Sebelipase alfa in children and adults with lysosomal acid lipase deficiency: Final results of the ARISE study.

BACKGROUND & AIMS: Children and adults with lysosomal acid lipase deficiency (LAL-D) experience cirrhosis and dyslipidemia from lysosomal accumulation of cholesteryl esters and triglycerides. Sebelipase alfa enzyme replacement therapy is indicated for individuals with LAL-D. We report final results from the phase III randomized ARISE study of sebelipase alfa in children aged ≥4 years and adults with LAL-D. METHODS: The study included a 20-week, double-blind, placebo-controlled period; a 130-week, open-label, extension period; and a 104-week, open-label, expanded treatment period. In the open-label periods, all patients received intravenous sebelipase alfa every other week. The primary outcome was alanine aminotransferase (ALT) level normalization; aspartate aminotransferase (AST) levels, lipid parameters, liver histology, liver and spleen volume and fat content, and safety were also assessed. RESULTS: Of 66 patients enrolled, 59 completed the study. Median (range) age at randomization was 13 (4.7-59) years. At the last open-label visit, ALT and AST levels had normalized in 47% and 66% of patients, respectively. Patients who switched from placebo to sebelipase alfa experienced sustained improvements in ALT and AST during the open-label periods that mirrored those observed in the sebelipase alfa group during the double-blind period. Median (IQR) percent changes in lipid levels included a 25% (39%, 6.5%) reduction in low-density lipoprotein cholesterol and a 27% (19%, 44%) increase in high-density lipoprotein cholesterol. Most adverse events during the open-label periods were mild to moderate in severity; 13 patients had infusion-associated reactions (serious in 1 patient). Six patients (9%) developed anti-drug antibodies. CONCLUSIONS: Early and rapid improvements in markers of liver injury and lipid abnormalities with sebelipase alfa were sustained, with no progression of liver disease, for up to 5 years. CLINICAL TRIAL NUMBER: NCT01757184; EudraCT Number: 2011-002750-31 LAY SUMMARY: Sebelipase alfa is used to treat lysosomal acid lipase deficiency (LAL-D), a rare, inherited disease of lipid metabolism. We report the final results of the phase III ARISE clinical study, which show that replacement of the defective LAL enzyme with sebelipase alfa for up to 5 years allows adults and children 4 years of age and older to maintain their initial improvements in liver and cholesterol parameters over the long term, without worsening of liver disease.

NAD(P)-dependent steroid dehydrogenase-like protein and neutral cholesterol ester hydrolase 1 serve as novel markers for early detection of gastric cancer identified using quantitative proteomics.

BACKGROUND: Gastric cancer (GC) is the third most common cause of cancer deaths worldwide. In the present study, we aimed to identify novel GC biomarkers by integrating isobaric tags of relative and absolute quantitation (iTRAQ) for aberrantly expressed proteins in GC patients. METHODS: Using stable isotope tags, we labeled an initial discovery group comprising four paired gastric cancer and adjacent gastric tissue samples, and subjected them to LC-ESI-MS/MS. We used a validation set comprising 129 paired gastric cancer and adjacent gastric tissues from patients and benign healthy controls to validate the candidate targets. RESULTS: We identified two proteins, NAD(P)-dependent steroid dehydrogenase-like (NSDHL) and neutral cholesterol ester hydrolase 1 (NCEH1), that were significantly overexpressed in GC tissues. The sensitivity and specificity of NSDHL were 80.6% and 74.4%, respectively, in GC compared with a sensitivity of 25.6% in adjacent tissues and 24% in benign healthy controls. The area under the ROC curve (AUC) for NSDHL was 0.810 for GC detection. Overexpression of NSDHL in GC was significantly correlated with local tumor invasion. The sensitivity and specificity of NCEH1 were 77.5% and 73.6%, respectively, in GC compared with a sensitivity of 26.4% in adjacent tissues and 20% in benign controls. The AUC for NSDHL was 0.792. Overexpression of NCEH1 was significantly associated with tumor histological classification and local invasion. Moreover, a combined analysis of NSDHL and NCEH1 achieved a sensitivity and specificity of 85.7% and 83%, respectively, and the AUC was 0.872. The combined analysis of NSDHL and NCEH1 was significantly correlated with histological grade and TNM Ⅱ-Ⅳ staging. CONCLUSIONS: iTRAQ-labeled quantitative proteomics represents a powerful method to identify novel cancer biomarkers. The present study identified NSDHL and NCEH1 as useful biomarkers for screening, diagnosis, and prognosis of patients with gastric cancer.

