Myonuclear alterations associated with exercise are independent of age in humans.

Age-related decline in skeletal muscle structure and function can be mitigated by regular exercise. However, the precise mechanisms that govern this are not fully understood. The nucleus plays an active role in translating forces into biochemical signals (mechanotransduction), with the nuclear lamina protein lamin A regulating nuclear shape, nuclear mechanics and ultimately gene expression. Defective lamin A expression causes muscle pathologies and premature ageing syndromes, but the roles of nuclear structure and function in physiological ageing and in exercise adaptations remain obscure. Here, we isolated single muscle fibres and carried out detailed morphological and functional analyses on myonuclei from young and older exercise-trained individuals. Strikingly, myonuclei from trained individuals were more spherical, less deformable, and contained a thicker nuclear lamina than those from untrained individuals. Complementary to this, exercise resulted in increased levels of lamin A and increased myonuclear stiffness in mice. We conclude that exercise is associated with myonuclear remodelling, independently of age, which may contribute to the preservative effects of exercise on muscle function throughout the lifespan. KEY POINTS: The nucleus plays an active role in translating forces into biochemical signals. Myonuclear aberrations in a group of muscular dystrophies called laminopathies suggest that the shape and mechanical properties of myonuclei are important for maintaining muscle function. Here, striking differences are presented in myonuclear shape and mechanics associated with exercise, in both young and old humans. Myonuclei from trained individuals were more spherical, less deformable and contained a thicker nuclear lamina than untrained individuals. It is concluded that exercise is associated with age-independent myonuclear remodelling, which may help to maintain muscle function throughout the lifespan.

Cardiorespiratory fitness not sedentary time or physical activity is associated with cardiometabolic risk in active older adults.

Sedentary time (ST) and moderate-to-vigorous physical activity (MVPA) are associated with cardiometabolic health. Cardiorespiratory fitness (CRF) is also implicated but often overlooked in health recommendations. This study assessed the relationships between ST, MVPA, CRF, and cardiometabolic health in highly active older individuals. 125 healthy amateur cyclists aged 55 to 79 years had their ST and MVPA levels assessed by actigraphy over a 7-day period. CRF was assessed using a maximal effort cycle ergometry test to determine VO2max with results normalized to both body mass and fat-free mass measured by DXA. Markers of cardiometabolic risk (blood glucose, triglycerides, cholesterol, HDL, LDL, Insulin, HOMA IR, blood pressure, and body fat) were assessed and used to determine cumulative cardiometabolic risk. Multiple linear regression was used to assess ST, MVPA, and CRF associations with cardiometabolic health with the relationship between activity levels and CRF determined. CRF was associated with training volume (P = .003), but not ST or MVPA. A high CRF was associated with lower cumulative cardiometabolic risk, body fat percentage, triglyceride, and HDL levels (P < .05 in all cases). MVPA was negatively associated with body fat percentage, while ST was not associated with any marker of cardiometabolic risk when adjusting for activity levels. An association between CRF and cardiometabolic risk even in a group of older individuals with high fitness levels highlights the importance that CRF may have in maintaining health.

Active GSK3ß and an intact ß-catenin TCF complex are essential for the differentiation of human myogenic progenitor cells.

Wnt-ß-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-ß-catenin signalling in myogenic differentiation. We show that dephosphorylated ß-catenin (active-ß-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of ß-catenin gain-of-function (GSK3 inhibition and ß-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-ß-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.

Human variability and noncancer risk assessment--an analysis of the default uncertainty factor.

A 10-fold uncertainty factor is used for noncancer risk assessments to allow for possible interindividual differences between humans in the fate of the chemical in the body (kinetics) and target organ sensitivity (dynamics). Analysis of a database on the variability in each of these aspects is consistent with an even subdivision of the 10-fold factor into 10(0.5) (3.16) for kinetics and 10(0.5) (3.16) for dynamics. Analysis of the number of subjects in a normally and log-normally distributed population which would not be covered by factors of 3.16 supports this subdivision and also the use of a 10-fold factor to allow for both aspects. Analysis of kinetic data for subgroups of the population indicates that the standard default value of 3.16 for kinetics will not be adequate for all routes of elimination and all groups of the population. A scheme is proposed which would allow the selection of appropriate default uncertainty factors based on knowledge of the biological fate and effects of the chemical under review.

