Differential glycosylation of tissue non-specific alkaline phosphatase in mesenchymal stromal cells differentiated into either an osteoblastic or adipocytic phenotype.

It has long been known that tissue non-specific alkaline phosphatase (TNAP) is essential for the correct formation of bone, as altered expression or function of this enzyme results in hypophosphatasia, a disease characterised by compromised bone structure, density and strength. However, recent evidence strongly suggests that the enzyme also has a role in lipid accrual and adipogenesis, a function that seems far removed from bone formation. Given that mesenchymal stromal cells (MSCs) are progenitors of both osteoblasts and adipocytes, the question arises of how TNAP is regulated to potentially have a different function when MSCs undergo either osteogenesis or adipogenesis. As the primary protein sequence is unchanged for the enzyme during both types of differentiation, any differences in function must be attributed to post-translational modification and/or localisation. We therefore examined the location of TNAP in bone- or adipose-derived MSCs differentiated into an adipocytic phenotype and compared the glycosylation state of the enzyme in MSCs differentiated into either osteoblasts or adipocytes. TNAP was found to co-locate with perilipin around lipid droplets in MSCs from bone, subcutaneous- and visceral adipose tissue during adipocytic differentiation. Treatment of TNAP with wheat germ lectin followed by electrophoresis showed minor differences in glycosylation between the phosphatase isolated from cells from these tissues, whereas electrophoresis after neuraminidase digestion highlighted differential glycosylation between cell types and during adipogenesis and osteoblastogenesis. This infers that post-translational modification of TNAP is altered during differentiation and is dependent on the eventual phenotype of the cells.

The Role and Interactions of Programmed Cell Death 4 and its Regulation by microRNA in Transformed Cells of the Gastrointestinal Tract.

Data from GLOBOCAN 2020 estimates that there were 19.3 million new cases of cancer and 10.0 million cancer-related deaths in 2020 and that this is predicted to increase by 47% in 2040. The combined burden of cancers of the gastrointestinal (GI) tract, including oesophageal-, gastric- and colorectal cancers, resulted in 22.6% of the cancer-related deaths in 2020 and 18.7% of new diagnosed cases. Understanding the aetiology of GI tract cancers should have a major impact on future therapies and lessen this substantial burden of disease. Many cancers of the GI tract have suppression of the tumour suppressor Programmed Cell Death 4 (PDCD4) and this has been linked to the expression of microRNAs which bind to the untranslated region of PDCD4 mRNA and either inhibit translation or target the mRNA for degradation. This review highlights the properties of PDCD4 and documents the evidence for the regulation of PDCD4 expression by microRNAs in cancers of the GI tract.

A new perspective on the function of Tissue Non-Specific Alkaline Phosphatase: from bone mineralization to intra-cellular lipid accumulation.

Tissue-nonspecific alkaline phosphatase (TNAP) is one of four isozymes, which include germ cell, placental and intestinal alkaline phosphatases. The TNAP isozyme has 3 isoforms (liver, bone and kidney) which differ by tissue expression and glycosylation pattern. Despite a long history of investigation, the exact function of TNAP in many tissues is largely unknown. Only the bone isoform has been well characterised during mineralization where the enzyme hydrolyses pyrophosphate to inorganic phosphate, which combines with calcium to form hydroxyapatite crystals deposited as new bone. The inorganic phosphate also increases gene expression of proteins that support tissue mineralization. Recent studies have shown that TNAP is expressed in preadipocytes from several species, and that inhibition of TNAP activity causes attenuation of intracellular lipid accumulation in these and other lipid-storing cells. The mechanism by which TNAP stimulates lipid accumulation is not known; however, proteins that are important for controlling phosphate levels in bone are also expressed in adipocytes. This review examines the evidence that inorganic phosphate generated by TNAP promotes transcription that enhances the expression of the regulators of lipid storage and consequently, that TNAP has a major function of lipid metabolism.

Model for Studying the Effects of Chronic Metabolic Disease on Endogenous Bone Marrow Stem Cell Populations.

