Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets

Background PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters. Methods We performed nutritional interventions in young healthy male volunteers and offspring of type 2 diabetic (OffT2D) patients that are more prone to develop insulin resistance, including: i) acute post-prandial hyperlipidemic challenge (n=10), ii) 4 days of high-fat (HF) or high-fat/high-protein (HFHP) (n=10), iii) 7 (HFruc1, n=16) or 6 (HFruc2, n=9) days of hypercaloric high-fructose diets. An acute oral fat load was also performed in two patients bearing the R104C-V114A loss-of-function (LOF) PCSK9 mutation. Plasma PCSK9 concentrations were measured by ELISA. For the HFruc1 study, intrahepatocellular (IHCL) and intramyocellular lipids were measured by 1H magnetic resonance spectroscopy. Hepatic and whole-body insulin sensitivity was assessed with a two-step hyperinsulinemic-euglycemic clamp (0.3 and 1.0 mU.kg-1.min-1). Findings HF and HFHP short-term diets, as well as an acute hyperlipidemic oral load, did not significantly change PCSK9 concentrations. In addition, post-prandial plasma triglyceride excursion was not altered in two carriers of PCSK9 LOF mutation compared with non carriers. In contrast, hypercaloric 7-day HFruc1 diet increased plasma PCSK9 concentrations by 28% (p=0.05) in healthy volunteers and by 34% (p=0.001) in OffT2D patients. In another independent study, 6-day HFruc2 diet increased plasma PCSK9 levels by 93% (p<0.0001) in young healthy male volunteers. Spearman’s correlations revealed that plasma PCSK9 concentrations upon 7-day HFruc1 diet were positively associated with plasma triglycerides (r=0.54, p=0.01) and IHCL (r=0.56, p=0.001), and inversely correlated with hepatic (r=0.54, p=0.014) and whole-body (r=−0.59, p=0.0065) insulin sensitivity. Conclusions Plasma PCSK9 concentrations vary minimally in response to a short term high-fat diet and they are not accompanied with changes in cholesterolemia upon high-fructose diet. Short-term high-fructose intake increased plasma PCSK9 levels, independent on cholesterol synthesis, suggesting a regulation independent of SREBP-2. Upon this diet, PCSK9 is associated with insulin resistance, hepatic steatosis and plasma triglycerides.

A 4-wk high-fructose diet alters lipid metabolism without affecting insulin sensitivity or ectopic lipids in healthy humans

BACKGROUND: High fructose consumption is suspected to be causally linked to the epidemics of obesity and metabolic disorders. In rodents, fructose leads to insulin resistance and ectopic lipid deposition. In humans, the effects of fructose on insulin sensitivity remain debated, whereas its effect on ectopic lipids has never been investigated. OBJECTIVE: We assessed the effect of moderate fructose supplementation on insulin sensitivity (IS) and ectopic lipids in healthy male volunteers (n = 7). DESIGN: IS, intrahepatocellular lipids (IHCL), and intramyocellular lipids (IMCL) were measured before and after 1 and 4 wk of a high-fructose diet containing 1.5 g fructose . kg body wt(-1) . d(-1). Adipose tissue IS was evaluated from nonesterified fatty acid suppression, hepatic IS from suppression of hepatic glucose output (6,6-2H2-glucose), and muscle IS from the whole-body glucose disposal rate during a 2-step hyperinsulinemic euglycemic clamp. IHCL and IMCL were measured by 1H magnetic resonance spectroscopy. RESULTS: Fructose caused significant (P < 0.05) increases in fasting plasma concentrations of triacylglycerol (36%), VLDL-triacylglycerol (72%), lactate (49%), glucose (5.5%), and leptin (48%) without any significant changes in body weight, IHCL, IMCL, or IS. IHCL were negatively correlated with triacylglycerol after 4 wk of the high-fructose diet (r = -0.78, P < 0.05). CONCLUSION: Moderate fructose supplementation over 4 wk increases plasma triacylglycerol and glucose concentrations without causing ectopic lipid deposition or insulin resistance in healthy humans.

