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Aims: GLP-1 receptor agonists (GLP-1 RAs) are proven therapies for type 2 diabetes mellitus (T2DM) and overweight or obesity. We performed non-clinical and first-in-human (FIH) evaluation of ECC5004/AZD5004, an oral small-molecule GLP-1 RA., Materials and Methods: ECC5004 was profiled in cell lines overexpressing human GLP-1R, in glucose-stimulated insulin secretion (GSIS) assays in a human β-cell line and non-human primates (NHPs). To evaluate safety, ECC5004 was orally administered to NHPs for 9 months and a phase I, double-blind, placebo-controlled FIH study was conducted. This study evaluated single doses of ECC5004 (1-300 mg) in healthy volunteers, and multiple daily doses (5, 10, 30 and 50 mg) in patients with T2DM for 28 days., Results: ECC5004 bound to the hGLP-1R (IC 50  = 2.4 nM) augmented cAMP signalling without β-arrestin-2 recruitment or receptor internalization. ECC5004 potentiated GSIS in both EndoC-βH5 cells (EC 50  = 5.9 nM) and in vivo in NHPs (EC 50  = 0.022 nM). Dose-dependent body weight changes compared to control were seen in the 9-month NHP toxicity study. In the first-in-human study, ECC5004 was well tolerated with no serious adverse events. Dose-dependent reductions in glucose and body weight were observed with a dose-proportional exposure at doses ≥25 mg., Conclusion: ECC5004 engaged the GLP-1R across the therapeutic dose range tested and had a safety and tolerability profile consistent with other GLP-1 RAs, along with a pharmacokinetic profile compatible with once-daily oral dosing. These data support continued development of ECC5004 as a potential therapy for T2DM and overweight or obesity., Clinical Trial Registration: NCT05654831., (© 2024 The Author(s). Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Excessive fructose intake is a risk factor for the development of obesity and its complications. Targeting ketohexokinase (KHK), the first enzyme of fructose metabolism, has been investigated for the management of MASLD. We compared the effects of systemic, small molecule inhibitor of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. We measured KHK enzymatic activity, extensively quantified glycogen accumulation, performed RNAseq analysis, and enumerated hepatic metabolites using mass spectrometry. Both KHK siRNA and KHK inhibitor led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown decreased the de novo lipogenesis pathway, whereas the inhibitor increased the fatty acid oxidation pathway. Moreover, KHK knockdown completely prevented hepatic fructolysis and improved glucose tolerance. Conversely, the KHK inhibitor only partially reduced fructolysis, but it also targeted triokinase, mediating the third step of fructolysis. This leads to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. Overexpression of wild-type, but not kinase-dead KHK in cultured hepatocytes increased hepatocyte injury and glycogen accumulation when treated with fructose. The differences between KHK inhibition and knockdown are, in part, explained by the kinase-dependent and independent effects of KHK on hepatic metabolism.

