Rebekka Pohl

Lipid abnormalities in alpha/beta2-syntrophin null mice are independent from ABCA1

The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2-/-) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2-/- mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2-/- mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2-/- mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity.

Ceramide and polyunsaturated phospholipids are strongly reduced in human hepatocellular carcinoma

Lipid composition affects membrane function, cell proliferation and cell death and is changed in cancer tissues. Hepatocellular carcinoma (HCC) is an aggressive cancer and this study aimed at a comprehensive characterization of hepatic and serum lipids in human HCC. Cholesteryl ester were higher in tumorous tissues (TT) compared to adjacent non-tumorous tissues (NT). Free cholesterol exerting cytotoxic effects was not changed. Phosphatidylethanolamine, -serine (PS) and -inositol but not phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) were reduced in HCC tissues. Saturated species mostly increased and polyunsaturated species were diminished in all of these phospholipids. Ceramide (Cer) was markedly reduced in HCC tissues and higher levels of sphingomyelin suggest impaired sphingomyelinase activity as one of the underlying mechanisms. Importantly, ceramide in NT increased in HCC stage T3. Ceramide released from hepatocytes attracts immune cells and a positive association of the macrophage specific receptor CD163 with NT ceramide was identified. HCC associated lipid changes did not differ in patients suffering from type 2 diabetes. Protein levels of p53 were induced in TT and negatively correlated with Cer d18:1/16:0 and PS 36:1. Of the lipid species changed in HCC tissues only TT Cer d18:1/16:0, Cer d18:1/24:1, PC 38:6 and LPC 22:6 correlated with the respective serum levels. Our study demonstrates a considerably altered hepatic lipidome in HCC tissues. Ceramide was markedly reduced in HCC tissues, and therefore, raising ceramide levels specifically in the tumor represents a reasonable therapeutic approach for the treatment of this malignancy.

Annexin A6 protein is downregulated in human hepatocellular carcinoma

Annexin A6 (AnxA6) is a lipid-binding protein highly expressed in the liver, regulating cholesterol homeostasis and signaling pathways with a role in liver physiology. Here, we analyzed whether hepatic AnxA6 levels are affected by pathological conditions that are associated with liver dysfunction and liver injury. AnxA6 levels in the fatty liver of mice fed a high-fat diet, in ob/ob and db/db animals and in human fatty liver are comparable to controls. Similarly, AnxA6 levels appear unaffected in murine nonalcoholic steatohepatitis and human liver fibrosis. Accordingly, adiponectin, lysophosphatidylcholine, palmitate, and TGFbeta, all of which have a role in liver injury, do not affect AnxA6 expression in human hepatocytes. Likewise, adiponectin and IL8 do not alter AnxA6 levels in primary human hepatic stellate cells. However, in hepatic tumors of 18 patients, AnxA6 protein levels are substantially reduced compared to nontumorous tissues. AnxA6 mRNA is even increased in the tumors suggesting that posttranscriptional mechanisms are involved herein. Lipidomic analysis shows trends toward elevated cholesteryl ester and sphingomyelin in the tumor samples, yet the ratio of tumor to nontumorous AnxA6 does not correlate with these lipids. The current study shows that AnxA6 is specifically reduced in human hepatocellular carcinoma suggesting a role of this protein in hepatocarcinogenesis.

Hepatic scavenger receptor BI is associated with type 2 diabetes but unrelated to human and murine non-alcoholic fatty liver disease.

Scavenger receptor, class B type I (SR-BI) is a physiologically relevant regulator of high density lipoprotein (HDL) metabolism. Low HDL is a common feature of patients with non-alcoholic fatty liver disease (NAFLD). Here, hepatic SR-BI expression was analyzed in human and murine NAFLD. In primary human hepatocytes NAFLD relevant factors like inflammatory cytokines, lipopolysaccharide and TGF-β did not affect SR-BI protein. Similarly, oleate and palmitate had no effect. The adipokines chemerin, adiponectin, leptin and omentin did not regulate SR-BI expression. Accordingly, hepatic SR-BI was not changed in human and murine fatty liver and non-alcoholic steatohepatits. SR-BI was higher in type 2 diabetes patients but not in those with hypercholesterolemia. The current study indicates a minor if any role of SR-BI in human and murine NAFLD.

Hepatic chemerin mRNA expression is reduced in human nonalcoholic steatohepatitis

Chemerin is associated with insulin resistance and is expressed in the liver. Nonalcoholic fatty liver disease (NAFLD) is related to impaired insulin sensitivity, but studies evaluating hepatic and serum chemerin in NAFLD resulted in discordant data.

Pro-Resolving Molecules-New Approaches to Treat Sepsis?

