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Aims/hypothesis: Insulin resistance in skeletal muscle and liver plays a major role in the pathophysiology of type 2 diabetes. The hyperinsulinaemic-euglycaemic clamp is considered the gold standard for assessing peripheral and hepatic insulin sensitivity, yet it is a costly and labour-intensive procedure. Therefore, easy-to-measure, cost-effective approaches to determine insulin sensitivity are needed to enable organ-specific interventions. Recently, evidence emerged that plasma cathepsin D (CTSD) is associated with insulin sensitivity and hepatic inflammation. Here, we aimed to investigate whether plasma CTSD is associated with hepatic and/or peripheral insulin sensitivity in humans., Methods: As part of two large clinical trials (one designed to investigate the effects of antibiotics, and the other to investigate polyphenol supplementation, on insulin sensitivity), 94 overweight and obese adults (BMI 25-35 kg/m 2 ) previously underwent a two-step hyperinsulinaemic-euglycaemic clamp (using [6,6- 2 H 2 ]glucose) to assess hepatic and peripheral insulin sensitivity (per cent suppression of endogenous glucose output during the low-insulin-infusion step, and the rate of glucose disappearance during high-insulin infusion [40 mU/(m 2 × min)], respectively). In this secondary analysis, plasma CTSD levels, CTSD activity and plasma inflammatory cytokines were measured., Results: Plasma CTSD levels were positively associated with the proinflammatory cytokines IL-8 and TNF-α (IL-8: standardised β = 0.495, p < 0.001; TNF-α: standardised β = 0.264, p = 0.012). Plasma CTSD activity was negatively associated with hepatic insulin sensitivity (standardised β = -0.206, p = 0.043), independent of age, sex, BMI and waist circumference, but it was not associated with peripheral insulin sensitivity. However, plasma IL-8 and TNF-α were not significantly correlated with hepatic insulin sensitivity., Conclusions/interpretation: We demonstrate that plasma CTSD activity, but not systemic inflammation, is inversely related to hepatic insulin sensitivity, suggesting that plasma CTSD activity may be used as a non-invasive marker for hepatic insulin sensitivity in humans.

Non-alcoholic steatohepatitis (NASH) is characterized by liver lipid accumulation and inflammation. The mechanisms that trigger hepatic inflammation are poorly understood and subsequently, no specific non-invasive markers exist. We previously demonstrated a reduction in the plasma lysosomal enzyme, cathepsin D (CatD), in children with NASH compared to children without NASH. Recent studies have raised the concept that non-alcoholic fatty liver disease (NAFLD) in adults is distinct from children due to a different histological pattern in the liver. Yet, the link between plasma CatD to adult NASH was not examined. In the current manuscript, we investigated whether plasma CatD in adults correlates with NASH development and regression. Biopsies were histologically evaluated for inflammation and NAFLD in three complementary cohorts of adults (total n = 248). CatD and alanine aminotransferase (ALT) were measured in plasma. Opposite to our previous observations with childhood NASH, we observed increased levels of plasma CatD in patients with NASH compared to adults without hepatic inflammation. Furthermore, after surgical intervention, we found a reduction of plasma CatD compared to baseline. Our observations highlight a distinct pathophysiology between NASH in children and adults. The observation that plasma CatD correlated with NASH development and regression is promising for NASH diagnosis.

Objectives: Nonalcoholic steatohepatitis (NASH) is the most severe form of a hepatic condition known as nonalcoholic fatty liver disease (NAFLD). NASH is histologically characterized by hepatic fat accumulation, inflammation, and ballooning, and eventually coupled with fibrosis that, in turn, may progress to end-stage liver disease even in young individuals. Hence, there is a critical need for specific noninvasive markers to predict hepatic inflammation at an early age. We investigated whether plasma levels of cathepsin D (CatD), a lysosomal protease, correlated with the severity of liver inflammation in pediatric NAFLD., Methods: Liver biopsies from children (n=96) with NAFLD were histologically evaluated according to the criteria of Kleiner (NAFLD activity score) and the Brunt's criteria. At the time of liver biopsy, blood was taken and levels of CatD, alanine aminotransferase (ALT), and cytokeratin-18 (CK-18) were measured in plasma., Results: Plasma CatD levels were significantly lower in subjects with liver inflammation compared with steatotic subjects. Furthermore, we found that CatD levels were gradually reduced and corresponded with increasing severity of liver inflammation, steatosis, hepatocellular ballooning, and NAFLD activity score. CatD levels correlated with pediatric NAFLD disease progression better than ALT and CK-18. In particular, CatD showed a high diagnostic accuracy (area under receiver operating characteristic curve (ROC-AUC): 0.94) for the differentiation between steatosis and hepatic inflammation, and reached almost the maximum accuracy (ROC-AUC: 0.998) upon the addition of CK-18., Conclusions: Plasma CatD holds a high diagnostic value to distinguish pediatric patients with hepatic inflammation from children with steatosis.

