Chemical toolbox to interrogate Heparanase-1 activity.

The development of a robust chemical toolbox to interrogate the activity of heparanase-1 (HPSE-1), an endo-ß-d-glucuronidase and the only known enzyme that cleaves heparan sulfate (HS), has become critically important. The primary function of HPSE-1, cleaving HS side chains from heparan sulfate proteoglycans (HSPGs), regulates the integrity of the extracellular matrix (ECM) and the bioavailability of active, heparan sulfate-binding partners such as enzymes, growth factors, chemokines, and cytokines. HPSE-1 enzymatic activity is strictly regulated and has been found to play fundamental roles in pathophysiological processes. HPSE-1 is significantly overexpressed under various conditions including cancer, metastasis, angiogenesis, and inflammation, making HPSE-1 a promising therapeutic and diagnostic target. Chemical tools that can detect and image HPSE-1 activity in vitro and/or in vivo can help drive the discovery of novel and efficacious anti-HPSE-1 drugs, investigate the basic biology of HPSE-1, and help serve as a diagnostic tool in clinical applications. Here, we will give an overview of the common chemical tools to detect HPSE-1 activity and highlight the novel heparanase probes recently developed in our lab.

A Fluorogenic Green Merocyanine-Based Probe to Detect Heparanase-1 Activity.

Heparanase-1 (HPSE-1), an endo-ß-D-glucuronidase, is an extracellular matrix (ECM) remodeling enzyme that degrades heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs). HPSE-1 functions to remodel the ECM and thereby disseminate cells, liberate HS-bound bioactive molecules, and release biologically active HS fragments. Being the only known enzyme for the cleavage of HS, HPSE-1 regulates a number of fundamental cellular processes including cell migration, cytokine regulation, angiogenesis, and wound healing. Overexpression of HPSE-1 has been discovered in most cancers, inflammatory diseases, viral infections, among others. As an emerging therapeutic target, the biological role of HPSE-1 remains to be explored but is hampered by a lack of research tools. To expand the chemical tool-kit of fluorogenic probes to interrogate HPSE-1 activity, we design and synthesized a fluorogenic green disaccharide-based HPSE-1 probe using our design strategy of tuning the electronic effect of the aryl aglycon. The novel probe exhibits a highly sensitive 278-fold fluorescence turn-on response in the presence of recombinant human HPSE-1, while emitting green light at 560 nm, enabling the fluorescence imaging of HPSE-1 activity in cells.

Short-Term Decreasing and Increasing Dietary BCAA Have Similar, but Not Identical Effects on Lipid and Glucose Metabolism in Lean Mice.

Branched-chain amino acids (BCAA) showed multiple functions in glycolipid metabolism and protein synthesis. However, the impacts on the metabolic health of low or high dietary BCAA remain controversial due to the various experimental conditions. Gradient levels of BCAA were supplemented in lean mice for four weeks: 0BCAA (without BCAA), 1/2BCAA (half BCAA), 1BCAA (regular BCAA), and 2BCAA (double BCAA). The results showed that the diet without BCAA caused energy metabolic disorders, immune defects, weight loss, hyperinsulinemia, and hyperleptinemia. 1/2BCAA and 2BCAA diets reduced body fat percentage, but 1/2 BCAA also decreased muscle mass. 1/2BCAA and 2BCAA groups improved lipid and glucose metabolism by affecting metabolic genes. Meanwhile, significant differences between low and high dietary BCAA were observed. The results of this study provide evidence and reference for the controversy about dietary BCAA levels, which indicates that the main difference between low and high BCAA dietary levels may present in the longer term.

A Universal and Modular Scaffold for Heparanase Activatable Probes and Drugs.

Heparanase (HPSE) is an endo-ß-glucuronidase involved in extracellular matrix remodeling in rapidly healing tissues, most cancers and inflammation, and viral infection. Its importance as a therapeutic target warrants further study, but such is hampered by a lack of research tools. To expand the toolkits for probing HPSE enzymatic activity, we report the design of a substrate scaffold for HPSE comprised of a disaccharide substrate appended with a linker, capable of carrying cargo until being cleaved by HPSE. Here exemplified as a fluorogenic, coumarin-based imaging probe, this scaffold can potentially expand the availability of HPSE-responsive imaging or drug delivery tools using a variety of imaging moieties or other cargo. We show that electronic tuning of the scaffold provides a robust response to HPSE while simplifying the structural requirements of the attached cargo. Molecular docking and modeling suggest a productive probe/HPSE binding mode. These results further support the hypothesis that the reactivity of these HPSE-responsive probes is predominantly influenced by the electron density of the aglycone. This universal HPSE-activatable scaffold will greatly facilitate future development of HPSE-responsive probes and drugs.

Threonine supplementation prevents the development of fat deposition in mice fed a high-fat diet.