Inflammation is a complex response of the body to exogenous and endogenous insults. Chronic and systemic diseases are attributed to uncontrolled inflammation. Molecules involved in the initiation of inflammation are very well studied while pathways regulating its resolution are insufficiently investigated. Approaches to down-modulate mediators relevant for the onset and duration of inflammation are successful in some chronic diseases, while all of them have failed in sepsis patients. Inflammation and immune suppression characterize sepsis, indicating that anti-inflammatory strategies alone are inappropriate for its therapy. Heme oxygenase 1 is a sensitive marker for oxidative stress and is upregulated in inflammation. Carbon monoxide, which is produced by this enzyme, initiates multiple anti-inflammatory and pro-resolving activities with higher production of omega-3 fatty acid-derived lipid metabolites being one of its protective actions. Pro-resolving lipids named maresins, resolvins and protectins originate from the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid while lipoxins are derived from arachidonic acid. These endogenously produced lipids do not simply limit inflammation but actively contribute to its resolution, and thus provide an opportunity to combat chronic inflammatory diseases and eventually sepsis.

The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth

The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knock-down of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role in cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity.

Resolvin E1 and chemerin C15 peptide do not improve rodent non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a progressive liver disease more commonly diagnosed in obesity. Therapeutic options to treat NASH are limited. Liver inflammation is a hallmark of NASH, and here it was tested whether the lipid mediator resolvin E1 (RvE1) and chemerin derived C15 peptide, which both exert potent anti-inflammatory activities, ameliorate NASH pathology. Male mice fed an atherogenic diet for 12 weeks, well described to induce NASH, received intraperitoneal injections of RvE1, C15 peptide or PBS as control for four days. Both treatments did not affect body weight or serum ALT. Liver triglycerides were neither reduced by the lipid nor the peptide. Hepatic expression of the macrophage marker F4/80 and the inflammatory mediators TNF and CCL2 was not changed. Further, fibrotic genes including TGFbeta, alphaSMA and CTGF were not affected by RvE1 or C15 injections. Serum adiponectin was comparable in the three groups. RvE1 and C15 are ligands of CMKLR1 whose expression was not reduced upon feeding the NASH inducing diet. This excludes low receptor levels as reason for therapeutic failure. In summary, current data demonstrate that RvE1 and chemerin derived C15 peptide do not ameliorate murine NASH.

Ex vivo analysis of serum chemerin activity in murine models of obesity

Objectives: Chemerin is an adipokine with established roles in inflammation, adipogenesis and the regulation of glucose and lipid homeostasis. Extracellular proteolytic processing of chemerin generates a spectrum of isoforms that differ significantly with respect to the ability to activate the cognate receptors chemokine‐like receptor 1 (CMKLR1) and G‐protein‐coupled receptor 1 (GPR1). Increased total serum chemerin has been widely reported in obese humans as well as in preclinical rodent models of adiposity. However, very little information is available regarding the correspondence, if any, of changes in total serum chemerin protein with chemerin bioactivity. Methods: Total serum chemerin and ex vivo CMKLR1 and GPR1 activation was compared using two widely used murine obesity models: high fat diet feeding (HFD) and leptin deficiency (ob/ob). Results: Total serum chemerin levels and ex vivo CMKLR1 and GPR1 activation were significantly induced in HFD. The bioactivity ratio (bioactive chemerin/total chemerin) was also increased when measured with CMKLR1, but not GPR1. In contrast, while ob/ob mice exhibited increased total serum chemerin protein, ex vivo receptor activation was observed with GPR1, but not CMKLR1. There was no change in bioactivity ratio for either receptor. Of note, GPR1 but not CMKLR1 bioactivity positively correlated with adipose tissue inflammation. Conclusions: While increased total serum chemerin is a consistent finding among rodent obesity models, this may not accurately reflect changes in chemerin bioactivity which is the major determinant of biological effects. HIGHLIGHTSEx vivo CMKLR1 activity/total chemerin increases in serum of mice fed high fat diet.Ex vivo GPR1 activity/total chemerin is unchanged in serum of mice fed high fat diet.There was no change in bioactivity ratio for either receptor in ob/ob mice.

Annexin A6 regulates adipocyte lipid storage and adiponectin release

Lipid storage and adipokine secretion are critical features of adipocytes. Annexin A6 (AnxA6) is a lipid-binding protein regulating secretory pathways and its role in adiponectin release was examined. The siRNA-mediated AnxA6 knock-down in 3T3-L1 preadipocytes impaired proliferation, and differentiation of AnxA6-depleted cells to mature adipocytes was associated with higher soluble adiponectin and increased triglyceride storage. The latter was partly attributed to reduced lipolysis. Accordingly, AnxA6 overexpression in 3T3-L1 adipocytes lowered cellular triglycerides and adiponectin secretion. Indeed, serum adiponectin was increased in AnxA6 deficient mice. Expression analysis identified AnxA6 protein to be more abundant in intra-abdominal compared to subcutaneous adipose tissues of mice and men. AnxA6 protein levels increased in white adipose tissues of obese mice and here, levels were highest in subcutaneous fat. AnxA6 protein in adipocytes was upregulated by oxidative stress which might trigger AnxA6 induction in adipose tissues and contribute to impaired fat storage and adiponectin release.