Niemann-Pick disease type C (NPC) is a lysosomal storage disorder characterized by liver disease and progressive neurodegeneration. Deficiency of either NPC1 or NPC2 leads to the accumulation of cholesterol and glycosphingolipids in late endosomes and early lysosomes. In order to identify pathological mechanisms underlying NPC and uncover potential biomarkers, we characterized liver gene expression changes in an Npc1 mouse model at six ages spanning the pathological progression of the disease. We identified altered gene expression at all ages, including changes in asymptomatic, 1-week-old mice. Biological pathways showing early altered gene expression included: lipid metabolism, cytochrome P450 enzymes involved in arachidonic acid and drug metabolism, inflammation and immune responses, mitogen-activated protein kinase and G-protein signaling, cell cycle regulation, cell adhesion and cytoskeleton remodeling. In contrast, apoptosis and oxidative stress appeared to be late pathological processes. To identify potential biomarkers that could facilitate monitoring of disease progression, we focused on a subset of 103 differentially expressed genes that encode secreted proteins. Further analysis identified two secreted proteins with increased serum levels in NPC1 patients: galectin-3 (LGALS3), a pro-inflammatory molecule, and cathepsin D (CTSD), a lysosomal aspartic protease. Elevated serum levels of both proteins correlated with neurological disease severity and appeared to be specific for NPC1. Expression of Lgals3 and Ctsd was normalized following treatment with 2-hydroxypropyl-β-cyclodextrin, a therapy that reduces pathological findings and significantly increases Npc1(-/-) survival. Both LGALS3 and CTSD have the potential to aid in diagnosis and serve as biomarkers to monitor efficacy in therapeutic trials.

Unspecific inflammatory lesions of the urinary tract are known to be a vital question in urology and nephrology of our country and in foreign ones. Used in experiments were rats of mixed population. Lysosomal enzymes were studied in homogenates of the liver and contralateral kidney tissues and in the blood serum as well 1, 3, 6 days after simulation of the pathology experimental model. Activities were measured of such lysosomal enzymes as acid DNA-ase and RNA-ase, cathepsin, and acid phosphatase. Results of the studies made showed that in unspecific purulent affection of the kidneys there occurs an activation of acid blood hydrolases followed by their activation in the liver. The contralateral kidney, because of it being linked in functioning with the liver, remains relatively "inert" with respect to activation of lysosomal enzymes.

Lysosomal acid hydrolases are thought to play an important role in inflammation associated with rheumatoid arthritis. A Siddha preparation of Semecarpus anacardium nut extract called Serankottai Nei was tested for its capacity to stabilize lysosomes obtained from liver and kidney of adjuvant-induced arthritic animals. Lysosomal membrane stability was measured by determining the release of acid hydrolases from the lysosomes. The drug was administered at a dose level of 150 mg/kg body weight for 14 days to arthritic animals after the adjuvant injection. The total and free activity of lysosomal enzymes were significantly increased in arthritic rats with concomitant increase in plasma levels of protein-bound carbohydrates. Significantly increased lysosomal membrane fragility as observed in arthritic condition was reduced in drug-treated animals. Antiarthritic activity of the drug through its stabilizing action on lysosomal membranes could be inferred from this study.

Adult male Wistar rats were divided in 3 groups. In the first group, infrarenal abdominal aorta was occluded for 6 hours and reperfused thereafter. In the second group, the reperfusion was not made, and the third group underwent a sham operation. Lysosomal enzymatic activities were assessed in serum tissues. Lysosomal membrane fragilities were estimated also in these tissues. Finally, 14C-aminopyrine breath test (ABT) was studied to define the possible liver dysfunction caused by limb ischemia. As a result, release of lysosomal enzymes from ischemic muscle was confirmed in vitro experiments, and the increase in the serum was statistically significant in the first and second groups as compared to the third group. Particularly prominent was a marked elevation of cathepsin-D in the first group which was observed immediately after the release of occlusion. Results of liver lysosomal enzymes and ABT revealed significant cellular damages and depression of microsomal function of the liver both in the first and second groups. These studies suggest that lysosomal enzymes derived from ischemic muscle exert a possible toxicity on the liver, and liver damage thus resulted may play a roll on the pathogenesis of whole body injury associated with acute and critical lower limb ischemia.

Impairments in structural integrity of liver, heart and skeletal muscle lysosomes followed by necrotic and necrobiotic alterations in the tissues and secretion of active hydrolases in blood were detected under conditions of exhaustive physical exercises in rats when dose-dependent physical loading was studied. At the same time, in patients with hypertrophic cardiomyopathy physical loading, carried out as stepwise spiroveloergometry up to the submaximal heart rate, led to release of lysosomal enzymes into blood, which was not observed in healthy persons under similar conditions. The higher level of enzymes was found in circulation of the patients with most severe forms of the disease.

Serum from patients with chronic liver diseases (chronic hepatitis, cirrhosis, and hepatomas) contained greater concentrations of calciferin (total 1.7-fold, free 3.3-fold) than that from normal subjects. There were also decreases in the concentration and affinity of the calciferin-binding protein(s) in the patients' sera, but the amount of cathepsin D (EC 3.4.23.5)-like acid protease (total and free) was within normal limits. In addition, rats with acute liver injuries (partial hepatectomy or CCl4 administration) showed increases in calciferin and acid protease in their serum. Rats subjected to continuous feeding of 3'-methyl-4-dimethylaminoazobenzene (a potent hepatocarcinogen) also showed an increase of both analytes in the earlier stages (two to eight weeks; acute phase). Later (13 weeks or more; chronic phase), however, only acid protease appeared to return to normal values.