Aim: To explore how introduction of the lower WHO gestational diabetes (GDM) glucose criteria in Sweden affected prediabetes/type-2-diabetes (T2D) incidence two years postpartum., Methods: Women included in the PREvention of PostPartum (PREPP) diabetes study were diagnosed with GDM according to EASD 1991 criteria (GDM OLD ; n = 93) or only WHO 2013 criteria (GDM WHO ; n = 174). Both groups were further stratified by BMI, and BMI-matched normoglycemic pregnancy controls were included (n = 88). Postpartum assessments included oral glucose tolerance tests (OGTT) and anthropometric measurements., Results: There was a higher postpartum incidence of T2D in GDM OLD versus GDM WHO (P < 0.001). Despite similar BMI, GDM OLD exhibited higher fasting and OGTT glucose levels, lower fat-free-mass, and hip circumference compared to GDM WHO . In normal-weight women, both GDM groups displayed higher HOMA-IR and lower fat-free-mass compared to controls, with GDM OLD additionally showing lower HOMA-β, slower insulin release during OGTT, and worse glucose tolerance than GDM WHO . Among obese women, the main differences were lower fat-free-mass and hip circumference in GDM OLD ., Conclusion: The lower glucose cut-offs during pregnancy resulted in lower postpartum incidence of T2D, irrespective of BMI. Fat-free-mass emerged as a key determinant in glucose levels across BMI categories, while lower beta-cell function played a significant role in normal-weight women., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ulrika Andersson-Hall reports financial support was provided by Emil and Wera Cornell Foundation. Agneta Holmang reports financial support was provided by The Swedish state under the ALF agreement between the Swedish government and the county councils. Verena Sengpiel reports financial support was provided by The Swedish state under the ALF agreement between the Swedish government and the county councils. Ulrika Andersson-Hall reports financial support was provided by Tore Nilson Foundation for Medical Research. Ulrika Andersson-Hall reports financial support was provided by Erik & Lily Philipsons foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Analogues of the hepatokine fibroblast growth factor 21 (FGF21) are in clinical development for type 2 diabetes and nonalcoholic steatohepatitis (NASH) treatment. Although their glucose-lowering and insulin-sensitizing effects have been largely unraveled, the mechanisms by which they alleviate liver injury have only been scarcely addressed. Here, we aimed to unveil the mechanisms underlying the protective effects of FGF21 on NASH using APOE*3-Leiden.CETP mice, a well-established model for human-like metabolic diseases. Liver-specific FGF21 overexpression was achieved in mice, followed by administration of a high-fat high-cholesterol diet for 23 weeks. FGF21 prevented hepatic lipotoxicity, accompanied by activation of thermogenic tissues and attenuation of adipose tissue inflammation, improvement of hyperglycemia and hypertriglyceridemia, and upregulation of hepatic programs involved in fatty acid oxidation and cholesterol removal. Furthermore, FGF21 inhibited hepatic inflammation, as evidenced by reduced Kupffer cell (KC) activation, diminished monocyte infiltration, and lowered accumulation of monocyte-derived macrophages. Moreover, FGF21 decreased lipid- and scar-associated macrophages, which correlated with less hepatic fibrosis as demonstrated by reduced collagen accumulation. Collectively, hepatic FGF21 overexpression limits hepatic lipotoxicity, inflammation, and fibrogenesis. Mechanistically, FGF21 blocks hepatic lipid influx and accumulation through combined endocrine and autocrine signaling, respectively, which prevents KC activation and lowers the presence of lipid- and scar-associated macrophages to inhibit fibrogenesis., Competing Interests: CL, MS, BS, JL, Hv, EZ, MT, BG, MB, YW, PR No competing interests declared, AA, AP, SO, MU, IA, YI, KW, XP employee of AstraZeneca, (© 2023, Liu et al.)

Circadian rhythms play a critical role in regulating metabolism, including daily cycles of feeding/fasting. Glucokinase (GCK) is central for whole-body glucose homeostasis and oscillates according to a circadian clock. GCK activators (GKAs) effectively reduce hyperglycemia, but their use is also associated with hypoglycemia, hyperlipidemia, and hepatic steatosis. Given the circadian rhythmicity and natural postprandial activation of GCK, we hypothesized that GKA treatment would benefit from being timed specifically during feeding periods. Acute treatment of obese Zucker rats with the GKA AZD1656 robustly increased flux into all major metabolic pathways of glucose disposal, enhancing glucose elimination. Four weeks of continuous AZD1656 treatment of obese Zucker rats improved glycemic control; however, hepatic steatosis and inflammation manifested. In contrast, timing AZD1656 to feeding periods robustly reduced hepatic steatosis and inflammation in addition to improving glycemia, whereas treatment timed to fasting periods caused overall detrimental metabolic effects. Mechanistically, timing AZD1656 to feeding periods diverted newly synthesized lipid toward direct VLDL secretion rather than intrahepatic storage. In line with increased hepatic insulin signaling, timing AZD1656 to feeding resulted in robust activation of AKT, mTOR, and SREBP-1C after glucose loading, pathways known to regulate VLDL secretion and hepatic de novo lipogenesis. In conclusion, intermittent AZD1656 treatment timed to feeding periods promotes glucose disposal when needed the most, restores metabolic flexibility and hepatic insulin sensitivity, and thereby avoids hepatic steatosis. Thus, chronotherapeutic approaches may benefit the development of GKAs and other drugs acting on metabolic targets.

Fibroblast growth factor 21 (FGF21) is a promising therapeutic agent for treatment of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). We show that therapeutic levels of FGF21 were achieved following subcutaneous (s.c.) administration of mRNA encoding human FGF21 proteins. The efficacy of mRNA was assessed following 2-weeks repeated s.c. dosing in diet-induced obese (DIO), mice which resulted in marked decreases in body weight, plasma insulin levels, and hepatic steatosis. Pharmacokinetic/pharmacodynamic (PK/PD) modelling of several studies in both lean and DIO mice showed that mRNA encoding human proteins provided improved therapeutic coverage over recombinant dosed proteins in vivo . This study is the first example of s.c. mRNA therapy showing pre-clinical efficacy in a disease-relevant model, thus, showing the potential for this modality in the treatment of chronic diseases, including T2D and NASH., Competing Interests: L. Brito, S.P., and K.B. were at the time of study conduct employees of Moderna, Inc. All other authors were at the time of study conduct and publication all employees of AstraZeneca., (© 2022 The Authors.)