Disease-associated impairment/dysfunction of stem cell populations is prominent in chronic metabolic and inflammatory diseases, such as type 2 diabetes mellitus (DM) where the multifunctional properties (viability, proliferation, paracrine secretion, multilineage differentiation) of bone marrow resident mesenchymal stem cells (MSCs) can be affected. The growth and viability impairments make it difficult to study the underlying molecular mechanisms related to the dysfunction of these cells in vitro. We have consequently optimized the isolation and culture conditions for impaired/dysfunctional bone marrow MSCs from B6.Cg-Lepob/J obese prediabetic mice. The method described here permits ex vivo investigations into disease-associated functional impairments and the dysregulated molecular mechanisms in these primary MSCs through direct comparisons with their healthy wild-type C57BL6/J control mouse counterparts.

Adipocyte-progenitor cell communication that influences adipogenesis.

Adipose tissue is located in discrete depots that are differentially associated with elevated risk of metabolic complications, with fat accretion in visceral depots being most detrimental to metabolic health. Currently, the regulation of specific adipose depot expansion, by adipocyte hypertrophy and hyperplasia and consequently fat distribution, is not well understood. However, a growing body of evidence from in vitro investigations indicates that mature adipocytes secrete factors that modulate the proliferation and differentiation of progenitor, adipose-derived stem cells (ADSCs). It is therefore plausible that endocrine communication between adipocytes and ADSCs located in different depots influences fat distribution, and may therefore contribute to the adverse health outcomes associated with visceral adiposity. This review will explore the available evidence of paracrine and endocrine crosstalk between mature adipocytes and ADSCs that affects adipogenesis, as a better understanding of the regulatory roles of the extracellular signalling mechanisms within- and between adipose depots may profoundly change the way we view adipose tissue growth in obesity and related comorbidities.

Isolation and Characterization of Different Mesenchymal Stem Cell Populations from Rat Femur.

Purified mesenchymal stem cells (MSCs) may be used for a multitude of applications, from the study of biological processes such as cell division and coordinated gene expression to tissue engineering and regenerative medicine. However, although highly similar, MSCs isolated and purified from different tissues may be biologically different in the ability of the cells to respond to environmental cues that instigate and propagate changes in cell fate such as differentiation, proliferation, apoptosis, and senescence. Selecting which MSC subtype to study may therefore profoundly influence the outcome of the investigation. Here we outline the isolation, purification, and differentiation of three different MSC subtypes derived from various depots within rat bone. These include MSCs from bone marrow, compact bone, and the proximal femur. Osteoblastic and adipogenic differentiation exemplify differences between these cells.

Systemic Factors During Metabolic Disease Progression Contribute to the Functional Decline of Adipose Tissue-Derived Mesenchymal Stem Cells in Reproductive Aged Females.

It is known that advanced metabolic disorders such as type 2 diabetes compromise the functional and regenerative capacity of endogenous adipose-tissue resident stem cells (ADSCs). It is, however, still unclear at which stage of disease progression ADSCs become compromised and whether systemic factors contribute to their functional decline. It was therefore hypothesized that inflammatory changes in the systemic microenvironment during distinct stages of disease progression negatively affect the functional capacity of ADSCs. A total of forty-seven (n = 47) black African reproductive aged females (32 ± 8 years; mean ± SD) were included in this study and subdivided into: (a) healthy lean (C; body mass index, BMI ≤ 25 kg/m2), (b) healthy overweight/obese (OB; BMI ≥ 25 kg/m2), (c) obese metabolic syndrome (MetS; BMI ≥ 30 kg/m2), and (d) type 2 diabetes mellitus (T2DM; previously diagnosed and on treatment) groups. Participants underwent anthropometric assessments and a DXA scan to determine their body composition and adipose indices. Each persons' systemic metabolic- (cholesterol, HDL, LDL, triglycerides, and blood glucose) and inflammatory profiles (CRP, SDF1α, TNFα, IL6, IL8, IL10, and IFNy) were also evaluated. Participant-derived serum was then used to treat an ADSC cell line in vitro and its effect on viability (MTT-based assay), proliferation (BrdU), migration (wound healing assay), and osteogenic differentiation assessed. When exposed to serum derived from overweight/obese individuals (with or without metabolic syndrome), both the proliferative and migratory responses of ADSCs were less pronounced than when exposed to healthy control serum. Serum IL6 concentrations were identified as a factor influencing the proliferation of ADSCs, suggesting that long-term disruption to the systemic cytokine balance can potentially disrupt the proliferative responses of ADSCs. Obese participant-derived serum (with and without metabolic syndrome) furthermore resulted in lipid accumulation during osteogenic differentiation. This study, therefore demonstrated that systemic factors in obese individuals, regardless of the presence of metabolic syndrome, can be detrimental to the multifunctional properties of ADSCs.

