Bile acids mediate fructose-associated liver tumour growth in mice.

High fructose diets are associated with an increased risk of liver cancer. Previous studies in mice suggest increased lipogenesis is a key mechanism linking high fructose diets to liver tumour growth. However, these studies administered fructose to mice at supraphysiological levels. The aim of this study was to determine whether liver tumour growth and lipogenesis were altered in mice fed fructose at physiological levels. To test this, we injected male C57BL/6 mice with the liver carcinogen diethylnitrosamine and then fed them diets without fructose or fructose ranging from 10 to 20 % total calories. Results showed mice fed diets with ≥15 % fructose had significantly increased liver tumour numbers (2-4-fold) and total tumour burden (∼7-fold) vs mice fed no-fructose diets. However, fructose-associated tumour burden was not associated with lipogenesis. Conversely, unbiased metabolomic analyses revealed bile acids were elevated in the sera of mice fed a 15 % fructose diet vs mice fed a no-fructose diet. Using a syngeneic ectopic liver tumour model, we show that ursodeoxycholic acid, which decreases systemic bile acids, significantly reduced liver tumour growth in mice fed the 15 % fructose diet but not mice fed a no-fructose diet. These results point to a novel role for systemic bile acids in mediating liver tumour growth associated with a high fructose diet. Overall, our study shows fructose intake at or above normal human consumption (≥15 %) is associated with increased liver tumour numbers and growth and that modulating systemic bile acids inhibits fructose-associated liver tumour growth in mice.

Class IIa HDACs inhibit cell death pathways and protect muscle integrity in response to lipotoxicity.

Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.

Effects of antipsychotic drugs on energy metabolism.

Schizophrenia (SCZ) is a complex neuropsychiatric disorder associated with altered bioenergetic pathways and mitochondrial dysfunction. Antipsychotic medications, both first and second-generation, are commonly prescribed to manage SCZ symptoms, but their direct impact on mitochondrial function remains poorly understood. In this study, we investigated the effects of commonly prescribed antipsychotics on bioenergetic pathways in cultured neurons. We examined the impact of risperidone, aripiprazole, amisulpride, and clozapine on gene expression, mitochondrial bioenergetic profile, and targeted metabolomics after 24-h treatment, using RNA-seq, Seahorse XF24 Flux Analyser, and gas chromatography-mass spectrometry (GC-MS), respectively. Risperidone treatment reduced the expression of genes involved in oxidative phosphorylation, the tricarboxylic acid cycle, and glycolysis pathways, and it showed a tendency to decrease basal mitochondrial respiration. Aripiprazole led to dose-dependent reductions in various mitochondrial function parameters without significantly affecting gene expression. Aripiprazole, amisulpride and clozapine treatment showed an effect on the tricarboxylic acid cycle metabolism, leading to more abundant metabolite levels. Antipsychotic drug effects on mitochondrial function in SCZ are multifaceted. While some drugs have greater effects on gene expression, others appear to exert their effects through enzymatic post-translational or allosteric modification of enzymatic activity. Understanding these effects is crucial for optimising treatment strategies for SCZ. Novel therapeutic interventions targeting energy metabolism by post-transcriptional pathways might be more effective as these can more directly and efficiently regulate energy production.

The impact of a single dose of whey protein on glucose flux and metabolite profiles in normoglycemic males: insights into glucagon and insulin biology.