We gathered recent (2010-2019) data on the VOC and formaldehyde levels in Finnish non-industrial indoor work environments. The data comprised 9789 VOC and 1711 formaldehyde samples collected from the indoor air of offices, schools, kindergartens, and healthcare offices. We assessed the health risks by comparing the measured concentrations to the health-based RW I/II and EU-LCI reference values. The concentrations of individual VOCs and formaldehyde in these work environments were generally very low and posed no health risks. Total VOC concentration (TVOC) as well as concentrations of several individual compounds, including aromatic compounds, alkanes, 2-ethyl-1-hexanol, and formaldehyde, showed clearly decreasing trends. In contrast, several aldehydes, acids, and a few other compounds showed increasing trends. However, the increasing trends did not seem to affect the higher ends of the distributions, as the 95th percentile values remained fairly stable or decreased over the years. The VOC patterns in the environments of the offices, schools, kindergartens, and healthcare offices varied, probably reflecting the differences in typical activities and the use of materials. However, we do not expect these differences to be relevant to health outcomes.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10- 3 H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (R a ), whole-body FFA oxidation (R ox ), and nonoxidative disposal (R st ). In the liver, clearance (K β-ox ) and flux (R β-ox ) of FFA into β-oxidation were estimated using [9,10- 3 H]-(R)-bromopalmitate/[U- 14 C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, R a , R ox , and R st with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to β-oxidation, with increased K β-ox , R β-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma β-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation., Competing Interests: Conflict of interest All authors are or were employed by AstraZeneca and have shares in the company., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive., Methods and Results: Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index., Conclusion: FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro . The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021)., Competing Interests: L.N. and H.-C.T. are employees of Alnylam Pharmaceuticals Inc. K.W., G.O.M., and J.B. are employees of AstraZeneca., (© 2021 The Author(s).)

Context: Insulin resistance (IR) is a risk factor for type 2 diabetes, diabetic kidney disease, cardiovascular disease and nonalcoholic steatohepatitis. Biliopancreatic diversion (BPD) is the most effective form of bariatric surgery for improving insulin sensitivity., Objective: To identify plasma proteins correlating with the early restoration of insulin sensitivity after BPD., Design: Prospective single-center study including 20 insulin-resistant men with morbid obesity scheduled for BPD. Patient characteristics and blood samples were repeatedly collected from baseline up to 4 weeks postsurgery. IR was assessed by homeostatic model assessment for insulin resistance (HOMA-IR), Matsuda Index, and by studying metabolic profiles during meal tolerance tests. Unbiased proteomic analysis was performed to identify plasma proteins altered by BPD. Detailed plasma profiles were made on a selected set of proteins by targeted multiple reaction monitoring mass spectrometry (MRM/MS). Changes in plasma proteome were evaluated in relation to metabolic and inflammatory changes., Results: BPD resulted in improved insulin sensitivity and reduced body weight. Proteomic analysis identified 29 proteins that changed following BPD. Changes in plasma levels of afamin, apolipoprotein A-IV (ApoA4), and apolipoprotein A-II (ApoA2) correlated significantly with changes in IR., Conclusion: Circulating levels of afamin, ApoA4, and ApoA2 were associated with and may contribute to the rapid improvement in insulin sensitivity after BPD., (© Endocrine Society 2020.)

GPR81 is a receptor for the metabolic intermediate lactate with an established role in regulating adipocyte lipolysis. Potentially novel GPR81 agonists were identified that suppressed fasting plasma free fatty acid levels in rodents and in addition improved insulin sensitivity in mouse models of insulin resistance and diabetes. Unexpectedly, the agonists simultaneously induced hypertension in rodents, including wild-type, but not GPR81-deficient mice. Detailed cardiovascular studies in anesthetized dogs showed that the pressor effect was associated with heterogenous effects on vascular resistance among the measured tissues: increasing in the kidney while remaining unchanged in hindlimb and heart. Studies in rats revealed that the pressor effect could be blocked, and the renal resistance effect at least partially blocked, with pharmacological antagonism of endothelin receptors. In situ hybridization localized GPR81 to the microcirculation, notably afferent arterioles of the kidney. In conclusion, these results provide evidence for a potentially novel role of GPR81 agonism in blood pressure control and regulation of renal vascular resistance including modulation of a known vasoeffector mechanism, the endothelin system. In addition, support is provided for the concept of fatty acid lowering as a means of improving insulin sensitivity.