Vanadate Impedes Adipogenesis in Mesenchymal Stem Cells Derived from Different Depots within Bone.

Glucocorticoid-induced osteoporosis (GIO) is associated with an increase in bone marrow adiposity, which skews the differentiation of mesenchymal stem cell (MSC) progenitors away from osteoblastogenesis and toward adipogenesis. We have previously found that vanadate, a non-specific protein tyrosine phosphatase inhibitor, prevents GIO in rats, but it was unclear whether vanadate directly influenced adipogenesis in bone-derived MSCs. For the present study, we investigated the effect of vanadate on adipogenesis in primary rat MSCs derived from bone marrow (bmMSCs) and from the proximal end of the femur (pfMSCs). By passage 3 after isolation, both cell populations expressed the MSC cell surface markers CD90 and CD106, but not the hematopoietic marker CD45. However, although variable, expression of the fibroblast marker CD26 was higher in pfMSCs than in bmMSCs. Differentiation studies using osteogenic and adipogenic induction media (OM and AM, respectively) demonstrated that pfMSCs rapidly accumulated lipid droplets within 1 week of exposure to AM, while bmMSCs isolated from the same femur only formed lipid droplets after 3 weeks of AM treatment. Conversely, pfMSCs exposed to OM produced mineralized extracellular matrix (ECM) after 3 weeks, compared to 1 week for OM-treated bmMSCs. Vanadate (10 µM) added to AM resulted in a significant reduction in AM-induced intracellular lipid accumulation and expression of adipogenic gene markers (PPARγ2, aP2, adipsin) in both pfMSCs and bmMSCs. Pharmacological concentrations of glucocorticoids (1 µM) alone did not induce lipid accumulation in either bmMSCs or pfMSCs, but resulted in significant cell death in pfMSCs. Our findings demonstrate the existence of at least two fundamentally different MSC depots within the femur and highlights the presence of MSCs capable of rapid adipogenesis within the proximal femur, an area prone to osteoporotic fractures. In addition, our results suggest that the increased bone marrow adiposity observed in GIO may not be solely due to direct effect of glucocorticoids on bone-derived MSCs, and that an increase in femur lipid content may also arise from increased adipogenesis in MSCs residing outside of the bone marrow niche.

Free fatty acid G-protein coupled receptor signaling in M1 skewed white adipose tissue macrophages.

Obesity is associated with the establishment and maintenance of a low grade, chronically inflamed state in the white adipose tissue (WAT) of the body. The WAT macrophage population is a major cellular participant in this inflammatory process that significantly contributes to the pathophysiology of the disease, with the adipose depots of obese individuals, relative to lean counterparts, having an elevated number of macrophages that are skewed towards a pro-inflammatory phenotype. Alterations in the WAT lipid micro-environment, and specifically the availability of free fatty acids, are believed to contribute towards the obesity-related quantitative and functional changes observed in these cells. This review specifically addresses the involvement of the five G-protein coupled free fatty acid receptors which bind exogenous FFAs and signal in macrophages. Particular focus is placed on the involvement of these receptors in macrophage migration and cytokine production, two important aspects that modulate inflammation.

Depot-specific and hypercaloric diet-induced effects on the osteoblast and adipocyte differentiation potential of adipose-derived stromal cells.

Adipose-derived stromal cells (ADSCs) can be differentiated in vitro into several mesenchyme-derived cell types. We had previously described depot-specific differences in the adipocyte differentiation of ADSCs, and consequently we hypothesized that there may also be depot-specific differences in osteoblast differentiation of ADSCs. For this study, the osteoblast differentiation potential of rat subcutaneous ADSCs (scADSCs) and perirenal visceral ADSCs (pvADSCs) was compared. Osteoblast differentiation media (OM) induced markers of the osteoblastic phenotype in scADSCs, but not in pvADSCs. ADSCs harvested from rats with diet-induced visceral obesity (DIO) exhibited reduced osteoinduction, compared to lean controls, but adipocyte differentiation was not affected. Expression of the pro-osteogenic transcription factor Msx2 was significantly higher in naïve scADSCs from lean and DIO rats than in pvADSCs. Our findings indicate that ADSCs from different anatomical sites are uniquely pre-programmed in vivo in a depot-specific manner, and that diet-induced metabolic disturbances translate into reduced osteoblast differentiation of ADSCs.

