Overexpression of APOC1 in obob mice leads to hepatic steatosis and severe hepatic insulin resistance

Obese obob mice with strong overexpression of the human apolipoprotein C1 (APOC1) exhibit excessive free fatty acid (FFA) and triglyceride (TG) levels and severely reduced body weight (due to the absence of subcutaneous adipose tissue) and skin abnormalities. To evaluate the effects of APOC1 overexpression on hepatic and peripheral insulin sensitivity in a less-extreme model, we generated obob mice with mild overexpression of APOC1 (obob/APOC1(+/-)) and performed hyperinsulinemic clamp analysis. Compared with obob littermates, obob/APOC1(+/-) mice showed reduced body weight (-25%) and increased plasma levels of TG (+632%), total cholesterol (+134%), FFA (+65%), glucose (+73%), and insulin (+49%). Hyperinsulinemic clamp analysis revealed severe whole-body and hepatic insulin resistance in obob/APOC1(+/-) mice and, in addition, increased hepatic uptake of FFA and hepatic TG content. Treatment of obob/APOC1(+/-) mice with rosiglitazone strongly improved whole-body insulin sensitivity as well as hepatic insulin sensitivity, despite a further increase of hepatic fatty acid (FA) uptake and a panlobular increase of hepatic TG accumulation. We conclude that overexpression of APOC1 prevents rosiglitazone-induced peripheral FA uptake leading to severe hepatic steatosis. Interestingly, despite rosiglitazone-induced hepatic steatosis, hepatic insulin sensitivity improves dramatically. We hypothesize that the different hepatic fat accumulation and/or decrease in FA intermediates has a major effect on the insulin sensitivity of the liver.     

Structure of complement component C2A: implications for convertase formation and substrate binding

C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix alpha7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix alpha7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.     

Mining the Giardia genome and proteome for conserved and unique basal body proteins

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.     

Effects of high fat diet on the Basal activity of the hypothalamus-pituitary-adrenal axis in mice: a systematic review

Hypothalamus-pituitary-adrenal-axis activity is suggested to be involved in the pathophysiology of the metabolic syndrome. In diet-induced obesity mouse models, features of the metabolic syndrome are induced by feeding high fat diet. However, the models reveal conflicting results with respect to the hypothalamus-pituitary-adrenal-axis activation. The aim of this review was to assess the effects of high fat feeding on the activity of the hypothalamus-pituitary-adrenal-axis in mice. PubMed, EMBASE, Web of Science, the Cochrane database, and Science Direct were electronically searched and reviewed by 2 individual researchers. We included only original mouse studies reporting parameters of the hypothalamus-pituitary-adrenal-axis after high fat feeding, and at least 1 basal corticosterone level with a proper control group. Studies with adrenalectomized mice, transgenic animals only, high fat diet for less than 2 weeks, or other interventions besides high fat diet, were excluded. 20 studies were included. The hypothalamus-pituitary-adrenal-axis evaluation was the primary research question in only 5 studies. Plasma corticosterone levels were unchanged in 40%, elevated in 30%, and decreased in 20% of the studies. The effects in the peripheral tissues and the central nervous system were also inconsistent. However, major differences were found between mouse strains, experimental conditions, and the content and duration of the diets. This systematic review demonstrates that the effects of high fat feeding on the basal activity of the hypothalamus-pituitary-adrenal-axis in mice are limited and inconclusive. Differences in experimental conditions hamper comparisons and accentuate the need for standardized evaluations to discern the effects of diet-induced obesity on the hypothalamus-pituitary-adrenal-axis.     