The current study extends previously reported PPARα agonist WY 14,643 (30 µmol/kg/day for 4 weeks) effects on circulating amino acid concentrations in rats fed a 48% saturated fat diet. Steady-state tracer experiments were used to examine in vivo kinetic mechanisms underlying altered plasma serine, glycine and arginine levels. Urinary urea and creatinine excretion were measured to assess whole-body amino acid catabolism. WY 14,643 treated animals demonstrated reduced efficiency to convert food consumed to body weight gain while liver weight was increased compared to controls. WY 14,643 raised total amino acid concentration (38%), largely explained by glycine, serine and threonine increases. 3H-glycine, 14C-serine and 14C-arginine tracer studies revealed elevated rates of appearance (Ra) for glycine (45.5 ± 5.8 versus 17.4 ± 2.7 µmol/kg/min) and serine (21.0 ± 1.4 versus 12.0 ± 1.0) in WY 14,643 versus control. Arginine was substantially decreased (-62%) in plasma with estimated Ra reduced from 3.1 ± 0.3 to 1.2 ± 0.2 µmol/kg/min in control versus WY 14,643. Nitrogen excretion over 24 hours was unaltered. Hepatic arginase activity was substantially decreased by WY 14,643 treatment. In conclusion, PPARα agonism potently alters metabolism of several specific amino acids in the rat. The changes in circulating levels of serine, glycine and arginine reflected altered fluxes into the plasma rather than changes in clearance or catabolism. This suggests that PPARα has an important role in modulating serine, glycine and arginine de novo synthesis.

Inhibition of AMP deaminase (AMPD) holds the potential to elevate intracellular adenosine and AMP levels and, therefore, to augment adenosine signaling and activation of AMP-activated protein kinase (AMPK). To test the latter hypothesis, novel AMPD pan inhibitors were synthesized and explored using a panel of in vitro, ex vivo, and in vivo models focusing on confirming AMPD inhibitory potency and the potential of AMPD inhibition to improve glucose control in vivo. Repeated dosing of selected inhibitors did not improve glucose control in insulin-resistant or diabetic rodent disease models. Mice with genetic deletion of the muscle-specific isoform Ampd1 did not showany favorable metabolic phenotype despite being challenged with high-fat diet feeding. Therefore, these results do not support the development of AMPD inhibitors for the treatment of type 2 diabetes.

Metabolic Flexibility Was Assessed in Male Zucker Rats: lean controls, obese controls, and obese rats treated with the dual peroxisome proliferator activated receptor (PPAR) α/γ agonist, tesaglitazar, 3  μ mol/kg/day for 3 weeks. Whole body glucose disposal rate (R d ) and hepatic glucose output (HGO) were assessed under basal fasting and hyperinsulinemic isoglycemic clamp conditions using [3,(3)H]glucose. Indices of tissue specific glucose utilization (R g ') were measured at basal, physiological, and supraphysiological levels of insulinemia using 2-deoxy-D-[2,6-(3)H]glucose. Finally, whole body and tissue specific FFA and glucose utilization and metabolic fate were evaluated under basal and hyperinsulinemic conditions using a combination of [U-(13)C]glucose, 2-deoxy-D-[U-(14)C]glucose, [U-(14)C]palmitate, and [9,10-(3)H]-(R)-bromopalmitate. Tesaglitazar improved whole body insulin action by greater suppression of HGO and stimulation of R d compared to obese controls. This involved increased insulin stimulation of R g ' in fat and skeletal muscle as well as increased glycogen synthesis. Tesaglitazar dramatically improved insulin mediated suppression of plasma FFA level, whole body turnover (R fa ), and muscle, liver, and fat utilization. At basal insulin levels, tesaglitazar failed to lower HGO or R fa compared to obese controls. In conclusion, the results demonstrate that tesaglitazar has a remarkable ability to improve insulin mediated control of glucose and FFA fluxes in obese Zucker rats.

We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.

