Hyperglycaemia and oxidative stress upregulate HSP60 & HSP70 expression in HeLa cells

Heat Shock Proteins 60 & 70 (HSP60 & HSP70) are intracellular protein that has been shown to be present at elevated levels in systemic circulation in Type 2 Diabetes mellitus (T2DM) patients. Conditions that lead to its secretion, and the mechanism of its translocation from cells, have not yet been defined. The aim of this study was to determine if specific cell stressors associated with T2DM, namely hyperglycaemia and oxidative stress, result in the upregulation of HSP60 in human cells in vitro. Human HeLa cells were grown in media supplemented with 100 mM glucose, 200 μM hydrogen peroxide (H₂O₂), and 50 μM sodium azide. Initially, the effect of these treatments on cell growth rate was examined, with each treatment significantly inhibiting growth rate. LDH and MTT assays were also used to successfully demonstrate that these treatments do not significantly increase cell lysis, but do significantly impair mitochondrial dehydrogenase activity. To confirm this mitochondria specific form of inhibition, DCFDA assay were used to investigate any increases in intracellular reactive oxygen species (ROS) generation. All three treatments resulted in significantly increased ROS generation, with greater ROS production occurring with a greater exposure time. Interestingly, when the protein levels of HSP60 and HSP70 were measured after 3 and 7 days of exposure of the HeLa cells to 100 mM glucose, 200 μM H₂O₂, and 50 μM sodium azide significant induction of these two molecular stress proteins were observed ranging from 2.43-5.08 fold compared to untreated control cells

Does Hsp60 provide a link between mitochondrial stress and inflammation in Diabetes Mellitus?

The focus of this review is to summarise the known relationships between the expression of Heat Shock Protein 60 (Hsp60) and its association with the pathogenesis of Type 1 and Type 2 Diabetes Mellitus. Hsp60 is a mitochondrial stress protein that is induced by mitochondrial impairment. It is known to be secreted from a number of cell types and circulating levels have been documented in both Type 1 and 2 Diabetes mellitus patients. The biological significance of extracellular Hsp60 however, remains to be established. We will examine the links between Hsp60 and cellular anti and pro- inflammatory processes and specifically address how Hsp60 appears to affect immune inflammation by at least two different mechanisms: as a ligand for innate immune receptors and as an antigen recognized by adaptive immune receptors. We will also look at the role of Hsp60 during immune cell activation in atherosclerosis, a significant risk factor during the pathogenesis of Diabetes Mellitus

Pterocellin A isolated from marine bryozoan Pterocella vesiculosa is cytotoxic to human HeLa cells via mitochondrial apoptotic processes

Pterocellin A is a novel bioactive alkaloid isolated from the New Zealand marine bryozoan Pterocella vesiculosa. It exhibits potent antitumour activity towards the P388 (murine leukaemia) cell line in vitro and is selectively sensitive towards certain non-small cell lung, melanoma, and breast cancer cell lines, however, the biological mode of action of pterocellin A is unknown. Using the human cervical cancer cell line HeLa, we show that pterocellin A exhibited cytotoxicity against HeLa cells with an IC50 of 886 ng/mL. Time-course MTT and LDH assays were carried out and the results showed only a low level of cytosolic LDH was detected in the supernatant after all the cells have died from pterocellin A treatment at 2000 ng/mL. This indicated the cells maintained membrane integrity upon cell death which suggested apoptotic cell death. Additionally, morphological changes were observed under the microscope after 6 h of treatment. Cell shrinkage and nucleus condensation were observed, as well as apparent membrane blebbing, a key feature of apoptosis. The MTT data was also indicative of mitochondria impairment which could suggest that pterocellin A targets the mitochondria. This idea was supported by the observed changes in the morphology and location of the mitochondria after exposure to pterocellin A. Furthermore, the level of activated caspase-3 in HeLa cells increased after treatment with pterocellin A; activated caspase-3 can only be detected after a series of signalling events following the induction of apoptosis. These data support the notion that pterocellin A is an inducer of apoptosis in HeLa cells possibly via mitochondria related processes