Protein ingestion concurrently stimulates euglycemic glucagon and insulin secretion, a response that is particularly robust with rapidly absorbing proteins. Previously, we have shown that ingestion of repeated doses of rapidly absorbing whey protein equally stimulated endogenous glucose production (EGP) and glucose disposal (Rd), thus explaining the preservation of euglycemia. Here, we aimed to determine if a smaller single dose of whey could elicit a large enough glucagon and insulin response to stimulate glucose flux. Therefore, in normoglycemic young adult males (n = 10; age ∼26; BMI ∼25), using [6,6-2H2] glucose tracing and quantitative targeted metabolite profiling, we determined the metabolic response to a single 25 g "standard" dose of whey protein. Whey protein ingestion did not alter glycemia, but increased circulating glucagon (peak 4-fold basal), insulin (peak 6-fold basal), amino acids, and urea while also reducing free fatty acid (FFA) and glycerol concentrations. Interestingly, the postprandial insulin response was driven by both a stimulation of insulin secretion and marked reduction in hepatic insulin clearance. Whey protein ingestion resulted in a modest stimulation of EGP and Rd, both peaking at ∼20% above baseline 1 h after protein ingestion. These findings demonstrate that the ingestion of a single standard serving of whey protein can induce a euglycemic glucagon and insulin response that stimulates glucose flux. We speculate on a theory that could potentially explain how glucagon and insulin synergistically provide hardwired control of nitrogen and glucose homeostasis.NEW & NOTEWORTHY Protein ingestion concurrently stimulates glucagon and insulin secretion. Here we show that in normoglycemic males, ingestion of a single "standard" 25 g serving of rapidly absorbing whey protein drives a sufficiently large glucagon and insulin response, such that it simultaneously increases endogenous glucose production and glucose disposal. We speculate on a novel theory that could potentially explain how the antagonistic/synergistic actions of glucagon and insulin simultaneously provide tight control of glucose and nitrogen homeostasis.

Insulin sensitivity is preserved in mice made obese by feeding a high starch diet.

Obesity is generally associated with insulin resistance in liver and muscle and increased risk of developing type 2 diabetes, however there is a population of obese people that remain insulin sensitive. Similarly, recent work suggests that mice fed high carbohydrate diets can become obese without apparent glucose intolerance. To investigate this phenomenon further, we fed mice either a high fat (Hi-F) or high starch (Hi-ST) diet and measured adiposity, glucose tolerance, insulin sensitivity, and tissue lipids compared to control mice fed a standard laboratory chow. Both Hi-ST and Hi-F mice accumulated a similar amount of fat and tissue triglyceride compared to chow-fed mice. However, while Hi-F diet mice developed glucose intolerance as well as liver and muscle insulin resistance (assessed via euglycaemic/hyperinsulinaemic clamp), obese Hi-ST mice maintained glucose tolerance and insulin action similar to lean, chow-fed controls. This preservation of insulin action despite obesity in Hi-ST mice was associated with differences in de novo lipogenesis and levels of C22:0 ceramide in liver and C18:0 ceramide in muscle. This indicates that dietary manipulation can influence insulin action independently of the level of adiposity and that the presence of specific ceramide species correlates with these differences.

Creatine supplementation reduces the cerebral oxidative and metabolic stress responses to acute in utero hypoxia in the late-gestation fetal sheep.

Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain intracellular ATP levels thereby reducing oxidative and metabolic stress responses during oxygen deprivation. Using microdialysis, we investigated the real-time in vivo effects of fetal creatine supplementation on cerebral metabolism following acute in utero hypoxia caused by umbilical cord occlusion (UCO). Fetal sheep (118 days' gestational age (dGA)) were implanted with an inflatable Silastic cuff around the umbilical cord and a microdialysis probe inserted into the right cerebral hemisphere for interstitial fluid sampling. Creatine (6 mg kg-1  h-1 ) or saline was continuously infused intravenously from 122 dGA. At 131 dGA, a 10 min UCO was induced. Hourly microdialysis samples were obtained from -24 to 72 h post-UCO and analysed for percentage change of hydroxyl radicals (• OH) and interstitial metabolites (lactate, pyruvate, glutamate, glycerol, glycine). Histochemical markers of protein and lipid oxidation were assessed at post-mortem 72 h post-UCO. Prior to UCO, creatine treatment reduced pyruvate and glycerol concentrations in the microdialysate outflow. Creatine treatment reduced interstitial cerebral • OH outflow 0 to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO presented with reduced levels of hypoxaemia ( PO2${P_{{{\rm{O}}_{\rm{2}}}}}$ and SO2${S_{{{\rm{O}}_{\rm{2}}}}}$ ) during UCO which associated with reduced interstitial cerebral pyruvate, lactate and • OH accumulation. No effects of creatine treatment on immunohistochemical markers of oxidative stress were found. In conclusion, fetal creatine treatment decreased cerebral outflow of • OH and was associated with an improvement in cerebral bioenergetics following acute hypoxia. KEY POINTS: Fetal hypoxia can cause persistent metabolic and oxidative stress responses that disturb energy homeostasis in the brain. Creatine in its phosphorylated form is an endogenous phosphagen; therefore, supplementation is a proposed prophylactic treatment for fetal hypoxia. Fetal sheep instrumented with a cerebral microdialysis probe were continuously infused with or without creatine-monohydrate for 10 days before induction of 10 min umbilical cord occlusion (UCO; 131 days' gestation). Cerebral interstitial fluid was collected up to 72 h following UCO. Prior to UCO, fetal creatine supplementation reduced interstitial cerebral pyruvate and glycerol concentrations. Fetal creatine supplementation reduced cerebral hydroxyl radical efflux up to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO and reduced levels of systemic hypoxaemia during UCO were associated with reduced cerebral interstitial pyruvate, lactate and • OH following UCO. Creatine supplementation leads to some improvements in cerebral bioenergetics following in utero acute hypoxia.

Baseline serum amino acid levels predict treatment response to augmentation with N-acetylcysteine (NAC) in a bipolar disorder randomised trial.

N-acetylcysteine (NAC) acts on glutamatergic and redox systems, two systems implicated in the pathophysiology of bipolar disorder (BD). This has led to the investigation of NAC as a potential candidate for the treatment of BD. The aim of this study was to investigate metabolomic markers to identify predictors of NAC response in a cohort of BD participants. This study is a secondary analysis of a 16-week, multi-site, randomized, double-blinded, parallel-group, placebo-controlled trial in BD participants with a current acute depressive episode. This study included trial participants who received either NAC 2000 mg/day, or placebo. Participants (NAC: n = 31, placebo: n = 29) were assessed at baseline and week 16 using the Montgomery Åsberg Depression Rating Scale (MADRS) and were dichotomised into "responders" (MADRS at week 16 < 50% of MADRS at baseline) and "non-responders" (MADRS at week 16 > 50% at baseline). Untargeted gas chromatography-mass spectrometry analysis was performed to analyse baseline levels of 68 serum metabolites. Of the nine metabolites that differentiated placebo and NAC groups, five were amino acids with lower levels in the NAC responder group compared with the NAC non-responders. Further analysis generated a predictive model of MADRS improvement including glycine, norleucine, threonine, proline, phenylalanine, tyrosine, glutamic acid, lysine and leucine (R2 = 0.853; adjusted R2 = 0.733). This prediction model predicted 85% of the variance in MADRS outcome after adjunctive treatment with NAC. BD participants with lower serum levels of free amino acids at baseline may be more likely to respond to adjunctive treatment with NAC.

Translating glucose tolerance data from mice to humans: Insights from stable isotope labelled glucose tolerance tests.