Nucleic acid-based community fingerprinting methods are valuable tools in microbial ecology, as they offer rapid and robust means to compare large series of replicates and references. To avoid the time-consuming and potentially subjective procedures of peak-based examination, we assessed the possibility to apply direct curve-based data analysis on community fingerprints produced with bacterial length heterogeneity PCR (LH-PCR). The dataset comprised 180 profiles from a 21-week rhizoremediation greenhouse experiment with three treatments and 10 sampling times. Curve-based analysis quantified the progressive effect of the plant (Galega orientalis) and the reversible effect of the contaminant (fuel oil) on bacterial succession. The major observed community shifts were assigned to changes in plant biomass and contamination level by canonical correlation analysis. A novel method to extract relative abundance data from the fingerprint curves for Shannon diversity index revealed contamination to reversibly decrease community complexity. By cloning and sequencing the fragment lengths, recognized to change in time in the averaged LH-PCR profiles, we identified Aquabacterium (Betaproteobacteria) as the putative r-strategic fuel oil degrader, and K-strategic Alphaproteobacteria growing in abundance later in succession. Curve-based community fingerprint analysis can be used for rapid data prescreening or as a robust alternative for the more heavily parameterized peak-based analysis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The objective of this investigation was to use a pharmacokinetic (PK)/pharmacodynamic (PD) approach to describe and evaluate a PK model of nicotinic acid (NiAc) in guinea pigs and a PD feedback model of changes in non-esterified fatty acid (NEFA) concentrations in rats following multiple intravenous infusions of NiAc at different rates and durations of inhouse and literature (NEFA after extravascular NiAc dosing) data. Serial arterial blood samples were taken for evaluation of NiAc exposure in guinea pigs and NEFA in rats. The biophase kinetics of NiAc was assumed to impact on NEFA turnover with feedback incorporated via an inhibitory moderator compartment. The response acted linearly on the production of moderator, which then acted inversely on the turnover rate of response. The potency, expressed as the amount of NiAc in the biophase causing a 50 % inhibitory effect (ID(50)), was 6.5 nmol +/- 31 % and the half-life of response (t(1/2, kout)) 2 min +/- 18 %. The half-life of tolerance (t(1/2, ktol)) was 9 min +/- 27 %. The model can be used to provide information about factors that determine the time course of NEFA response following different rates and routes of administration of NiAc or NiAc analogues.

IL-6 is produced and released in large amounts from skeletal muscle during prolonged exercise in both mice and humans, but there are few data indicating the biological significance of this. IL-6 exerts metabolic effects such as stimulating energy expenditure and reducing body fat mass. We have now investigated the effects of IL-6 deficiency on exercise endurance and energy expenditure in preobese and obese IL-6-deficient (IL-6(-/-)) mice. Four-month-old preobese and 7-month-old obese IL-6(-/-) male mice backcrossed to C57BL/6 and their littermate controls were exercised on a treadmill, and energy expenditure was measured as oxygen consumption with the use of indirect calorimetry. The preobese IL-6(-/-) mice were significantly leaner than the control mice, whereas the older IL-6(-/-) mice, as expected, had developed obesity. Resting young, but not older, IL-6(-/-) mice had an elevated respiratory exchange ratio (RER), indicating that they oxidize carbohydrates rather than fat for energy utilization. During exercise, the young and older IL-6(-/-) mice had a reduced endurance and a progressive decrease in oxygen consumption compared with control mice. There was no difference in RER in young IL-6(-/-) mice, whereas RER was enhanced in older IL-6(-/-), mice during exercise. In summary, IL-6(-/-) mice have reduced endurance and energy expenditure during exercise, suggesting that IL-6 is necessary for normal exercise capacity.

Abstract A non-indigenous wild-type strain Rhizobium galegae HAMBI 540, which specifically nodulates perennial goat's rue (Galega orientalis), and its marker gene-tagged derivatives R. galegae HAMBI 2363(luc), R. galegae HAMBI 2368(gusA21) and R. galegae HAMBI 2364(gusA30) were used to evaluate the persistence, population dynamics and competitiveness for nodulation of rhizobia under field conditions in Finland. The lysimeters were filled with clean or diesel oil-polluted (3000 mug g(-1)) agricultural soil. During the first 2 years of the field release luc- and gusA21-tagged strains could be effectively detected by cultivation, reinforced with colony polymerase chain reaction. The population densities remained relatively stable from 10(4) to 10(5) cfu g(-1) dry soil from spring until late autumn. Replicate limiting dilution polymerase chain reaction analysis gave comparable results with cultivation with strain HAMBI 2363 until 49 weeks after inoculation. GUS activity of strain HAMBI 2368 could be stably detected in nodules and soil. On the other hand, luc activity weakened clearly in cold conditions along with decreased metabolic activity of rhizobia. The competitive ability for nodulation of the gusA30-tagged strain decreased slowly with time compared to the wild-type strain. Moderate soil pollution did not have significant effects on target bacteria or plant growth. Limited vertical movement of target bacteria outside the rhizosphere was detected from percolated water.