Effects of four-week high-fructose diet on gene expression in skeletal muscle of healthy men

AIMS: A high-fructose diet (HFrD) may play a role in the obesity and metabolic disorders epidemic. In rodents, HFrD leads to insulin resistance and ectopic lipid deposition. In healthy humans, a four-week HFrD alters lipid homoeostasis, but does not affect insulin sensitivity or intramyocellular lipids (IMCL). The aim of this study was to investigate whether fructose may induce early molecular changes in skeletal muscle prior to the development of whole-body insulin resistance. METHODS: Muscle biopsies were taken from five healthy men who had participated in a previous four-week HFrD study, during which insulin sensitivity (hyperinsulinaemic euglycaemic clamp), and intrahepatocellular lipids and IMCL were assessed before and after HFrD. The mRNA concentrations of 16 genes involved in lipid and carbohydrate metabolism were quantified before and after HFrD by real-time quantitative PCR. RESULTS: HFrD significantly (P<0.05) increased stearoyl-CoA desaturase-1 (SCD-1) (+50%). Glucose transporter-4 (GLUT-4) decreased by 27% and acetyl-CoA carboxylase-2 decreased by 48%. A trend toward decreased peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was observed (-26%, P=0.06). All other genes showed no significant changes. CONCLUSION: HFrD led to alterations of SCD-1, GLUT-4 and PGC-1alpha, which may be early markers of insulin resistance.

Plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, e-selectin and C-reactive protein levels in response to 4-week very-high-fructose or -glucose diets.

BACKGROUND/OBJECTIVES High intake of added sweeteners is considered to have a causal role in the pathogenesis of cardiometabolic disorders. Especially, high-fructose intake is regarded as potentially harmful to cardiometabolic health. It may cause not only weight gain but also low-grade inflammation, which represents an independent risk factor for developing type 2 diabetes and cardiovascular disease. In particular, fructose has been suggested to induce plasminogen activator inhibitor-1 (PAI-1) expression in the liver and to increase circulating inflammatory cytokines. We therefore aimed to investigate, whether high-fructose diet has an impact on PAI-1, monocyte chemoattractant protein-1 (MCP-1), e-selectin and C-reactive protein (CRP) concentrations in healthy humans. SUBJECTS/METHODS We studied 20 participants (12 males and 8 females) of the TUebingen FRuctose Or Glucose study. This is an exploratory, parallel, prospective, randomized, single-blinded, outpatient, hypercaloric, intervention study. The participants had a mean age of 30.9 ± 2.1 years and a mean body mass index of 26.0 ± 0.5 kg/m(2) and they received 150 g of either fructose or glucose per day for 4 weeks.Results:There were neither significant changes of PAI-1, MCP-1, e-selectin and CRP after fructose (n=10) and glucose (n=10) intervention nor treatment effects (all P>0.2). Moreover, we did not observe longitudinal associations of the inflammatory parameters with triglycerides, liver fat, visceral fat and body weight in the fructose group. CONCLUSIONS Temporary high-fructose intake does not seem to cause inflammation in apparently healthy people in this secondary analysis of a small feeding trial.

Role of proton MR for the study of muscle lipid metabolism

1H-MR spectroscopy (MRS) of intramyocellular lipids (IMCL) became particularly important when it was recognized that IMCL levels are related to insulin sensitivity. While this relation is rather complex and depends on the training status of the subjects, various other influences such as exercise and diet also influence IMCL concentrations. This may open insight into many metabolic interactions; however, it also requires careful planning of studies in order to control all these confounding influences. This review summarizes various historical, methodological, and practical aspects of 1H-MR spectroscopy (MRS) of muscular lipids. That includes a differentiation of bulk magnetic susceptibility effects and residual dipolar coupling that can both be observed in MRS of skeletal muscle, yet affecting different metabolites in a specific way. Fitting of the intra- (IMCL) and extramyocellular (EMCL) signals with complex line shapes and the transformation into absolute concentrations is discussed. Since the determination of IMCL in muscle groups with oblique fiber orientation or in obese subjects is still difficult, potential improvement with high-resolution spectroscopic imaging or at higher field strength is considered. Fat selective imaging is presented as a possible alternative to MRS and the potential of multinuclear MRS is discussed. 1H-MRS of muscle lipids allows non-invasive and repeated studies of muscle metabolism that lead to highly relevant findings in clinics and patho-physiology.