OBJECTIVE: The glucose tolerance test (GTT) is widely used in human and animal biomedical and pharmaceutical research. Despite its prevalent use, particularly in mouse metabolic phenotyping, to the best of our knowledge we are not aware of any studies that have attempted to qualitatively compare the metabolic events during a GTT in mice with those performed in humans. METHODS: Stable isotope labelled oral glucose tolerance tests (siOGTTs; [6,6-2H2]glucose) were performed in both human and mouse cohorts to provide greater resolution into postprandial glucose kinetics. The siOGTT allows for the partitioning of circulating glucose into that derived from exogenous and endogenous sources. Young adults spanning the spectrum of normal glucose tolerance (n = 221), impaired fasting (n = 14), and impaired glucose tolerance (n = 19) underwent a 75g siOGTT, whereas a 50 mg siOGTT was performed on chow (n = 43) and high-fat high-sucrose fed C57Bl6 male mice (n = 46). RESULTS: During the siOGTT in humans, there is a long period (>3hr) of glucose absorption and, accordingly, a large, sustained insulin response and robust suppression of lipolysis and endogenous glucose production (EGP), even in the presence of glucose intolerance. In contrast, mice appear to be highly reliant on glucose effectiveness to clear exogenous glucose and experience only modest, transient insulin responses with little, if any, suppression of EGP. In addition to the impaired stimulation of glucose uptake, mice with the worst glucose tolerance appear to have a paradoxical and persistent rise in EGP during the OGTT, likely related to handling stress. CONCLUSIONS: The metabolic response to the OGTT in mice and humans is highly divergent. The potential reasons for these differences and their impact on the interpretation of mouse glucose tolerance data and their translation to humans are discussed.

An Ethanolic Extract of Artemisia dracunculus L. Enhances the Metabolic Benefits of Exercise in Diet-induced Obese Mice.

PURPOSE: The purpose of this study was to determine the effect of an ethanolic extract of Artemisia dracunculus L. (5011) combined with exercise on in vivo glucose and fat metabolism in diet-induced obese male mice. METHODS: After 8 wk of high-fat diet (HFD) feeding, 52 mice were randomly allocated to a voluntary wheel running group (HFD Ex), a 5011 + HFD sedentary group (5011 Sed), a 5011 + HFD Ex (5011 Ex), or an HFD sedentary group (HFD Sed) for 4 wk. Real-time energy expenditure and substrate utilization were measured by indirect calorimetry. A stable isotope glucose tolerance test was performed before and after the 4-wk wheel running period to determine changes in endogenous glucose production and glucose disposal. We also performed an analysis of genes and proteins associated with the early response to exercise and exercise adaptations in skeletal muscle and liver. RESULTS: When compared with HFD Ex mice, 5011 Ex mice had increased fat oxidation during speed- and distance-matched wheel running bouts. Both HFD Ex and 5011 Ex mice had reduced endogenous glucose during the glucose tolerance test, whereas only the 5011 Sed and the 5011 Ex mice had improved glucose disposal after the 4-wk experimental period when compared with HFD Sed and HFD Ex mice. 5011 Ex mice had increased Pgc1-α and Tfam expression in skeletal muscle when compared with HFD Ex mice, whereas Pdk4 expression was reduced in the liver of HFD Ex and 5011 Ex mice. CONCLUSIONS: Our study demonstrates that 5011, an ethanolic extract of A. dracunculus L., with a history of medicinal use, enhances the metabolic benefits of exercise to improve in vivo fat and glucose metabolism.

Autophagy is not involved in lipid accumulation and the development of insulin resistance in skeletal muscle.

OBJECTIVE: To investigate the effect of high fat diet-induced insulin resistance on autophagy markers in the liver and skeletal muscle of mice in the fasted state and following an oral glucose bolus. METHODS: Forty C57BL/6J male mice were fed either a high fat, high sucrose (HFSD, n = 20) or standard chow control (CON, n = 20) diet for 16 weeks. Upon trial completion, mice were gavaged with water or glucose and skeletal muscle and liver were collected 15 min post gavage. Protein abundance and gene expression of autophagy markers and activation of related signalling pathways were assessed. RESULTS: Compared to CON, the HFSD intervention increased LC3B-II and p62/SQSTM1 protein abundance in the liver which is indicative of elevated autophagosome content via reduced clearance. These changes coincided with inhibitory autophagy signalling through elevated p-mTOR S2448 and p-ULK1S758. HFSD did not alter autophagy markers in skeletal muscle. Administration of an oral glucose bolus had no effect on autophagy markers or upstream signalling responses in either tissue regardless of diet. CONCLUSION: HFSD induces tissue-specific autophagy impairments, with autophagosome accumulation indicating reduced lysosomal clearance in the liver. In contrast, autophagy markers were unchanged in skeletal muscle, indicating that autophagy is not involved in the development of skeletal muscle insulin resistance.