Obesity is the main factor involved in the onset of many diseases. Threonine supplementation has been demonstrated to reduce fat mass and serum triglycerides in already obese mice. However, it is unclear whether threonine could inhibit the development of obesity in mice without previous high-fat diet induction. In the present study, mice were fed a chow diet (CD) or a high-fat diet (HFD), supplemented or not with threonine (3.0% in drinking water) for 15 weeks. Results showed that mice subjected to chronic threonine supplementation showed decreased body weight, epididymal white adipose tissue weight, serum low-density lipoprotein cholesterol, and total cholesterol in comparison with HFD-fed mice. In the epididymal adipose tissue, gene expressions of sterol regulatory element-binding protein 1c and fatty acid synthase were up-regulated, while hormone sensitive lipase, adiponectin and fibroblast growth factor 21 were down-regulated. In the liver tissue, gene expressions of sirtuin1, adenosine monophosphate-activated protein kinase and peroxisome proliferator activated receptor γ co-activator 1α were up-regulated by threonine supplementation in HFD-fed mice. These results suggest that long-term threonine supplementation inhibited fat mass and improved lipid metabolism, making it a potential agent to prevent the development of diet-induced obesity.

Leucine, but not isoleucine or valine, affects serum lipid profiles and browning of WAT in mice.

Branched chain amino acids (BCAA), especially leucine (Leu), have been reported to decrease fat deposition. However, opposite effects of BCAA on lipid metabolism have been observed. To determine the role of BCAA in lipid metabolism, an amino acid-defined diet was formulated and C57BL/6J mice were assigned into the following groups: amino acid-defined control diet and control diet supplemented with Leu, isoleucine, or valine. Nitrogen was balanced by proportionally mixed amino acids except BCAA. Results showed that dietary Leu supplementation significantly increased the levels of serum triglycerides, total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol and urea nitrogen. Metabolomics showed that biosynthesis of unsaturated fatty acids was altered by Leu supplementation. Leu treatment up-regulated the expression of genes related to fat synthesis and down-regulated the expression of genes related to fatty acid synthesis. Furthermore, the genes and proteins of selective markers involved in browning of white adipose tissue (WAT) were up-regulated by dietary supplementation with Leu. This study indicated that dietary supplementation with Leu, but not isoleucine or valine, significantly affected lipid metabolism by regulating lipid metabolism-related genes and serum fatty acid concentration, providing a new tool in the management of obesity and metabolic disorders.

Dietary supplementation with aromatic amino acids decreased triglycerides and alleviated hepatic steatosis by stimulating bile acid synthesis in mice.

Emerging evidence shows that amino acids can modulate lipid metabolism. Aromatic amino acids (AAAs) serve as important precursors of several neurotransmitters and metabolic regulators that play a vital role in regulating nutrient metabolism. But whether AAAs have a lipid-lowering function remains unknown. Here mice were fed amino acid-defined diets containing AAAs at 1.82% and 3.64% for 3 weeks. We demonstrated that double AAA intake significantly decreased the serum and hepatic triglycerides and serum low-density lipoprotein cholesterol, but increased the high-density lipoprotein cholesterol as well as insulin tolerance. Combined metabolomic and transcriptomic analysis showed that the hepatic acidic pathway of bile acid synthesis was responsible for the improvement in lipid metabolism by AAA treatment. This study suggests that AAAs have the potential to ameliorate steatosis and provides a new alternative to improve lipid metabolism.

Valine Supplementation Does Not Reduce Lipid Accumulation and Improve Insulin Sensitivity in Mice Fed High-Fat Diet.

Branched-chain amino acids (BCAAs), particularly leucine, were reported to decrease obesity and relevant metabolic syndrome. However, whether valine has a similar effect has rarely been investigated. In the present study, mice were assigned into four treatments (n = 10): chow diet supplemented with water (CW) or valine (CV) and high-fat diet supplemented with water (HW) or valine (HV). Valine (3%, w/v) was supplied in the drinking water. The results showed that valine treatment markedly increased serum triglyceride and insulin levels of chow diet-fed mice. The body weight, serum triglyceride level, white adipose tissue weight, and glucose and insulin intolerance were significantly elevated by valine supplementation in high-fat diet-fed mice. Metabolomics and transcriptomics showed that several genes related to fat oxidation were downregulated, and arachidonic acid and linoleic acid metabolism were altered in the HV group compared to the HW group. In conclusion, valine supplementation did not suppress lipid deposition and metabolic disorders in mice, which provides a new understanding for BCAAs in the modulation of lipid metabolism.

Splitting failure in side walls of a large-scale underground cavern group: a numerical modelling and a field study.

Vertical splitting cracks often appear in side walls of large-scale underground caverns during excavations owing to the brittle characteristics of surrounding rock mass, especially under the conditions of high in situ stress and great overburden depth. This phenomenon greatly affects the integral safety and stability of the underground caverns. In this paper, a transverse isotropic constitutive model and a splitting failure criterion are simultaneously proposed and secondly programmed in FLAC3D to numerically simulate the integral stability of the underground caverns during excavations in Dagangshan hydropower station in Sichuan province, China. Meanwhile, an in situ monitoring study on the displacement of the key points of the underground caverns has also been carried out, and the monitoring results are compared with the numerical results. From the comparative analysis, it can be concluded that the depths of splitting relaxation area obtained by numerical simulation are almost consistent with the actual in situ monitoring values, as well as the trend of the displacement curves, which shows that the transverse isotropic constitutive model combining with the splitting failure criterion is appropriate for investigating the splitting failure in side walls of large-scale underground caverns and it will be a helpful guidance of predicting the depths of splitting relaxation area in surrounding rock mass.