Lysosomal acid lipase promotes cholesterol ester metabolism and drives clear cell renal cell carcinoma progression.

OBJECTIVES: Clear cell renal cell carcinoma (ccRCC) is characterized histologically by accumulation of cholesterol esters, cholesterol and other neutral lipids. Lysosomal acid lipase (LAL) is a critical enzyme involved in the cholesterol ester metabolism. Here, we sought to determine whether LAL could orchestrate metabolism of cholesterol esters in order to promote ccRCC progression. MATERIALS AND METHODS: Quantitative reverse-transcription PCR and western blots were conducted to assess the expression of LAL in human ccRCC tissues. We analysed the relationship between LAL levels and patient survival using tissue microarrays. We used cell proliferation assays, colony formation assays, cell death assays, metabolic assays and xenograft tumour models to evaluate the biological function and underlying mechanisms. RESULTS: LAL was up-regulated in ccRCC tissue. Tissue microarray analysis revealed higher levels of LAL in advanced grades of ccRCC, and high LAL expression indicated lower patient survival. Suppressing LAL expression not only blocked the utilization of cholesterol esters but also impaired proliferation and cellular survival. Furthermore, immunohistochemistry staining showed that LAL expression was correlated with Akt phosphorylation. Suppressing LAL expression decreased the phosphorylation level of Akt and Src and reduced the level of 14,15-epoxyeicosatrienoic acids in ccRCC cells. Supplement of 14,15-epoxyeicosatrienoic acids rescued proliferation in vitro and in vivo. CONCLUSIONS: LAL promoted cell proliferation and survival via metabolism of epoxyeicosatrienoic acids and activation of the Src/Akt pathway.

Aging-associated reductions in lipolytic and mitochondrial proteins in mouse adipose tissue are not rescued by metformin treatment.

Mitochondrial enzyme expression is reduced in adipose tissue from old mice, yet little is known regarding mechanisms that could be mediating, or interventions that could be used, to reverse these changes. The purpose of this study was to examine the relationship between lipolytic and fatty acid reesterification enzymes, 5' adenosine monophosphate-activated protein kinase and mitochondrial proteins in adipose tissue from young versus old mice. A second aim was to determine whether metformin treatment could rescue the age-associated decline in adipose tissue mitochondrial proteins. Approximately 22-month-old male C57BL/6 mice were fed a diet with or without 0.5% metformin for 8 weeks. Compared with young mice (~11 wk of age), the protein content/phosphorylation of hormone-sensitive lipase, adipose tissue triglyceride lipase, and phosphoenolpyruvate carboxykinase were reduced in old mice. This was paralleled by increases in the plasma nonesterified fatty acid:glycerol ratio and reductions in adipose tissue 5' adenosine monophosphate-activated protein kinase activity and select mitochondrial proteins in old mice. There were no differences in these variables when comparing adipose tissue from young and 6-month-old mice. While metformin improved glucose homeostasis, it did not increase 5' adenosine monophosphate-activated protein kinase phosphorylation or mitochondrial enzymes. Our findings demonstrate a co-ordinated down regulation of lipolytic, reesterification, and mitochondrial enzymes in adipose tissue with aging that is unresponsive to metformin treatment.

Postweaning low-calcium diet promotes later-life obesity induced by a high-fat diet.

The aim of this study was to investigate the effects of a postweaning low-calcium diet on later obesity and explore the underlying mechanisms. Ninety-six male rats were weaned at 3 weeks of age, fed standard (STD: 0.50% calcium, n=48) and low-calcium (LC: 0.15% calcium, n=48) diets for 3 weeks, and then fed the standard diet for a 3-week washout period successively. Finally, the STD rats were divided into STD control and high-fat diet (HFD) groups, and the LC ones into LC control and LC+HFD (LCHF) groups. The STD and LC rats were fed the standard diet, while the HFD control and LCFD ones were fed a high-fat diet for 6 weeks to induce obesity. During the three feeding periods, adenosine-monophosphate-activated protein kinase (AMPK) and its responsive proteins phospho-acetyl-coA carboxylase, carnitine palmitoyltransferase 1 and uncoupling protein 3 were persistently down-regulated in the LC group (decreased by 18%, 24%, 18% and 20%, respectively) versus the STD group, and these effects were significantly more pronounced in the LCHFD group (decreased by 21%, 30%, 23% and 25%, respectively) than the HFD group by a later high-fat stimuli, causing more fat and body weight in adulthood. However, lipolysis enzymes, serum leptin, insulin and lipids were not significantly affected until the body weight and fat content changed at 15 weeks of age. The results suggest that the low-calcium diet after weaning promotes rat adult-onset obesity induced by high-fat diet, which might be achieved by programming expressions of genes involved in AMPK pathway.