Loss of protein kinase D activity demonstrates redundancy in cardiac glucose metabolism and preserves cardiac function in obesity.

OBJECTIVE: Protein kinase D (PKD) signaling has been implicated in stress-induced cardiac remodeling and function as well as metabolic processes including contraction-mediated cardiac glucose uptake. PKD has recently emerged as a nutrient-sensing kinase that is activated in high-lipid environments, such as in obesity. However, the role of PKD signaling in cardiac glucose metabolism and cardiac function in both normal and obese conditions remains unknown. METHODS: A cardiac-specific and inducible dominant negative (DN) PKD mouse model was developed. Echocardiography was used to assess cardiac function, while metabolic phenotyping was performed, including stable isotope metabolomics on cardiac tissue in mice fed either regular chow or a high-fat diet (43% calories from fat). RESULTS: Cardiac PKD activity declined by ∼90% following DN PKD induction in adult mice. The mice had diminished basal cardiac glucose clearance, suggesting impaired contraction-mediated glucose uptake, but normal cardiac function. In obesity studies, systolic function indices were reduced in control mice, but not in cardiac DN PKD mice. Using targeted stable isotope metabolomic analyses, no differences in glucose flux through glycolysis or the TCA cycle were observed between groups. CONCLUSIONS: The data show that PKD contributes to cardiac dysfunction in obesity and highlight the redundancy in cardiac glucose metabolism that maintains cardiac glucose flux in vivo. The data suggest that impairments in contraction-mediated glucose uptake are unlikely to drive cardiac dysfunction in both normal and metabolic disease states.

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions.

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and 13 C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly 13 C3 labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. 13 C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies.

Ultrahigh-Resolution Mass Spectrometry Method for Resolving 13C-Enrichment Patterns in a Microalgal Lipidome.

The analysis of 13C-labeled lipids by mass spectrometry is challenging due to the complexity from labeling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionization and in-source fragmentation, which can create complex overlapping isotope patterns that can only be resolved using high-resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13C-labeled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labeling information. Mass resolving powers of 240000 full width half-maximum (fwhm) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts, and unresolved lipid species after chromatographic separation. This enabled the percentage of 13C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labeling within the headgroup vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and remodeling processes and can be extended to other biological systems.

Mapping the Associations of the Plasma Lipidome With Insulin Resistance and Response to an Oral Glucose Tolerance Test.

CONTEXT: Insulin resistance (IR) remains a global health challenge. Lipidomics offers an opportunity to identify biomarkers and better understand mechanisms of IR associated with abnormal lipid metabolism. OBJECTIVE: The objective of this article is to determine plasma lipid species associated with indices of IR and evaluate the lipidome response to an oral glucose tolerance test (OGTT). DESIGN AND SETTING: This study was community based and cross-sectional. PARTICIPANTS AND SAMPLE: Plasma samples (collected at 0 and 120 min during an OGTT) from nonobese, young adults age 18 to 34 years (n = 246) were analyzed using liquid chromatography-tandem mass spectrometry. MAIN OUTCOME MEASURES: The associations between indices of IR and lipid classes and species (with a sex interaction term), or changes in lipid levels during an OGTT, were tested using linear models (adjusted for age, sex, body mass index, total cholesterol, high-density lipoprotein cholesterol, and triglycerides). RESULTS: Some (213) and (199) lipid species were associated with the homeostatic model assessment of insulin resistance and insulin area under curve (AUC), respectively. Alkylphosphatidylcholine (10), alkenylphosphatidylcholine (23), and alkylphosphatidylethanolamine (6) species were associated with insulin AUC in men only. Species of phosphatidylcholine (7) and sphingomyelin (5) were associated in women only. In response to an OGTT, a perturbation in the plasma lipidome, particularly in acylcarnitine species, was observed; and the changes in many lipid species were associated with insulin AUC. CONCLUSIONS: The plasma lipidome and changes in lipid levels during an OGTT were associated with indices of IR. These findings underlie the involvement of molecular lipid species in the pathogenesis of IR and possibly crosstalk between IR and sex-specific regulation of lipid metabolism.

