The hexosamine biosynthetic pathway can mediate myocardial apoptosis in a rat model of diet-induced insulin resistance.

AIMS: Type 2 diabetes is characterized by deranged metabolic pathways that may result in cardiovascular complications. For example, hyperglycaemia promotes flux through the hexosamine biosynthetic pathway (HBP) leading to greater O-GlcNAcylation of target proteins, with pathophysiological outcomes. This study investigated mechanisms whereby increased HBP flux elicits myocardial apoptosis in a rat model of diet-induced hyperglycaemia/insulin resistance. METHODS: Four-week-old male Wistar rats were fed a high-fat diet (86 days) after which insulin resistance was assessed vs. matched controls. Oxidative stress was evaluated, and apoptotic peptide levels, BAD phosphorylation and overall O-GlcNAcylation assessed by immunoblotting. Protein-specific O-GlcNAcylation and BAD-Bcl-2 dimerization were determined by immunoprecipitation and Western blotting. RESULTS: Rats consuming the high-fat diet exhibited a moderate elevation in body weight, higher fasting insulin and glucose levels, and insulin resistance vs. controls. Overall protein O-GlcNAcylation was increased in hyperglycaemic/insulin-resistant hearts. In parallel, myocardial peptide levels of apoptotic markers (caspase-3, cytochrome-c, BAD) were significantly higher with insulin resistance. To gain mechanistic insight into our findings, we evaluated O-GlcNAcylation of BAD, a pro-apoptotic Bcl-2 homolog. Here we found increased BAD O-GlcNAcylation and decreased BAD phosphorylation (Ser136) in hyperglycaemic/insulin-resistant rat hearts. These data are in agreement with competition by phosphorylation and O-GlcNAcylation for the same or neighbouring site(s) on target proteins. Moreover, we observed increased BAD-Bcl-2 dimerization in hyperglycaemic/insulin-resistant hearts. CONCLUSION: The main finding of this study is that increased apoptosis in hyperglycaemic/insulin-resistant hearts can also be mediated through HBP-induced BAD O-GlcNAcylation and greater formation of BAD-Bcl-2 dimers (pro-apoptotic).

Proteomic analysis of mitochondrial proteins in a mouse model of type 2 diabetes.

OBJECTIVE: Impaired mitochondrial function may contribute to the onset of contractile dysfunction with insulin resistance/type 2 diabetes. Our aim was therefore to determine alterations in the mitochondrial proteome of a mouse model of obesity/type 2 diabetes. METHODS: Mitochondrial proteins were isolated from hearts collected from 18- to 20-week-old female db/db mice and compared to matched controls. We performed two-dimensional polyacrylamide gel electrophoresis to determine differentially expressed proteins. Peptides of interest were further analysed by mass spectrometry and Mascot software was employed to identify protein matches. RESULTS: Our data showed that ATP synthase D chain, ubiquinol cytochrome-C reductase core protein 1 and electron transfer flavoprotein subunit alpha peptide levels were altered with obesity. Moreover, we found coordinate downregulation of contractile proteins in the obese heart, i.e. α-smooth muscle actin, α-cardiac actin, myosin heavy-chain α and myosin-binding protein C. CONCLUSION: We propose that decreased contractile protein levels may contribute to contractile dysfunction of hearts from diabetic mice.

Impaired contractile function and mitochondrial respiratory capacity in response to oxygen deprivation in a rat model of pre-diabetes.

AIM: Obesity is a major contributor to the global burden of disease and is closely associated with the development of type 2 diabetes and cardiovascular diseases. This study tested the hypothesis that mitochondrial respiratory capacity of the pre-diabetic heart is decreased leading to impaired contractile function and tolerance to ischaemia/reperfusion. METHODS: Eight-week-old male Wistar rats were fed a high caloric diet for 16 weeks after which anthropometric, metabolic, cardiac and mitochondrial parameters were evaluated vs. age-matched lean controls. Cardiac function (working heart perfusions) and mitochondrial respiratory capacity were assessed at baseline and in response to acute oxygen deprivation. RESULTS: Rats fed the high caloric diet exhibited increased body weight and visceral fat vs. the control group. Heart weights of obese rats were also increased. Triglyceride, fasting plasma insulin and free fatty acid levels were elevated, while high-density lipoprotein cholesterol levels were reduced in the obese group. Contractile function was attenuated at baseline and further decreased after subjecting hearts to ischaemia-reperfusion. Myocardial infarct sizes were increased while ADP phosphorylation rates were diminished in obese rats. However, no differences were found for mtDNA levels and the degree of oxidative stress-induced damage. CONCLUSIONS: These data show that decreased mitochondrial bioenergetic capacity in pre-diabetic rat hearts may impair respiratory capacity and reduce basal contractile function and tolerance to acute oxygen deprivation.