Chronic TNFalpha and cAMP pre-treatment of human adipocytes alter HSL, ATGL and perilipin to regulate basal and stimulated lipolysis.

We examined the effects of chronic TNFalpha and dibutyryl-cAMP (Db-cAMP) pre-treatment on the lipolytic machinery of human hMADS adipocytes. TNFalpha decreased adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein content and triglycerides (TG)-hydrolase activity but increased basal lipolysis due to a marked reduction in perilipin (PLIN) protein content. Conversely, Db-cAMP increased ATGL and HSL protein content but prevented PLIN phosphorylation, the net result being accentuated basal lipolysis. In forskolin-stimulated conditions, TNFalpha and Db-cAMP pre-treatment decreased stimulated TG-hydrolase activity and impaired PLIN phosphorylation. Together, this resulted in a severely attenuated response to forskolin-stimulated lipolysis.

Medium-chain Fatty acids attenuate agonist-stimulated lipolysis, mimicking the effects of starvation.

OBJECTIVE: To test the hypothesis that incorporation of medium-chain fatty acids (FAs) into adipocyte triglycerides alters intracellular lipolysis. RESEARCH METHODS AND PROCEDURES: 3T3-L1 adipocytes were pretreated with octanoate for various incubation periods. After the removal of exogenous FAs, cells were incubated with different lipolytic agonists. To determine the effects on lipolysis, we measured the following: the release of glycerol and FAs, lipase activity, protein levels of hormone-sensitive lipase (HSL), and perilipin A; translocation of HSL; phosphorylation of perilipin A; and levels of cellular adenosine triphosphate, cyclic adenosine monophosphate, and H2O2. To compare the effects of starvation with those caused by octanoate pretreatment, we measured glycerol release and H2O2 generation in rat adipocytes of starved donors. RESULTS: Pretreatment of adipocytes with octanoate in vitro increased basal lipolysis but decreased the cellular response for agonists. The same effects were seen in starvation in vivo. Preincubation with octanoate for 48 hours did not affect basal lipase activity, HSL, and perilipin protein levels, but it reduced agonist-stimulated perilipin phosphorylation and HSL translocation toward fat droplets. This was associated with a reduction in basal cellular adenosine triphosphate levels and agonist-stimulated cyclic adenosine monophosphate generation. Starvation and octanoate pretreatment both increased intracellular H2O2 concentrations, which might also contribute to the inhibition on agonist-stimulated lipolysis. DISCUSSION: Pretreatment with octanoate seems to induce changes in adipocyte lipolysis in a pattern mimicking the effects of starvation. Such changes could contribute, in part, to weight loss in animals and humans associated with dietary medium-chain FAs.

Mechanisms behind lipolytic catecholamine resistance of subcutaneous fat cells in the polycystic ovarian syndrome.

Lipolytic catecholamine resistance in sc fat cells is observed in polycystic ovarian syndrome (PCOS). The mechanisms behind this lipolysis defect were explored in vitro; sc fat cells were obtained from 10 young, nonobese PCOS women and from 14 matched, healthy control women. Fasting plasma glycerol levels were reduced by one third in PCOS (P < 0.05). Adipocytes of PCOS women were about 25% larger than in the controls (P < 0.05) and had 40% reduced noradrenaline-induced lipolysis (P < 0.05), which could be attributed to a 10-fold decreased beta(2)-adrenoceptor sensitivity (P < 0.05) and low ability of cAMP to activate the protein kinase A (PKA)/hormone-sensitive lipase (HSL) complex (P < 0.05). In PCOS, the adipocyte protein content of beta(2)-adrenoceptors, HSL, and the regulatory II beta-component of PKA were 70%, 55%, and 25% decreased, respectively (P < 0.001); but there was no change in the amount of the catalytic subunit of PKA or of beta(1)-adrenoceptors. Thus, lipolytic catecholamine resistance of sc adipocytes in PCOS is probably attributable to a combination of decreased amounts of beta(2)-adrenergic receptors, the regulatory II beta-component of PKA, and HSL. This may cause low in vivo lipolytic activity and enlarged sc fat cell size and promote later development of obesity in PCOS.

Lipolysis in adipocytes isolated from deep and superficial subcutaneous adipose tissue.

OBJECTIVE: Abdominal subcutaneous adipose tissue (SAT) occurs in two depots separated by a fascial plane: deep SAT and superficial SAT. In a recent study it was demonstrated that the amount of deep SAT has a much stronger relationship to insulin resistance than does superficial SAT. Because insulin resistance may be related to fatty acid release from adipose tissue, we hypothesized that the two SAT depots may have a different lipolytic activity. RESEARCH METHODS AND PROCEDURES: To test this hypothesis, we obtained samples of deep and superficial SAT from patients undergoing elective abdominal surgery. The rate of lipolysis was determined in the collagenase-digested adipocytes obtained from the two fat depots by measuring glycerol release in the presence and absence of isoproterenol. In addition, the relative concentration of hormone-sensitive lipase was determined in both SAT depots by Western blot analysis. RESULTS: Our results showed that the rate of isoproterenol-stimulated lipolysis was approximately 20% higher in cells from deep SAT compared with those from superficial SAT, indicating that the deep SAT is more lipolytically active. The concentration of hormone-sensitive lipase did not differ between the two adipose tissue depots. DISCUSSION: These findings suggest that the higher lipolytic activity of deep SAT may account for its stronger association with insulin resistance. The mechanism seems to be independent of differences in hormone-sensitive lipase concentration.

Cholesterol esterase activities in commercial pancreatic enzyme preparations and implications for use in pancreatic insufficient cystic fibrosis.

BACKGROUND: Although clinical symptoms in pancreatic insufficiency are often dramatically improved by pancreatic preparations, these often fail to normalize biochemical indicators of malabsorption. It seemed relevant, therefore, to investigate the amounts of cholesterol esterase in these preparations and, using in-vitro methods, some of the activities of this enzyme. The enzyme is just as physiologically important as lipase in accomplishing lipid digestion and absorption. METHODS: Cholesterol esterase was assayed in commercial pancreatic extract preparations, lyophilized pig pancreas and human duodenal fluid. The in-vitro activities of the enzyme were also investigated on single and mixed dietary substrates. RESULTS: Other than Creon, the commercial preparations showed negligible cholesterol esterase activities, whereas considerable activities were found in pancreatic tissue and duodenal fluids. In-vitro, pig cholesterol esterase was confirmed to be dependent on 3-hydroxy bile salt concentration for hydrolysis and synthesis and that the rate for hydrolysis greatly exceeds that of synthesis in normal concentrations of bile salts. However, with mixed lipid substrates, no bile salt concentration was found at which hydrolysis or synthesis predominates. CONCLUSIONS: When pancreatic or hepato-biliary function is compromised, optimum lipid hydrolysis may not be achieved in therapeutic use, and the pig enzyme may perform differently to the human enzyme. In-vivo trials may reveal whether augmentation of the commercial products with this enzyme would be worthwhile.

The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets.

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body's major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the beta-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the beta-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation.

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.

Effects of perturbations in hepatic free and esterified cholesterol pools on bile acid synthesis, cholesterol 7 alpha-hydroxylase, HMG-CoA reductase, acyl-CoA:cholesterol acyltransferase and cytosolic cholesteryl ester hydrolase.