Bovine blastocyst development depends on threonine catabolism

Increasing evidence suggests that pluripotency is a metabolically specialised state. In mouse, inner cell mass (ICM) cells and ICM-derived pluripotent stem cells (PSCs) critically depend on catabolising the amino acid threonine, while human PSCs require leucine, lysine, methionine and tryptophan. However, little is known about the specific amino acid requirements of putative pluripotent cells in bovine. We selectively depleted candidate essential amino acids (EAAs) from individually cultured bovine embryos to study their role in blastocyst development. Depleting one (-T, -M), two (-MT, -CM, -CT, -IL, -IK, -KL) or three (-CMT, -IKL) EAAs from chemically defined protein-free culture medium did not affect the morula-to-blastocyst transition from day five (D5) to D8 in vitro. By contrast, removing six (-CIKLMT, -FHRYVW), nine (+CMT, +IKL), eleven EAAs (+T, +M) or all twelve EAAs increasingly impaired blastocyst development. As no clear candidate emerged from this targeted screen, we focussed on threonine dehydrogenase (TDH), which catalyses threonine catabolism. TDH mRNA and protein was present at similar levels in trophectoderm (TE) and ICM but absent from several adult somatic tissues. We then treated morulae with an inhibitor (Qc1) that blocks TDH from catabolising threonine. Continuous exposure to Qc1 reduced total and high-quality blastocyst development from 37% to 26% and 18% to 8%, respectively (P<0.005). This was accompanied by ~2-fold decrease in ICM, TE and total cell numbers (P<0.005), which was due to increased autophagy (P<0.05). At the same time, ICM- (NANOG) and TE-restricted (KRT8) genes were up- and down-regulated, respectively (P<0.05). In summary, bovine blastocyst viability depended on TDH-mediated threonine catabolism. However, ICM and TE cells did not metabolically differ in this regard, highlighting species-specific connections between metabolism and pluripotency regulation in mouse vs cattle

Biochemical studies on animal models of ceroid-lipofuscinoses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Veterinary Pathology, Massey University

The ceroid-lipofuscinoses are recessively inherited lysosomal storage diseases of children and animals, characterised by brain and retinal atrophy and the accumulation of lipopigment in a variety of cells. A systematic study of isolated lipopigment from an ovine form of the disease had shown the major stored components to be proteinaceous. This thesis presents further characterisation and identification of the stored ovine lipopigment proteins. Separation of the lipopigment proteins by LDS-PAGE showed the presence of the 3.5 kDa and 14.8 kDa proteins noted in earlier studies, and an additional band at 24 kDa. The 14.8 and 24 kDa bands varied between preparations and from different gels of the same isolate. Radioiodination of lipopigment and silver staining of the proteins separated by LDS-PAGE indicated that the 3.5 kDa protein was the dominant protein component. As these proteins were unable to be separated from each other, exploitation of the molar dominance of the 3.5 kDa protein led to its identification by a non traditional sequencing approach. The major stored protein was shown to be the full proteolipid subunit c of the mitochondrial ATP synthase complex. The 14.8 and 24 kDa proteins were shown to be stable oligomers of subunit c. Quantitaion of the sequence data showed that subunit c accounted for at least 50% of the lipopigment mass. No other mitochondrial protein was detected. Analyses of isolated mitochondria showed that they were functionally normal and did not contain excess amounts of subunit c. Subunit c is classified as a proteolipid, due to its lipid-like solubility in chloroform/methanol mixtures. Its storage in lysosome derived lipopigment bodies explained many of the described physical characteristics of lipopigment in the ceroid-lipofuscinoses. Application of the same methodology showed that a bovine, and two distinct canine forms of the ceroid-lipofuscinoses were also subunit c storage diseases. It is postulated that the lesions in the ceroid-lipofuscinoses involve defects in the degradative pathway of subunit c at some point after its incorporation into the inner mitochondrial membrane

A comparative proteomics study of skeletal muscle mitochondria from myostatin null mice

Myostatin is a secreted protein which is known to be a negative regulator of skeletal muscle growth. Numerous studies have demonstrated that down-regulating the expression of myostatin results in an increase in muscle mass. This increase in skeletal muscle is accompanied by a marked change in the muscle fibre composition from one reliant on mitochondrial oxidative metabolism to one more focused on glycolytic metabolism. A comparative proteomics study was undertaken to investigate the effect of this altered metabolism has on the mitochondria from the gastrocnemius muscle of myostatin null mice, compared with those from wild type mice. The majority of the proteins identified showed no significant modulation between the two phenotypes, but gives an interesting insight into observations made in previous studies. Several proteins were shown to be modulated; however, only one of these was able to be identified. This protein which had sequence similarity to aldehyde reductase, was up-regulated in myostatin null mitochondria. The significance of this observation remains to be established. Interestingly, this protein has been implicated in detoxification of harmful products of lipid peroxidation