The Effects of Early-Onset Pre-Eclampsia on Placental Creatine Metabolism in the Third Trimester.

Creatine is a metabolite important for cellular energy homeostasis as it provides spatio-temporal adenosine triphosphate (ATP) buffering for cells with fluctuating energy demands. Here, we examined whether placental creatine metabolism was altered in cases of early-onset pre-eclampsia (PE), a condition known to cause placental metabolic dysfunction. We studied third trimester human placentae collected between 27-40 weeks' gestation from women with early-onset PE (n = 20) and gestation-matched normotensive control pregnancies (n = 20). Placental total creatine and creatine precursor guanidinoacetate (GAA) content were measured. mRNA expression of the creatine synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT), the creatine transporter (SLC6A8), and the creatine kinases (mitochondrial CKMT1A & cytosolic BBCK) was assessed. Placental protein levels of arginine:glycine aminotransferase (AGAT), GAMT, CKMT1A and BBCK were also determined. Key findings; total creatine content of PE placentae was 38% higher than controls (p < 0.01). mRNA expression of GATM (p < 0.001), GAMT (p < 0.001), SLC6A8 (p = 0.021) and BBCK (p < 0.001) was also elevated in PE placentae. No differences in GAA content, nor protein levels of AGAT, GAMT, BBCK or CKMT1A were observed between cohorts. Advancing gestation and birth weight were associated with a down-regulation in placental GATM mRNA expression, and a reduction in GAA content, in control placentae. These relationships were absent in PE cases. Our results suggest PE placentae may have an ongoing reliance on the creatine kinase circuit for maintenance of cellular energetics with increased total creatine content and transcriptional changes to creatine synthesizing enzymes and the creatine transporter. Understanding the functional consequences of these changes warrants further investigation.

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.

Mechanisms of hyperinsulinaemia in apparently healthy non-obese young adults: role of insulin secretion, clearance and action and associations with plasma amino acids.

AIMS/HYPOTHESIS: This study aimed to examine the metabolic health of young apparently healthy non-obese adults to better understand mechanisms of hyperinsulinaemia. METHODS: Non-obese (BMI < 30 kg/m2) adults aged 18-35 years (N = 254) underwent a stable isotope-labelled OGTT. Insulin sensitivity, glucose effectiveness and beta cell function were determined using oral minimal models. Individuals were stratified into quartiles based on their insulin response during the OGTT, with quartile 1 having the lowest and quartile 4 the highest responses. RESULTS: Thirteen per cent of individuals had impaired fasting glucose (IFG; n = 14) or impaired glucose tolerance (IGT; n = 19), allowing comparisons across the continuum of insulin responses within the spectrum of normoglycaemia and prediabetes. BMI (~24 kg/m2) was similar across insulin quartiles and in those with IFG and IGT. Despite similar glycaemic excursions, fasting insulin, triacylglycerols and cholesterol were elevated in quartile 4. Insulin sensitivity was lowest in quartile 4, and accompanied by increased insulin secretion and reduced insulin clearance. Individuals with IFG had similar insulin sensitivity and beta cell function to those in quartiles 2 and 3, but were more insulin sensitive than individuals in quartile 4. While individuals with IGT had a similar degree of insulin resistance to quartile 4, they exhibited a more severe defect in beta cell function. Plasma branched-chain amino acids were not elevated in quartile 4, IFG or IGT. CONCLUSIONS/INTERPRETATION: Hyperinsulinaemia within normoglycaemic young, non-obese adults manifests due to increased insulin secretion and reduced insulin clearance. Individual phenotypic characterisation revealed that the most hyperinsulinaemic were more similar to individuals with IGT than IFG, suggesting that hyperinsulinaemic individuals may be on the continuum toward IGT. Furthermore, plasma branched-chain amino acids may not be an effective biomarker in identifying hyperinsulinaemia and insulin resistance in young non-obese adults.

