visualization-quantification-mesenchymal-cell-adipogenic

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

The University of Auckland

Human milk cholesterol esterase (bile salt-activated lipase) plays a role in the dietary uptake of triacylglyceride and cholesteryl ester. The activities toward these substrates are mediated through a unique bile salt-activated mechanism. Previously, it has been proposed that a necessary step in this process is prior protein dimerisation in the presence of primary bile salts. In this study, we addressed the role of protein dimerisation by investigating bile salt interactions on full length and truncated recombinant forms, as analysed by size exclusion chromatography and concanavalin A Sepharose binding experiments. The present findings demonstrate that protein dimerisation is not an obligatory component of the bile salt-activated pathway. A new functional role for the glycosylated C-terminal domain in cholesterol esterase is also demonstrated in the prevention of non-specific hydrophobic interactions.

Bile salt activation of human cholesterol esterase does not require protein dimerisation.

Mucins carry out a number of protective roles, some of which are more easily studied than others. One mucin function is believed to be the protection of the mucosal epithelium against acidic and proteolytic damage in the stomach and intestines. In the present work, a portion of stomach mucin tandem repeat sequence (Muc6) was joined to the catalytic domain of a reporter enzyme [human milk cholesterol esterase (CE)] to determine whether the former can protect the latter protein from damage. This Muc6 domain replaced a unique series of glycosylated C-terminal repeats normally present in CE. The chimeric protein (CE/Muc6) was expressed in two different cell lines and its properties compared to recombinant full-length CE and a truncated version of CE which contained only the catalytic domain (CE/trunc). Results showed that both CE and CE/Muc6 were resistant to denaturation by acid and to proteolysis by pepsin at low pH values or by pancreatic proteases compared to CE/trunc. Thus, a stomach Muc6 domain is sufficient to confer stability on the CE catalytic domain, demonstrating a protective effect by a glycosylated mucin sequence.

Functional protective role for mucin glycosylated repetitive domains.

The neuropeptide, α-calcitonin gene-related peptide (α-CGRP), is expressed from sensory nerves that innervate fat. However, how α-CGRP may act in adipose tissue is unclear. Using 3T3-L1 adipocytes we observed that rat α-CGRP (rα-CGRP) evoked either a biphasic or monophasic reduction in intracellular free fatty acid (FFA) content. cAMP production was always monophasic and occurred when FFA responses were absent. Taken together with the observed potencies, these findings suggest that adipose tissue is a physiological target for α-CGRP. However, uncoupling of the FFA and CGRP-signaling responses with increasing passage number limits 3T3-L1 adipocytes as a suitable cellular model.

α-Calcitonin gene related peptide (α-CGRP) mediated lipid mobilization in 3T3-L1 adipocytes.

Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol.

Renal depletion of myo-inositol (MI) is associated with the pathogenesis of diabetic nephropathy in animal models, but the underlying mechanisms involved are unclear. We hypothesized that MI depletion was due to changes in inositol metabolism and therefore examined the expression of genes regulating de novo biosynthesis, reabsorption, and catabolism of MI. We also extended the analyses from diabetes mellitus to animal models of dietary-induced obesity and hypertension. We found that renal MI depletion was pervasive across these three distinct disease states in the relative order: hypertension (-51%)>diabetes mellitus (-35%)>dietary-induced obesity (-19%). In 4-wk diabetic kidneys and in kidneys derived from insulin-resistant and hypertensive rats, MI depletion was correlated with activity of the MI-degrading enzyme myo-inositol oxygenase (MIOX). By contrast, there was decreased MIOX expression in 8-wk diabetic kidneys. Immunohistochemistry localized the MI-degrading pathway comprising MIOX and the glucuronate-xylulose (GX) pathway to the proximal tubules within the renal cortex. These findings indicate that MI depletion could reflect increased catabolism through MIOX and the GX pathway and implicate a common pathological mechanism contributing to renal oxidative stress in metabolic disease.

Renal depletion of myo-inositol is associated with its increased degradation in animal models of metabolic disease.

Ma̅nuka honey, made from the nectar of Leptospermum scoparium, has garnered scientific and economical interest due to its nonperoxide antibacterial activity. Biomarkers for genuine ma̅nuka honey are increasingly in demand due to the presence of counterfeit ma̅nuka honey. This work reports the identification of a compound previously unreported in ma̅nuka honey by HPLC, and determination of the structure of the as 3,6,7-trimethyllumazine using NMR, MS, IR, and UV/vis spectroscopy. This assignment was confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in ma̅nuka honeys and could potentially serve as a biomarker for genuine ma̅nuka honey.

Isolation, Structural Elucidation, and Synthesis of Lepteridine From Ma̅nuka (Leptospermum scoparium) Honey.

d-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease.

Synthesis and biological activities of d-chiro-inositol analogues with insulin-like actions.

New Zealand manuka (Leptospermum scoparium) honey exhibits two unique fluorescence signatures that distinguish it from other honey types. One of these is the MM1 fluorescence marker (270-365nm excitation-emission) which we show is due to a Leptospermum nectar-derived compound, leptosperin. Synthetic or honey-purified leptosperin not only displayed an identical fluorescence spectrum, but supplementation of leptosperin into clover or artificial honeys generated the MM1 fluorescence signature. There was a quenching effect of the honey matrix on leptosperin fluorescence but otherwise leptosperin was chemically stable over prolonged storage at 37°C. Leptosperin was also present in the woody-fruited Australian Leptospermum species at elevated concentrations but virtually absent in Leptospermum subtenue suggesting its elevated expression developed following the mid-Miocene separation of the genus. These findings suggest that fluorescence spectroscopy could offer a rapid and high-throughput screening method for identification of Leptospermum honeys using the MM1 fluorescence marker.

Kerry Loomes

School of Biological Sciences

The University of Auckland

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker