Donald P. Cameron

Tumor Necrosis Factor-α Induces Apoptosis of Human Adipose Cells

Tumor necrosis factor-α (TNF-α) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-α mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-α has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-α in limiting increase in fat mass. The aim of the present study was to determine if TNF-α could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-α at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-α induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-α induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-α in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-α.

Apoptosis Is the Mode of β-Cell Death Responsible for the Development of IDDM in the Nonobese Diabetic (NOD) Mouse

The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of β-cell death responsible for the development of IDDM. Apoptotic cells were present within the islets of Langerhans in hematoxylin and eosin–stained sections of pancreases harvested from 3- to 18-week-old female NOD/Lt mice (a range of 11–50 apoptotic cells per 100 islets). Immunohistochemical localization of insulin to the dying cells confirmed the β-cell origin of the apoptosis. Although some islets from age-matched control female NOD/scid mice contained apoptotic cells, virtually all of these cells were insulin negative as determined by immunohistochemistry. The small number of apoptotic insulin-positive cells identified in islets from NOD/scid mice (a range of 0–1 apoptotic cells per 100 islets) was not statistically significant, compared with the numbers recorded in NOD/Lt mice. All dying cells showed the morphological changes characteristic of cell death by apoptosis and stained positively with the TUNEL method for end-labeling DNA strand breaks. The maximum mean amount of β-cell apoptosis occurring in NOD/Lt mice was at week 15 (50 apoptotic cells per 100 islets), which coincided with the earliest onset of diabetes as determined by blood glucose, urine glucose, and pancreatic immunoreactive insulin measurements. While there was no peak incidence of β-cell apoptosis throughout the time period studied (weeks 3–18), the incidence of apoptosis decreased at week 18, by which time 50% of the animals had overt diabetes. The low levels of β-cell apoptosis observed is indicative of a gradual deletion of the β-cell population throughout the extensive preclinical period seen in this model and would be sufficient to account for the β-cell loss resulting in IDDM. Apoptosis of β-cells preceded the appearance of T-cells (CD3-positive by immunohistochemistry) in islets. Lymphocytic infiltration of islets (insulitis) was not detected until week 6. The results show that β-cell apoptosis is responsible for the development of IDDM in the NOD/Lt mouse and that its onset precedes lymphocytic infiltration of the islets.

Beta cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model

Although insulin‐dependent diabetes mellitus (IDDM) results from irreversible loss of beta cells, the mode of cell death responsible for this loss has not previously been categorized. In this study, the multiple low‐dose streptozotocin (stz) model (intraperitoneal injection of stz at a concentration of 40 mg/kg body weight per day for five consecutive days) was used to investigate beta‐cell death during the development of IDDM in male C57B1/6 mice. Apoptotic cells were evident by light microscopy within the islets of Langerhans of treated animals from day 2 (the day of the second stz injection) until day 17. Immunohistochemical localization of insulin to the dying cells confirmed the beta‐cell origin of the apoptosis. Two peaks in the incidence of beta‐cell apoptosis occurred: the first at day 5, which corresponded to an increase in blood glucose concentration, and the second at day 11, when lymphocytic infiltration of the islets (insulitis) was maximal. Insulitis did not begin until day 9, by which time treated animals had developed overt diabetes as revealed by blood glucose and pancreatic immunoreactive insulin (IRI) measurements. Beta‐cell apoptosis preceded the appearance of T‐cells in the islets and continued throughout the period of insulitis. Thus, whether induced by stz or a subsequent immune response, apoptosis is the mode of cell death responsible for beta‐cell loss in the multiple low‐dose stz model of IDDM.

Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1.

The ability of ß cells to endure assaults by various environmental agents, including toxins and viruses, may be relevant to the development of diabetes. We have examined the mode of cell death caused by streptozotocin (STZ) in a murine pancreatic ß cell line, INS‐1. Apoptosis was identified by detection of initial endonuclease‐mediated DNA strand breaks by DNA gel electrophoresis. Apoptosis and necrosis were distinguished morphologically by light and electron microscopy. Higher rates of apoptosis, as compared to necrosis, were observed when cells were exposed to 15 mM STZ for 1 hr followed by a 24 hrs recovery period. Higher doses of STZ (30 mM) caused the cells to undergo necrosis (22%) as well as apoptosis (17%). These results suggest that the cytotoxic effect of STZ, at low doses, on ß cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of ß cell death is predominantly necrosis.

Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ERα gene but not ERβ by reverse transcriptase-polymerase chain reaction, possessed ERα protein on Western blotting, and displayed specific 17β-estradiol (E2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ERα complement for males or females. There were no gender differences in ERα complement for subcutaneous or visceral samples. We conclude that ERα but not ERβ is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ERα.

Effects of rosiglitazone and linoleic acid on human preadipocyte differentiation

Background Peroxisome proliferator activated receptor gamma (PPARγ) is a ligand‐activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro.

Familial Paget's disease of bone: nonlinkage to the PDB1 and PDB2 loci on chromosomes 6p and 18q in a large pedigree.

Pagets disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Pagets disease but the molecular basis remains largely unknown. Susceptibility loci for Pagets disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q21.1‐q22 (PDB2) in different pedigrees. We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Pagets disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log10 of the odds ratio (LOD) scores < −2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < −2.0) centered on D18S42. These data confirm the genetic heterogeneity of Pagets disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Pagets disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.

Defective Immunoreactive Insulin Secretion in the Acomys Cahirinus

In vivo studies of immunoreactive insulin (IRI) secretion and electron microscopic studies of pancreatic morphology have been performed in the spiny mouse, acomys cahirinus. A defective IRI release was demonstrated in response to glucose 1.0 gm./kg., arginine 200 mg./kg., glucagon 1 mg./kg., isoprenaline 20 μg./kg., aminophylline 240 mg./kg. and dibutyryl cyclic AMP 60 mg./kg., administered intraperitoneally when compared to normal Swiss white mice. The defect appeared to involve both phases of IRI release. Electron microscopic evidence supported the concept of defective IRI release in this species. Of particular interest was the frequent occurrence in B cells of autophagic vacuoles comparable to those described for other cells undergoing secretory arrest. It is suggested that the defect of IRI release involves some basic mechanism concerned with the transport of insulin out of the B cell.

Nicotinamide prevents the development of diabetes in the cyclophosphamide-induced NOD mouse model by reducing beta-cell apoptosis

The development of diabetes in non‐obese diabetic (NOD) mice, which normally takes between 3 and 7 months, can be accelerated by cyclophosphamide (CY) injections, with rapid progression to diabetes within only 2–3 weeks. This insulin‐dependent diabetes mellitus (IDDM) can be prevented or delayed in CY‐treated NOD mice by nicotinamide (NA). The present study was undertaken to determine the mode of cell death responsible for the development of IDDM in CY‐treated male NOD mice and to investigate the effect of NA on beta‐cell death. Apoptotic beta cells were present within the islets of Langerhans in haematoxylin and eosin‐stained sections of the pancreata harvested from 3‐ and 12‐week‐old male NOD mice, from 8 h until 14 days after a single intraperitoneal injection of CY (150 mg/kg body weight). The maximum amount of beta‐cell apoptosis in 3‐week‐old animals occurred 1–2 days after CY treatment (20 apoptotic cells per 100 islets), after which time levels of apoptosis declined steadily throughout the 14‐day period studied. The incidence of beta‐cell apoptosis in 12‐week‐old male NOD mice occurred in two peaks; the first was recorded 8–24 h after CY treatment (30 apoptotic cells/100 islets), while the second, at 7 days (36 apoptotic cells per 100 islets), coincided with increased insulitis. Administration of NA 15 min before CY treatment, and thereafter daily, substantially reduced the amount of apoptosis and effectively eliminated (4 apoptotic cells per 100 islets) the second wave of beta‐cell apoptosis seen at day 7 in 12‐week‐old animals given CY alone. These results show that apoptosis is the mode of beta‐cell death responsible for the development of CY‐induced IDDM and that prevention of IDDM by NA is associated with a reduction in beta‐cell apoptosis. Copyright

Post-prandial glucose and insulin responses to different types of spaghetti and bread

The glycemic response following ingestion of carbohydrate in various forms is different. The factors involved are not fully elucidated. In this study the glycemic and insulin responses to 50 g of carbohydrate in the form of white bread (WB), semolina bread (SB), white spaghetti (WS) and wholemeal spaghetti (BS) were compared in ten noninsulin-dependent diabetics. The responses were assessed by calculating the area under the curve. WB and SB had significantly higher glycemic responses compared with WS and BS (P less than 0.01). There was no difference in glycemic response between either form of bread, or either type of spaghetti. Similarly WB and SB had greater insulin responses than WS and BS (P less than 0.05). There was no difference in insulin response between WB and SB but BS had a greater response than WS (P less than 0.01) attributed to the higher protein content of BS. Thus, in this study the physical form of the food was a major factor influencing the glycemic response, and other factors such as particle size and fibre content had negligible effects.