Placental creatine metabolism in cases of placental insufficiency and reduced fetal growth.

Creatine is a metabolite involved in cellular energy homeostasis. In this study, we examined placental creatine content, and expression of the enzymes required for creatine synthesis, transport and the creatine kinase reaction, in pregnancies complicated by low birthweight. We studied first trimester chorionic villus biopsies (CVBs) of small for gestational age (SGA) and appropriately grown infants (AGA), along with third trimester placental samples from fetal growth restricted (FGR) and healthy gestation-matched controls. Placental creatine and creatine precursor (guanidinoacetate-GAA) levels were measured. Maternal and cord serum from control and FGR pregnancies were also analyzed for creatine concentration. mRNA expression of the creatine transporter (SLC6A8); synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT); mitochondrial (mtCK) and cytosolic (BBCK) creatine kinases; and amino acid transporters (SLC7A1 & SLC7A2) was assessed in both CVBs and placental samples. Protein levels of AGAT (arginine:glycine aminotransferase), GAMT, mtCK and BBCK were also measured in placental samples. Key findings; total creatine content of the third trimester FGR placentae was 43% higher than controls. The increased creatine content of placental tissue was not reflected in maternal or fetal serum from FGR pregnancies. Tissue concentrations of GAA were lower in the third trimester FGR placentae compared to controls, with lower GATM and GAMT mRNA expression also observed. No differences in the mRNA expression of GATM, GAMT or SLC6A8 were observed between CVBs from SGA and AGA pregnancies. These results suggest placental creatine metabolism in FGR pregnancies is altered in late gestation. The relevance of these changes on placental bioenergetics should be the focus of future investigations.

Phosphatidylserine decarboxylase is critical for the maintenance of skeletal muscle mitochondrial integrity and muscle mass.

OBJECTIVE: Phosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo. METHODS: To determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry. RESULTS: PSD knockdown disrupted muscle phospholipid homeostasis leading to an ∼25% reduction in PtdEtn and an ∼45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content. CONCLUSIONS: The PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass.

Modest changes to glycemic regulation are sufficient to maintain glucose fluxes in healthy young men following overfeeding with a habitual macronutrient composition.

Currently, it is unclear whether short-term overfeeding in healthy people significantly affects postprandial glucose regulation, as most human overfeeding studies have utilized induced experimental conditions such as the euglycemic-hyperinsulinemic clamp technique to assess glucoregulation. The aim of this study was to quantify glucose fluxes [rates of meal glucose appearance (Ra), disposal (Rd), and endogenous glucose production (EGP)] in response to 5 and 28 days of overfeeding (+45% energy) while maintaining habitual macronutrient composition (31.0 ± 1.9% fat, 48.6 ± 2.2% carbohydrate, 16.7 ± 1.4% protein) in healthy, lean young men. Meal tolerance testing was combined with the triple-stable isotope glucose tracer approach. Visceral adipose volume increased by ~15% with 5 days of overfeeding, while there was no further change at 28 days. In contrast, body mass (+1.6 kg) and fat mass (+1.3 kg) were significantly increased only after 28 days of overfeeding. Fasting EGP, Rd, and insulin were increased at 5 but unchanged after 28 days. Postprandial glucose and insulin responses were unaltered by 5 days of overfeeding but were modestly increased after 28 days (P < 0.05). However, meal Ra and glucose Rd were significantly increased after both 5 and 28 days of overfeeding (P < 0.05). Despite this, overfeeding did not lead to alterations to postprandial EGP suppression. Thus, in contrast to findings from euglycemic-hyperinsulinemic clamp studies, chronic overfeeding did not affect the ability to suppress EGP or stimulate Rd under postprandial conditions. Rather, glucose flux was appropriately maintained following 28 days of overfeeding through modest increases in postprandial glycemia and insulinemia.