Introduction of N-linked glycans in the lectin domain of surfactant protein D: impact on interactions with influenza A viruses

Porcine surfactant protein D (pSP-D) displays distinctively strong, broad-range inhibitory activity against influenza A virus (IAV). N-Linked glycosylation of the carbohydrate recognition domain (CRD) of pSP-D contributes to the high affinity of this collectin for IAV. To investigate the role of the N-linked glycan further, HEK293E protein expression was used to produce recombinant pSP-D (RpSP-D) that has similar structural and antiviral properties as NpSP-D. We introduced an additional N-linked glycan in the CRD of RpSP-D but this modification did not alter the antiviral activity. Human SP-D is unglycosylated in its CRD and less active against IAV compared with pSP-D. In an attempt to modify its antiviral properties, several recombinant human SP-D (RhSP-D) mutants were constructed with N-linked glycans introduced at various locations within its CRD. To retain lectin activity, necessary for the primary interactions between SP-D and IAV, N-linked glycosylation of RhSP-D was shown to be restricted to the corresponding position in the CRD of either pSP-D or surfactant protein A (SP-A). These N-glycosylated RhSP-D mutants, however, did not show increased neutralization activity against IAV. By developing RhSP-D mutants that also have the pSP-D-specific Ser-Gly-Ala loop inserted in the CRD, we could demonstrate that the N-linked glycan-mediated interactions between pSP-D and IAV involves additional structural prerequisites of the pSP-D CRD. Ultimately, these studies will help to develop highly effective SP-D-based therapeutic and prophylactic drugs against IAV.     

Adrenal Function in Females with Low Plasma HDL-C Due to Mutations in ABCA1 and LCAT

Introduction

Adrenal steroidogenesis is essential for human survival and depends on the availability of the precursor cholesterol. Male subjects with low plasma levels of high density lipoprotein (HDL) cholesterol are characterized by decreased adrenal function. Whether this is also the case in female subjects with low plasma HDL-C levels is unresolved to date.

Findings

15 female ATP binding cassette transporter AI (ABCAI) and 14 female lecithin-cholesterol acyltransferase (LCAT) were included in the study. HDL-C levels were 38% and 41% lower in ABCA1 and LCAT mutation carriers compared to controls, respectively. Urinary steroid excretion of 17-ketogenic steroids or 17-hydroxy corticosteroids did not differ between 15 female ABCA1 mutation carriers (p = 0.27 vs 0.30 respectively) and 30 matched normolipidemic controls or between 14 female LCAT mutation carriers and 28 matched normolipidemic controls (p = 0.10 and 0.14, respectively). Cosyntropin testing in an unselected subgroup of 8 ABCA1 mutation carriers and 3 LCAT mutation carriers did not reveal differences between carriers and controls.

Conclusion

Adrenal function in females with molecularly defined low HDL-C levels is not different from controls. The discrepancy with the finding of impaired steroidogenesis in males with molecularly defined low HDL-C levels underscores the importance of gender specific analyses in cholesterol-related research.     

GLP-1 Receptor Activation Inhibits VLDL Production and Reverses Hepatic Steatosis by Decreasing Hepatic Lipogenesis in High-Fat-Fed APOE*3-Leiden Mice

Objective

In addition to improve glucose intolerance, recent studies suggest that glucagon-like peptide-1 (GLP-1) receptor agonism also decreases triglyceride (TG) levels. The aim of this study was to evaluate the effect of GLP-1 receptor agonism on very-low-density lipoprotein (VLDL)-TG production and liver TG metabolism.

Experimental Approach

The GLP-1 peptide analogues CNTO3649 and exendin-4 were continuously administered subcutaneously to high fat diet-fed APOE*3-Leiden transgenic mice. After 4 weeks, hepatic VLDL production, lipid content, and expression profiles of selected genes involved in lipid metabolism were determined.

Results

CNTO3649 and exendin-4 reduced fasting plasma glucose (up to −30% and −28% respectively) and insulin (−43% and −65% respectively). In addition, these agents reduced VLDL-TG production (−36% and −54% respectively) and VLDL-apoB production (−36% and −43% respectively), indicating reduced production of VLDL particles rather than reduced lipidation of apoB. Moreover, they markedly decreased hepatic content of TG (−39% and −55% respectively), cholesterol (−30% and −55% respectively), and phospholipids (−23% and −36% respectively), accompanied by down-regulation of expression of genes involved in hepatic lipogenesis (Srebp-1c, Fasn, Dgat1) and apoB synthesis (Apob).