Interleukin (IL)-6 is a multifunctional immune-modulating cytokine that has been suggested to have important functions in glucose and lipid metabolism. It is secreted from adipose tissue during resting conditions and from muscle during strenuous exercise. Recently, the authors reported that mice deficient of IL-6 develop mature-onset obesity, which was reversed by IL-6 replacement. The IL-6-deficient mice had increased glucose levels and decreased glucose tolerance, and blood lipids were increased in females. Furthermore, it was found that intracerebroventricular (ICV) IL-6 treatment acutely increased energy expenditure in rats and led to loss of fat mass following prolonged treatment, without causing symptoms of sickness behaviour or increased levels of acute-phase reactants. Thus, these data indicate a role for IL-6 in the regulation of energy homeostasis in rodents. In humans, several single nucleotide polymorphisms in the IL-6 gene promoter are known, one of which (174 C) is associated with reduced IL-6 transcription as well as decreased basal metabolic rate and insulin sensitivity in healthy male subjects. Furthermore, it was found that IL-6 levels in cerebrospinal fluid in obese humans were inversely correlated with more severe obesity, suggesting that severe obesity is coupled to a relative central IL-6 deficiency. Taken together, these data suggest that endogenous IL-6 has antiobesity effects and, therefore, it is possible that low endogenous IL-6 production contributes to obesity in humans.

Recently, we demonstrated that intracerebroventricular injection of IL-6 increases energy expenditure and decreases body fat in rodents. Therefore, IL-6 may play a role in appetite and body weight control in the central nervous system. In the present study we evaluated cerebrospinal fluid (CSF) and serum IL-6 levels in humans in relation to body fat content and to CSF and serum levels of leptin. Thirty-two healthy overweight/obese male subjects with a body mass index range of 29.3-36.0 kg/m(2) were studied. Total and sc body fat were measured by dual energy x-ray absorptiometry and computed tomography, respectively. CSF IL-6 levels were in some individuals higher than serum IL-6 levels and correlated negatively with total body weight, sc and total body fat. In contrast, CSF leptin levels were 30-60 times lower than serum leptin levels and correlated positively with serum leptin, body weight, sc and total body fat. Furthermore, there was a negative correlation between CSF IL-6 and leptin. In conclusion, CSF IL-6 differs in many ways from CSF leptin. CSF IL-6 may be locally produced rather than serum derived, and body fat-regulating regions in the central nervous system may be exposed to insufficient IL-6 levels in more severe obesity.

We conducted an experimental study examining the site and mechanism of action of the anti-obesity effect of interleukin-6 (IL-6) in mice and rats. We used dual energy X-ray absorptiometry (DEXA) and computerized tomography to investigate the body composition of mice with knockout of the IL-6 gene and wild-type control mice. Rats were treated with IL-6 or vehicle through intracerebroventricular (ICV) cannulae. Energy expenditure was measured as oxygen consumption by indirect calorimetry in metabolic chambers. Results showed that the mice lacking IL-6 increased in body weight compared with wild-type mice from 6 months of age onward, although there was no marked difference in food intake between the pre-obese IL-6 knockout mice and the wild-type mice. IL-6 given as a single ICV injection to rats stimulated oxygen consumption; whereas, the same doses were ineffective when given peripherally. Chronic ICV IL-6 treatment decreased body weight and fat mass in rodents. Administration of IL-6 may decrease fat mass in mice and rats by stimulating energy expenditure at the CNS level, possibly in the hypothalamus.

Insulin-like growth factor (IGF) I is an important regulator of both skeletal growth and adult bone metabolism. To better understand the relative importance of systemic IGF-I versus locally expressed IGF-I we have developed a transgenic mouse model with inducible specific IGF-I gene inactivation in the liver (LI-IGF-I-/-). These mice are growing normally up to 12 weeks of age but have a disturbed carbohydrate and lipid metabolism. In this study, the long-term effects of liver-specific IGF-I inactivation on skeletal growth and adult bone metabolism were investigated. The adult (week 8-55) axial skeletal growth was decreased by 24% in the LI-IGF-I-/- mice whereas no major reduction of the adult appendicular skeletal growth was seen. The cortical cross-sectional bone area, as measured in the middiaphyseal region of the long bones, was decreased in old LI-IGF-I-/- mice. This reduction in the amount of cortical bone was caused mainly by decreased periosteal circumference and was associated with a weaker bone determined by a decrease in ultimate load. In contrast, the amount of trabecular bone was not decreased in the LI-IGF-I-/- mice. DNA microarray analysis of 30-week-old LI-IGF-I-/- and control mice indicated that only four genes were regulated in bone whereas approximately 40 genes were regulated in the liver, supporting the hypothesis that liver-derived IGF-I is of minor importance for adult bone metabolism. In summary, liver-derived IGF-I exerts a small but significant effect on cortical periosteal bone growth and on adult axial skeletal growth while it is not required for the maintenance of the trabecular bone in adult mice.