Effect of GH on human skeletal muscle lipid metabolism in GH deficiency

Adult-onset growth hormone (GH) deficiency (GHD) is associated with insulin resistance and decreased exercise capacity. Intramyocellular lipids (IMCL) depend on training status, diet, and insulin sensitivity. Using magnetic resonance spectroscopy, we studied IMCL content following physical activity (IMCL-depleted) and high-fat diet (IMCL-repleted) in 15 patients with GHD before and after 4 mo of GH replacement therapy (GHRT) and in 11 healthy control subjects. Measurements of insulin resistance and exercise capacity were performed and skeletal muscle biopsies were carried out to assess expression of mRNA of key enzymes involved in skeletal muscle lipid metabolism by real-time PCR and ultrastructure by electron microscopy. Compared with control subjects, patients with GHD showed significantly higher difference between IMCL-depleted and IMCL-repleted. GHRT resulted in an increase in skeletal muscle mRNA expression of IGF-I, hormone-sensitive lipase, and a tendency for an increase in fatty acid binding protein-3. Electron microscopy examination did not reveal significant differences after GHRT. In conclusion, variation of IMCL may be increased in patients with GHD compared with healthy control subjects. Qualitative changes within the skeletal muscle (i.e., an increase in free fatty acids availability from systemic and/or local sources) may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I on skeletal muscle.

Hepatic gene expression involved in glucose and lipid metabolism in transition cows: effects of fat mobilization during early lactation in relation to milk performance and metabolic changes.

Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation.

Rapid Communication: Cholesterol deficiency-associated APOB mutation impacts lipid metabolism in Holstein calves and breeding bulls.

During the last months, the number of reports on Holstein calves suffering from incurable idiopathic diarrhea dramatically increased. Affected calves showed severe hypocholesterolemia and mostly died within days up to a few months after birth. This new autosomal monogenic recessive inherited fat metabolism disorder, termed cholesterol deficiency (CD), is caused by a loss of function mutation of the bovine gene. The objective of the present study was to investigate specific components of lipid metabolism in 6 homozygous for the mutation (CDS) and 6 normal Holstein calves with different genotypes. Independent of sex, CDS had significantly lower plasma concentrations of total cholesterol (TC), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), triacylglycerides (TAG), and phospholipids (PL) compared with homozygous wild-type calves ( < 0.05). Furthermore, we studied the effect of the genotype on cholesterol metabolism in adult Holstein breeding bulls of Swissgenetics. Among a total of 254 adult males, the homozygous mutant genotype was absent, 36 bulls were heterozygous carriers (CDC), and 218 bulls were homozygous wild-type (CDF). In CDC bulls, plasma concentrations of TC, FC, HDL-C, LDL-C, VLDL-C, TAG, and PL were lower compared with CDF bulls ( < 0.05). The ratios of FC:cholesteryl esters (CE) and FC:TC were higher in CDC bulls compared with CDF bulls, whereas the ratio of CE:TC was lower in CDC bulls compared with CDF bulls ( < 0.01). In conclusion, the CD-associated mutation was shown to affect lipid metabolism in affected Holstein calves and adult breeding bulls. Besides cholesterol, the concentrations of PL, TAG, and lipoproteins also were distinctly reduced in homozygous and heterozygous carriers of the mutation. Beyond malabsorption of dietary lipids, deleterious effects of apolipoprotein B deficiency on hepatic lipid metabolism, steroid biosynthesis, and cell membrane function can be expected, which may result in unspecific symptoms of reduced fertility, growth, and health.

Lipid metabolism in Trypanosoma brucei

Trypanosoma brucei membranes consist of all major eukaryotic glycerophospholipid and sphingolipid classes. These are de novo synthesized from precursors obtained either from the host or from catabolised endocytosed lipids. In recent years, substantial progress has been made in the molecular and biochemical characterisation of several of these lipid biosynthetic pathways, using gene knockout or RNA interference strategies or by enzymatic characterization of individual reactions. Together with the completed genome, these studies have highlighted several possible differences between mammalian and trypanosome lipid biosynthesis that could be exploited for the development of drugs against the diseases caused by these parasites.

Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling

There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here, we report the findings of a plasma metabolomic investigation of HCC patients by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm, and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate, and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also conducted by ultraperformance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). By this method, we found that HCC was also associated with reduced levels of lysophosphocholines and in 4 of 20 patients with increased levels of lysophosphatidic acid [LPA(16:0)], where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GC-MS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GC-MS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Differential effect of pioglitazone (PGZ) and rosiglitazone (RGZ) on postprandial glucose and lipid metabolism in patients with type 2 diabetes mellitus: a prospective, randomized crossover study

Postprandial metabolism is impaired in patients with type 2 diabetes (T2Dm). Two thiazolidinediones pioglitazone (PGZ) and rosiglitazone (RGZ) have similar effects on glycaemic control but differ in their effects on fasting lipids. This study investigated the effects of RGZ and PGZ on postprandial metabolism in a prospective, randomized crossover trial.

Liver fat content and lipid metabolism in dairy cows during early lactation and during a mid-lactation feed restriction.

During the transition period, the lipid metabolism of dairy cows is markedly affected by energy status. Fatty liver is one of the main health disorders after parturition. The aim of this study was to evaluate the effects of a negative energy balance (NEB) at 2 stages in lactation [NEB at the onset of lactation postpartum (p.p.) and a deliberately induced NEB by feed restriction near 100 d in milk] on liver triglyceride content and parameters of lipid metabolism in plasma and liver based on mRNA abundance of associated genes. Fifty multiparous dairy cows were studied from wk 3 antepartum to approximately wk 17 p.p. in 2 periods. According to their energy balance in period 1 (parturition to wk 12 p.p.), cows were allocated to a control (CON; n=25) or a restriction group (RES; 70% of energy requirements; n=25) for 3 wk in mid lactation starting at around 100 d in milk (period 2). Liver triglyceride (TG) content, plasma nonesterified fatty acids (NEFA), and β-hydroxybutyrate were highest in wk 1 p.p. and decreased thereafter. During period 2, feed restriction did not affect liver TG and β-hydroxybutyrate concentration, whereas NEFA concentration was increased in RES cows as compared with CON cows. Hepatic mRNA abundances of tumor necrosis factor α, ATP citrate lyase, mitochondrial glycerol-3-phosphate acyltransferase, and glycerol-3-phosphate dehydrogenase 2 were not altered by lactational and energy status during both experimental periods. The expression of fatty acid synthase was higher in period 2 compared with period 1, but did not differ between RES and CON groups. The mRNA abundance of acetyl-coenzyme A-carboxylase showed a tendency toward higher expression during period 2 compared with period 1. The solute carrier family 27 (fatty acid transporter), member 1 (SLC27A1) was upregulated in wk 1 p.p. and also during feed restriction in RES cows. In conclusion, the present study shows that a NEB has different effects on hepatic lipid metabolism and TG concentration in the liver of dairy cows at early and later lactation. Therefore, the homeorhetic adaptations during the periparturient period trigger excessive responses in metabolism, whereas during the homeostatic control of endocrine and metabolic systems after established lactation, as during the period of feed restriction in the present study, organs are well adapted to metabolic and environmental changes.

The impact of antioxidant supplements and endurance exercise on genes of the carbohydrate and lipid metabolism in skeletal muscle of mice

To ascertain whether reactive oxygen species (ROS) contribute to training-induced adaptation of skeletal muscle, we administered ROS-scavenging antioxidants (AOX; 140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% N-acetyl-cysteine) via drinking water to 16 C57BL/6 mice. Sixteen other mice received unadulterated tap water (CON). One cohort of both groups (CON(EXE) and AOX(EXE) ) was subjected to treadmill exercise for 4 weeks (16-26 m/min, incline of 5°-10°). The other two cohorts (CON(SED) and AOX(SED) ) remained sedentary. In skeletal muscles of the AOX(EXE) mice, GSSG and the expression levels of SOD-1 and PRDX-6 were significantly lower than those in the CON(EXE) mice after training, suggesting disturbance of ROS levels. The peak power related to the body weight and citrate synthase activity was not significantly influenced in mice receiving AOX. Supplementation with AOX significantly altered the mRNA levels of the exercise-sensitive genes HK-II, GLUT-4 and SREBF-1c and the regulator gene PGC-1alpha but not G6PDH, glycogenin, FABP-3, MCAD and CD36 in skeletal muscle. Although the administration of AOX during endurance exercise alters the expression of particular genes of the ROS metabolism, it does not influence peak power or generally shift the metabolism, but it modulates the expression of specific genes of the carbohydrate and lipid metabolism and PGC-1alpha within murine skeletal muscle.