Conclusion

GLP-1 receptor agonism reduces VLDL production and hepatic steatosis in addition to an improvement of glycemic control. These data suggest that GLP-receptor agonists could reduce hepatic steatosis and ameliorate dyslipidemia in patients with type 2 diabetes mellitus.     

Long-term beneficial effect of a 16-week very low calorie diet on pericardial fat in obese type 2 diabetes mellitus patients

Pericardial fat accumulation has been associated with an increased cardiovascular risk. A very low calorie diet (VLCD) improves the cardiovascular risk profile in patients with type 2 diabetes mellitus (T2DM), by improving the metabolic profile, heart function, and triglyceride (TG) stores in (non)adipose tissues. However, long-term effects of a VLCD on pericardial fat volume and tissue-specific TG accumulation have not been documented. The aim of this study was therefore to assess the effects of a 16-week VLCD and of subsequent 14 months follow-up on a regular diet on pericardial fat in relation to other TG stores in obese T2DM patients. We included 14 obese patients with insulin-treated T2DM (mean ± s.e.m.: age 53 ± 2 years; BMI 35 ± 1 kg/m(2)). Pericardial fat and other (non)adipose TG stores were measured using magnetic resonance (MR) imaging and proton spectroscopy before and after a 16-week VLCD and after a 14-month follow-up without dietary interventions. A 16-week VLCD reduced body weight, pericardial fat, hepatic TG content, visceral and subcutaneous abdominal fat volumes to 78, 83, 16, 40, and 53% of baseline values respectively, (all P < 0.05). After an additional 14 months of follow-up on a regular diet, the reduction in pericardial fat volume sustained, despite a substantial regain in body weight, visceral abdominal fat, and hepatic TG content (respectively 90, 83 and 73% of baseline values). In conclusion, VLCD-induced weight loss in obese T2DM patients is accompanied by a substantial decrease in pericardial fat volume, which is sustained even after subsequent weight regain.     

Oxyntomodulin ameliorates glucose intolerance in mice fed a high-fat diet

We evaluated the acute effects of OXM on glucose metabolism in diet-induced insulin-resistant male C57Bl/6 mice. To determine the effects on glucose tolerance, mice were intraperitoneally injected with OXM (0.75, 2.5, or 7.5 nmol) or vehicle prior to an ip glucose tolerance test. OXM (0.75 nmol/h) or vehicle was infused during a hyperinsulinemic euglycemic clamp to quantify insulin action on glucose production and disposal. OXM dose-dependently improved glucose tolerance as estimated by AUC for glucose (OXM: 7.5 nmol, 1,564 +/- 460, P < 0.01; 2.5 nmol, 1,828 +/- 684, P < 0.01; 0.75 nmol, 2,322 +/- 303, P < 0.05; control: 2,790 +/- 222 mmol.l(-1).120 min). Insulin levels in response to glucose administration were higher in 7.5 nmol OXM-treated animals compared with controls. In basal clamp conditions, OXM increased EGP (82.2 +/- 14.7 vs. 39.9 +/- 5.7 micromol.min(-1).kg(-1), P < 0.001). During insulin infusion, insulin levels were twice as high in OXM-treated mice compared with controls (10.6 +/- 2.8 vs. 4.4 +/- 2.2 ng/ml, P < 0.01). Consequently, glucose infusion rate (118.6 +/- 30.8 vs. 38.8 +/- 26.4 microl/h, P < 0.001) and glucose disposal (88.1 +/- 13.0 vs. 45.2 +/- 6.9 micromol.min(-1).kg(-1), P < 0.001) were enhanced in mice that received OXM. In addition, glucose production was more suppressed during OXM infusion (35.7 +/- 15.5 vs. 15.8 +/- 11.4% inhibition, P < 0.05). However, if these data were expressed per unit concentration of circulating insulin, OXM did not affect insulin action on glucose disposal and production. These results indicate that OXM beneficially affects glucose metabolism in diet-induced insulin-resistant C57Bl/6 mice. It ameliorates glucose intolerance, most likely because it elevates glucose-induced plasma insulin concentrations. OXM does not appear to impact on insulin action.     

Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.