Recently we found that interleukin-6 (IL-6) knockout mice develop mature-onset obesity and that a single intracerebroventricular (ICV) injection of IL-6 increases energy expenditure. In the present study we investigated if chronic ICV treatment with IL-6 can suppress body fat mass. IL-6 was injected ICV daily for two weeks to rats fed a high-fat diet. IL-6 treatment but not saline treatment decreased body weight by 8.4% and decreased the relative weights of mesenteric and retroperitoneal fat pads. Consistent with this, circulating leptin levels were decreased by 40% after IL-6 treatment but not after saline treatment. Average food intake per day was decreased in the IL-6 treated group compared to the saline treated rats. IL-6 treatment did not change hepatic expression of the acute-phase protein haptoglobin, serum levels of insulin or insulin-like growth factor-I, or the weights of the heart, liver, kidneys, adrenals, and spleen. We conclude that centrally administered IL-6 can decrease body fat in rats without causing acute-phase reaction.

The immune-modulating cytokine interleukin-6 (IL-6) is expressed both in adipose tissue and centrally in hypothalamic nuclei that regulate body composition. We investigated the impact of loss of IL-6 on body composition in mice lacking the gene encoding IL-6 (Il6-/- mice) and found that they developed mature-onset obesity that was partly reversed by IL-6 replacement. The obese Il6-/- mice had disturbed carbohydrate and lipid metabolism, increased leptin levels and decreased responsiveness to leptin treatment. To investigate the possible mechanism and site of action of the anti-obesity effect of IL-6, we injected rats centrally and peripherally with IL-6 at low doses. Intracerebroventricular, but not intraperitoneal IL-6 treatment increased energy expenditure. In conclusion, centrally acting IL-6 exerts anti-obesity effects in rodents.

We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I-/-) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I-/- mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I-/- mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I-/- mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I-/- mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by increasing expression of pituitary GHRF and GHS receptors. The enhanced GH release in turn affects several liver parameters, in line with the existence of a pituitary-liver axis.

IGF-I is important for postnatal body growth and exhibits insulin-like effects on carbohydrate metabolism. The function of liver-derived IGF-I is still not established, although we previously demonstrated that liver-derived IGF-I is not required for postnatal body growth. Mice whose IGF-I gene in the liver was inactivated at 24 days of age were used to investigate the long-term role of liver-derived IGF-I for carbohydrate and lipid metabolism. Serum levels of leptin in these mice were increased by >100% at 3 months of age, whereas the fat mass of the mice was decreased by 25% at 13 months of age. The mice became markedly hyperinsulinemic and yet normoglycemic, indicating an adequately compensated insulin resistance. Furthermore, they had increased serum levels of cholesterol. We conclude that liver-derived IGF-I is of importance for carbohydrate and lipid metabolism.

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.

The liver size in adult mammals is tightly regulated in relation to body weight, but the hormonal control of this is largely unknown. We investigated the roles of interleukin-6 (IL-6) and tumor necrosis factor (TNF) receptor-1 in the regulation of intact liver weight in adult mice. The relative liver wet and dry weights of older adult (5- to 10-month-old) IL-6 knockout (IL-6(-/-)) mice were decreased by 22-28%, and total contents of DNA and protein were decreased compared with those in age-matched wild-type mice. Weights of other visceral organs were unaffected. Older adult (6- to 8-month-old) TNF receptor-1 knockout (TNFR1(-/-)) mice displayed decreased relative liver weight. Treatment with a single injection of IL-6 increased liver wet and dry weights in IL-6(-/-) and wild-type mice, but not TNFR1(-/-) mice. Treatment with TNFalpha enhanced liver weight and DNA synthesis of nonparenchymal liver cells at 24 h in wild-type, but not IL-6(-/-), mice. At 48 h, TNFalpha induced DNA synthesis in nonparenchymal cells and hepatocytes of both wild-type and IL-6(-/-) mice. In conclusion, TNF receptor-1 stimulation and IL-6 production are both necessary for normal liver weight gain in older adult mice. The results of TNFalpha and IL-6 treatment further indicate that the effects of TNF receptor-1 and IL-6 depend on each other for full stimulation of liver growth.

