ghrelin, GH, and IGF-1 concentrations mediate the fasting-induced decrease in 24hEE that characterizes thriftiness. METHODS: In 47 healthy individuals, 24hEE was measured in a whole-room indirect calorimeter both during 24-hour eucaloric and fasting conditions. Plasma total ghrelin, GH, and IGF-1 concentrations were measured by enzyme-linked immunosorbent assay after an overnight fast the morning before and after each 24-hour session. RESULTS: During 24-hour fasting, on average 24hEE decreased by 8.0% (P < .001), GH increased by ~5-fold (P < .001), whereas ghrelin (mean +23 pg/mL) and IGF-1 were unchanged (both P ≥ .19) despite a large interindividual variability in ghrelin change (SD 150 pg/mL). Greater fasting-induced increase in ghrelin was associated with a greater decrease in 24hEE during 24-hour fasting (r = -0.42, P = .003), such that individuals who increased ghrelin by 200 pg/mL showed an average decrease in 24hEE by 55 kcal/day. CONCLUSION: Short-term fasting induced selective changes in the ghrelin/GH/IGF-1 axis, specifically a ghrelin-independent GH hypersecretion that did not translate into increased IGF-1 concentrations. Greater increase in ghrelin after 24-hour fasting was associated with greater decrease in 24hEE, indicating ghrelin as a novel biomarker of increased energy efficiency of the thrifty phenotype.