Glucocorticoid administration and brief occlusion of the main pancreatic duct are likely to increase islet mass by a similar mechanism.

OBJECTIVES: Both glucocorticoid (GC) administration and brief occlusion of the main pancreatic duct result in an increase in total islet mass. Consequently, it was questioned whether these 2 stimuli would produce similar islet growth, indicating commonality in the mechanism of expansion. To test this, we assessed the effects on morphology after single and dual stimulation of the pancreas. METHODS: Rat pancreata were harvested 56 days after (1) brief occlusion of the main pancreatic duct, (2) daily GC administration, (3) GC administration and brief occlusion, or (4) sham operation without GC administration or occlusion. The pancreata were weighed, fixed, wax embedded, and sectioned for morphologic analysis. The endocrine to exocrine ratio, endocrine mass, and the contribution that small, medium, and large islets made to increased pancreatic endocrine mass were assessed. Blood was taken immediately before termination, after overnight fasting, for analysis of serum glucose, amylase, and insulin. RESULTS: GC treatment resulted in increased total pancreatic mass and exocrine mass, which were dissimilar to increases elicited by brief occlusion. However, there was no significant difference in the increase in the total endocrine mass or the increased mass of small, medium, or large islets between the GC, occluded, and dually stimulated pancreata. There were also no significant differences in the mean number of cells per islet between these groups. GC administration increased both circulating glucose and insulin in both occluded and nonoccluded groups, whereas occlusion alone had no effect on these parameters. CONCLUSIONS: Glucocorticoid administration and brief occlusion of the main pancreatic duct result in a similar expansion of islet mass. This is reflected in nonsignificant increases in endocrine mass/body weight and the percentage contribution of small, medium, and large islets to this increase. The majority of additional islet mass is from the expansion of the large islet population, although extra large islets are not found after either pancreatic treatment. The effects of GC treatment and occlusion are not additive, indicating that there is commonality in the mechanism of expansion. Because occlusion does not result in elevated glucose or insulin levels and gives rise to increased islet mass equivalent to GC administration and dual stimulation, it is unlikely that the increased islet mass after GC treatment is caused by the accompanying hyperinsulinemia as previously hypothesized.

The increase in pancreatic endocrine mass after brief occlusion of the main pancreatic duct is primarily due to islet expansion and does not solely originate from islet neogenesis.

OBJECTIVES: We have developed a method for stimulating an increase in total pancreatic endocrine mass to study the initial signaling events occurring during islet neogenesis and found that brief occlusion of the main pancreatic duct transiently stimulates ERK1/2. As transient activation is predominantly associated with differentiation rather than proliferation, we investigated whether increased mass in this model was derived from neogenesis or from expansion of preexisting islets. METHODS: The main pancreatic duct in rats was briefly occluded, the pancreas excised and weighed, and immunocytochemical analysis performed after 56 days. Changes in endocrine-to-exocrine ratio; the percentage of small, medium, and large islets; and endocrine mass were assessed. Proliferation was measured in islets cells at 3, 7, 14, 21, and 56 days post-occlusion. In vitro kinase assays and Western immunoblot analysis were performed on pancreatic lysates to assess ERK1/2 and JNK activation. RESULTS: Briefly occluding the main pancreatic duct results in rapid transient activation of ERK1/2 and a slower activation of JNK, followed by an 80% increase in endocrine mass 56 days post-occlusion. The majority of this additional endocrine mass was due to an increase in the large (51%) rather than the small (8%) islet population. Islet cell proliferation and islet cell size of occluded pancreata were equivalent to those of unaffected controls. CONCLUSION: Neogenesis makes only a minor contribution to the overall increase in pancreatic endocrine mass. The additional endocrine mass is mainly derived from preexisting islets, but this is unlikely to be due to islet cell proliferation or hypertrophy in this model.