Background/aims: Almost all circulating insulin-like growth factor-1 (IGF-1) is produced and secreted from the liver. However, the possible role of IGF-1 in local regulation of liver functions including liver growth is unclear. In the present study, we investigated the role of IGF-1 on liver growth in vivo and in hepatic stellate cell function in vitro., Results: Liver-specific knock-out of the IGF-1 gene by use of the cre-loxP system caused enhanced liver growth, possibly reflecting increased growth hormone (GH) secretion due to decreased negative feedback by IGF-1. Studies on cultured rat hepatic stellate cells (HSC) showed that IGF-1 and hepatocyte-conditioned medium (PCcM) time- and dose-dependently increased hepatocyte growth factor (HGF) mRNA and HGF immunoreactivity. IGF-1 and PCcM also enhanced DNA synthesis in the HSC cultures. The PCcM did not contain bioactive IGF-1 and was also able to stimulate proliferation when prepared under serum- and hormone-free conditions., Conclusion: In vivo results show that IGF-1 is not essential for normal growth of the intact liver. The in vitro results indicate that both IGF-1 and IGF- 1-independent factor(s) from hepatocytes can stimulate HGF production by HSC. It remains to be investigated whether these effects are of importance for liver regeneration or pathological conditions., (Copyright 2001 S. Karger AG, Basel.)

Background/aims: In the absence of liver damage, rapid liver growth can be induced pharmacologically by so-called primary liver growth promoters. The importance of the acute-phase cytokines interleukin-6 and tumor necrosis factor-alpha for the actions of these compounds is not clear. This study aimed to investigate the importance of IL-6 and TNF-receptor-1 in pharmacologically-induced liver growth., Methods: IL-6 knockout (IL-6(-/-)), TNF-receptor-1 knockout (TNFR1(-/-)) and wild-type mice were treated with the peroxisome proliferator nafenopin and the anti-androgen cyproterone acetate (CPA) in one single injection or for 6 days with daily injections, and examined at 24 or 48 h after treatment. In a control experiment, IL-6(-/-) mice were subjected to two-thirds partial hepatectomy., Results: Nafenopin treatment increased relative liver weight and DNA synthesis similarly in IL-6(-/-), TNFR1(-/-) and wild-type mice. CPA increased liver weight similarly in all groups, but did not increase DNA synthesis. Expression of peroxisome proliferator activated receptor-alpha mRNA was increased in both IL-6(-/-) and wild-type mice by nafenopin treatment, but not by CPA treatment. After hepatectomy DNA synthesis was suppressed in IL-6(-/-) mice compared to wild-type mice., Conclusions: Liver growth induced by nafenopin and CPA was not dependent on the presence of IL-6 or TNF receptor-1, whereas liver regeneration was decreased in IL-6(-/-) mice.

Background/aims: Hepatic stellate cells (HSC) are located in close proximity to hepatocytes in Disse's space. Hepatocyte derived factors have earlier been implicated in the paracrine regulation of HSC proliferation. The aim of the present study was to further characterize this mitogenic activity of the parenchymal cell conditioned medium (PCcM)., Methods: Primary rat HSC were cultured for 4 days. DNA synthesis was measured by 3H-thymidine incorporation. TGFbeta1 immunoreactivity was quantified by ELISA. PCcM was obtained from hepatocytes cultured in medium without serum or hormones for two days., Results: Incubation of 4-day-old HSC on plastic surface with PCcM for 2 days increased DNA synthesis, while no effect was seen in HSC cultured on Matrigel. Heat-, acid-, and protease-treatment of PCcM abolished its stimulatory effect. Size fractionations with spin columns indicated that the stimulatory effect was contained in the fractions of a molecular size between 30 and 100 kD. The addition of LY 294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, dose-dependently inhibited the PCcM induced increase in DNA synthesis to about 9% of the control values. The specific MAP kinase (MAPK) inhibitor, PD 98059 only suppressed the PCcM induced DNA synthesis to 35% of control cultures at the highest dose (10 microM). DNA content in the cultures was not affected by either blocker. HSC seemed to produce immunoreactive TGFbeta1. However, addition of latency-associated peptide (LAP), a potent TGFbeta1 blocker, stimulated DNA synthesis to a much less extent than PCcM., Conclusions: The factor(s) that stimulate DNA synthesis in HSC from hepatocytes are most likely protein(s) with a molecular size between 30-100 kD. These factor(s) rely more on PI3-K than on MAPK for their mitogenic effect and are probably not acting via TGFbeta1 inhibition.

Using strain-guaged duplicate maxillary dentures, the deformation patterns and magnitude of functional strain were studied on five complete denture wearing subjects who had varying amounts of soft mobile tissue over their maxillary edentulous alveolar ridge so called flabby ridges. The thickness of mobile tissue--expressed as depth--was measured by a sharpened periodontal probe with aid of a plastic base plate as a locator. The functional test included maximum biting, and the chewing of three test food samples. The results did not point on any direct relationship between the thickness of the soft tissue and the midline deformation pattern and/or magnitude of functional strain. It seems, however, as if the main influence of mobile tissue is on the strain behaviour in the lateral part of the denture.