Effects of expansion of the hepatic free cholesterol pool on bile acid and cholesterol metabolism and homeostasis were examined in rats fed cholesterol in high-fat diets or treated with oleyl-p-(n-decyl)-benzenesulfonate (ODS) or progesterone. Cholesterol feeding for 10-16 days, which increased free (33%) and esterified (6-fold) cholesterol, had no effect on cholate synthesis, total bile acid synthesis, or cholate turnover, whereas these activities were increased 60-80% by ODS and progesterone, which produced only small increases (19%) in free cholesterol. Cholesterol feeding reduced beta-hydroxy-beta-methylglutaryl (HMG)-CoA reductase (72%) and cholesteryl ester hydrolase (48%) and increased acyl-CoA:cholesterol acyltransferase (184%), whereas ODS and progesterone reversed these compensatory responses in cholesterol-fed rats. Cholesterol 7 alpha-hydroxylase was changed no more than 22% by any treatment. A bolus of ODS elevated biliary cholesterol output 41% and shifted biliary bile acid synthesis and composition toward 12-deoxy bile acids. These effects were not seen in ODS-fed or progesterone-treated rats, in which cholesteryl ester stores were depleted. It is concluded that effects of free cholesterol on bile acid synthesis and biliary cholesterol are probably mediated by specific precursor or regulatory pools which can be independently regulated and which represent a relatively small fraction of hepatic free cholesterol.

Human adipose tissue hormone-sensitive lipase: identification and comparison with other species.

The mRNA for human hormone-sensitive lipase (HSL) was identified using Northern blot analysis and a cDNA-probe for rat HSL. As in the rat, human adipose tissue expresses a single mRNA species of 3.3 kb. Using Western blotting with a polyclonal rabbit antibody towards rat adipose tissue HSL, the corresponding enzyme in human adipose tissue was identified with an apparent 88 kDa polypeptide, thus slightly larger than the rat and bovine 84 kDa, and the mouse and guinea-pig 82 kDa species. Additional evidence for the identification was provided by the inhibition of HSL diacylglycerol lipase activity by the anti-rat HSL antibody, and by NaF, DFP and Hg2+, known inhibitors of HSL. The concentration of the enzyme, as reflected by its activity per g tissue and the specific activity was about two thirds of that in the rat adipose tissue (200 g rats). The identification of the human enzyme protein made it possible to directly demonstrate its phosphorylation by cAMP-dependent protein kinase, thus extending the previous report regarding activation of the lipase with this kinase and ATP-Mg2+ in human adipose tissue extracts (Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131).

Hormone-sensitive lipase is responsible for the neutral cholesterol ester hydrolase activity in macrophages.

Anti-hormone-sensitive lipase (HSL) immunoglobulin selectively immunoprecipitates a single 84 kDa 32P-phosphoprotein from macrophage homogenates previously phosphorylated by cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP-Mg. This immunoglobulin also completely removes the neutral cholesterol ester hydrolase activity from macrophage homogenates. These data demonstrate that HSL is responsible for the neutral cholesterol ester hydrolase activity in macrophages and hence plays a key role in cholesterol metabolism in these cells.

Hormone-sensitive lipase in brown adipose tissue: identification and effect of cold exposure.

Hormone-sensitive lipase (HSL) in brown adipose tissue from mice was identified through immunoprecipitation with a polyclonal antibody (anti-HSL) towards rat white fat HSL and Western blotting. An 82 kDa polypeptide, slightly smaller than the rat white fat HSL 84 kDa subunit, was detected and its identity as HSL verified by inhibition properties. The HSL concentration per g tissue was several-fold higher in the mouse brown adipose tissue than in the rat white adipose tissue, but the specific activities per mg protein were similar. Cold-exposure (4 degrees C) of the mice for 24 h approximately doubled the HSL concentration but this increase parallelled the overall protein increase and did not reflect a specific effect on the HSL.

Localization and origin at rat intestinal cholesterol esterase determined by immunocytochemistry.

Monospecific rabbit antisera to rat pancreas cholesterol esterase were employed in the unlabeled antibody enzyme method of immunocytochemistry in combination with the horseradish peroxidase--antihorseradish peroxidase complex to localize this pancreatic enzyme within the wall of rat small intestine. Intestinal rings were fixed in paraformaldehyde with satisfactory preservation of structure and retention of cholesterol esterase antigenic determinants. Fixed sections, 6 micrometers thick, were stained. In the light microscope, specific reaction product, represented by intense brown areas, was uniformly distributed within the absorptive cells but was notably absent from the microvillar membrane. Reaction product was also seen within the laminapropria and submucosa. In contrast, reaction product was absent from sections of proximal intestine surgically deprived of pancreatic juice for 72 hours. Furthermore, the intensity of staining in sections of normal intestine decreased with increasing distance from the pancreatic duct. These observations support the concept that "intestinal" cholesterol esterase is of pancreatic origin. This enzyme is localized within the cells as opposed to the absorptive surface.