The effect of complement C5a on mitochondrial functions of PC12 cells

C5a is thought to play a role during complement-activated neuronal apoptotic cell death in the central nervous system. The mechanisms responsible are however not well-understood. As mitochondria play a key role during apoptosis, we investigated mitochondria as a potential target for C5a. Using PC12 cells, we demonstrated that exposure to C5a led to inhibition of mitochondrial respiration, dehydrogenase and cytochrome c oxidase activities. Interestingly, an increase in expression of the mitochondrial stress protein chaperonin 60 was also observed, confirming a marked effect of C5a on mitochondrial functions. These observations are the first documented intracellular effects noted for the complement molecule C5a in in-vitro cultured cells

Angiopoietin-like protein 2, a chronic inflammatory mediator, is a new target induced by TGF-β1 through a Smad3-dependent mechanism

Angiopoietin-like protein 2 (Angptl2) levels are increased by obesity and obesity-related pathological conditions, and it is considered to be an important adipocyte-derived inflammatory mediator. In contrast, the multifunctional cytokine TGF-β1 has been reported to be augmented in obesity of rodents and humans, but inhibits adipocyte differentiation in vitro. Here we demonstrate that TGF-β1 induces expression of the Angptl2 gene through a Smad3-dependent pathway in RAW264.7 macrophage cells, primary peritoneal macrophages, and differentiated 3T3-L1 adipocytes. Transcriptional induction of the Angptl2 gene by TGF-β1 was dependent on the Smad3 protein which binds to the Smad Binding Element (SBE) region located on the Angptl2 promoter. Macrophages with Smad3 knocked down by small interfering RNA showed reduction of TGF-β1-induced Angptl2 expression. These findings may provide insight into the molecular mechanisms of the increased expression of Angptl2 and TGF-β1 in obesity

Metformin induced expression of Hsp60 in human THP-1 monocyte cells

Metformin is in widespread clinical use for the treatment of diabetes mellitus in patients. It has been shown to inhibit mitochondrial bioenergetic functions by inhibiting complex I of the electron transport chain. The expression of mitochondrial-specific molecular stress protein Hsp60 is a key consequence of mitochondrial impairment. Since this protein has important immune-modulatory properties, we have investigated the expression of Hsp60 in human THP-1 monocyte cells exposed to metformin. In this study, we demonstrate significant up-regulation of Hsp60 at both mRNA and protein levels when these cells were exposed to metformin at therapeutic dosage levels. Interestingly, there was also an increase in expression of CD14 mRNA in these cells. This suggested a possible modulation of the differentiation rates of the THP-1 cells during exposure to metformin. As monocyte differentiation marks a critical step in atherosclerosis, these observations suggest that long-term exposure to metformin could have important implications for the diabetic patient

Detection of Hsp60 in saliva and serum from type 2 diabetic and non-diabetic control subjects

There is increasing evidence that mitochondrial dysfunction and oxidative stress may be integral to the pathogenesis of type 2 diabetes mellitus. Heat shock protein (Hsp60) is a mitochondrial stress protein known to be induced under conditions of mitochondrial impairment. Although this intracellular protein is normally found in the mitochondrion, several studies have shown that this protein is also present in systemic circulation. In this study, we report the presence of elevated levels of Hsp60 in both saliva and serum of type 2 diabetic patients compared to non-diabetic controls. Hsp60 was detectable in the saliva of 10% of control and 93% of type 2 diabetic patients. Levels detected were in the range of 3–7 ng/ml in control and 3–75 ng/ml in type 2 diabetic patients. Serum Hsp60 levels in the range of 3–88 ng/ml were detected in 33% of control subjects, and levels in the range of 28–1,043 ng/ml were detected in 100% of type 2 diabetic patients. This is the first reporting of the presence of mitochondrial stress protein in salivary secretions. The serum Hsp60 levels were 16-fold higher compared to those in saliva, and there was a good positive correlation between salivary and serum Hsp60 levels (r = 0.55). While the exact mechanisms responsible for the secretion of Hsp60 into biological fluids such as saliva and blood are not yet known. The presence of this molecular marker of mitochondrial stress in saliva offers a non-invasive route to further investigate the biological functions of extracellular Hsp60 in type 2 diabetes mellitus and other conditions