Improved prognosis of Epstein-Barr virus associated childhood Hodgkin's lymphoma: study of 47 South African cases.

AIM: To study the distribution of Hodgkin's lymphoma in South African children and report the incidence of Epstein-Barr virus (EBV) as regards age, race, sex, and histological subtype; to investigate whether EBV is relevant to survival. METHODS: Immunohistochemistry (IHC) and in situ hybridisation (ISH) to detect EBV were performed on 47 South African children with classical Hodgkin's lymphoma, ranging in age from 3 to 14 years and coming from different ethnic backgrounds. The correlation between the presence of the virus and clinical outcome was assessed. RESULTS: The nodular sclerosing subtype predominated, comprising 89% of cases; the remaining 11% were of the mixed cellularity subtype. EBV was present in 68%. Full clinical data were available for 36 cases; EBV positive patients presented with less aggressive symptoms at diagnosis and had a significantly longer median survival than EBV negative patients. CONCLUSIONS: The distribution of EBV in South African childhood Hodgkin's lymphoma follows a pattern intermediate to that of industrialised and non-industrialized countries. Furthermore, our data suggest that there is an association between poor prognosis and the non-detection of EBV products in South African childhood Hodgkin's lymphoma.

Detection of the T(14;18) translocation in follicle centre cell lymphomas in South African ethnic groups using PCR.

PCR was used to detect the t(14;18) translocation in 64 South African cases with follicle centre cell lymphoma. DNA was purified from paraffin-embedded tissue collected from different ethnic groups namely white, black and "mixed" race patients, and primers used to detect both mbr and mcr. The overall incidence of the translocation was 45%, which is similar to that of Caucasian and Chinese patients. The ratio of rearrangements occurring at the mbr and mcr was 7:1 which may be an overestimation. The ratio was three times higher for the "mixed" race group compared to whites, and this suggests that there may be ethnic variation in breakpoints in South African patients.

Analysis of low and high grade B-cell lymphoma subtypes using semi-nested PCR and two primer sets.

Immunoglobulin gene rearrangements in B-cell lymphoma subtypes may not always be detected by PCR if only one primer set is applied. We therefore analysed a range of low and high grade B-cell lymphoma subtypes for monoclonality using PCR, to determine appropriate primer selection strategies for routine diagnostic use. Semi-nested PCR was performed on 70 unequivocal B-cell lymphoma cases using paraffin-embedded tissue (PET). Consensus primers directed at the framework 3 (Fr3) and framework 2 (Fr2) regions of the immunoglobulin heavy chain (IgH) gene were used to detect monoclonality. Monoclonality was found in 77% of cases using primer Fr3, 58% using Fr2, and in 93% of cases when data were combined for both primers. In 89% of the 38 low grade and 97% of the 31 high grade B-cell lymphomas monoclonality could be detected when combining both primers. Fr3 gave superior results in most of the lymphoma subtypes analysed. We conclude that both Fr3 and Fr2 are useful, in a routine histopathology laboratory, for detecting monoclonality in most B-cell lymphoma subtypes. Certain subtypes, however, are sometimes not targeted by these primers and therefore require additional analyses.

Diagnosis of an anemone cell tumor as a B-cell lymphoma by molecular analysis.

An 83-year-old man presented with mediastinal and axillary lymphadenopathy. Immunophenotyping and electron microscopy performed on an excision biopsy were inconclusive. On a repeat biopsy a year later the ultrastructural features typical of an anemone cell tumor were seen. A panel of monoclonal antibodies showed positivity for vimentin only. The diagnosis of a high-grade B-cell lymphoma could be made only after detection of immunoglobulin gene rearrangement by polymerase chain reaction gene amplification.