Population heterogeneity and its impact on the risk management of food safety.

Successful risk management of possible toxicological effects of chemicals in food, including novel foods, requires diverse strategies. This paper concentrates on the advisability of introducing post marketing surveillance as a key stratagem in the risk management of selected foods and food chemicals. It is argued that this stratagem is particularly applicable to those occasions when the population at risk is relatively easily identified. A key element therefore in the successful use of post marketing surveillance in selected foods, requires those agencies responsible for risk management to introduce a more thorough analysis of population heterogeneity into their strategies.

B-cell responses to intravenous glucose and glucagon in non-diabetic twins of patients with type 1 (insulin-dependent) diabetes mellitus.

The B-cells of patients with recently diagnosed Type 1 (insulin-dependent) diabetes may have no response to glucose when the response to glucagon is present but attenuated. This observation suggests that the recognition of glucose is more severely affected than that for non-glucose stimulants. To determine whether a similar selective decrease in glucose response was present before the onset of diabetes we studied two groups of non-diabetic identical twins of patients with recently diagnosed Type 1 diabetes: one group with complement-fixing islet cell antibodies who were at high risk of developing diabetes (four of the five have already developed diabetes) and a group without such antibodies at low risk of developing diabetes. In addition, a group of patients with chronic pancreatitis were studied to control for non-specific damage to the B-cell. Responses to i.v. glucose and i.v. glucagon were compared. Patients with chronic pancreatitis has similar responses to both glucose and glucagon and the responses did not differ from control subjects. The B-cells of the immune positive group showed evidence of pathology because the insulin and C-peptide responses to both stimuli were reduced when compared to either their control subjects or the immune negative twin group. However, the B-cell response to both glucose and glucagon in the immune positive twins was similar. Because the B-cell response to glucose was not less than that to glucagon, a selective destruction of the glucose recognition system cannot be a characteristic of all twins throughout the period before they develop Type 1 diabetes.

Aetiology of insulin-dependent diabetes.

Two major types of diabetes are recognized, insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM). In this review we discuss some of the underlying factors which play a role in the events leading to IDDM.

N-terminal sequence analysis of human placental protein 14, purified in high yield from decidual cytosol.

Human placental protein 14 (PP14) has been purified in high yield from first trimester decidual cytosol. High-performance liquid chromatography on anion exchange, gel filtration and reverse-phase chromatography were used. The protein obtained is approximately 97% pure with an overall recovery of about 50% from the original tissue extract. The first 24 amino acids of the N-terminal were found to be Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-Gly-Thr-Glu-His - Glu-Met-Ala-Met. PP14 has been characterized in this study to be a dimeric glycoprotein of Mr 60,000, with homologous subunits having an Mr of 28,000.

Characterization and quantification of copper sulfate-induced vascularization of the rabbit cornea.

A model of angiogenesis in the rabbit cornea, with reproducible onset and duration of responses, was developed by using CuSO4 as the angiogenic stimulus. The vascularization of the cornea was quantified by means of an image analyzer. In addition, the effects of antiinflammatory compounds, dexamethasone and flurbiprofen, on the angiogenic response to CuSO4 were examined. Elvax pellets containing 10-75 micrograms of CuSO4 implanted in the corneal stroma dose-dependently induced neovascularization, which persisted for more than 64 days at the highest dose. Manual measurements of blood vessel lengths and image analysis measurements of blood vessel areas were comparable during the growth phase of vascularization, but only the image analysis measurements detected a subsequent regression phase. Therefore, the length method of measurement is only useful during the growth phase, whereas the image analysis method is useful during both the growth and regression phases of vascularization. Dexamethasone (50 micrograms, applied topically, three times a day) and flurbiprofen (100 micrograms, applied topically, three times a day) suppressed the inflammation produced by corneal implants containing 75 micrograms CuSO4. However, each drug only inhibited vascular growth by 50% during the 14 days of treatment. Although CuSO4 is not an endogenous angiogenic factor, the model presented in this report may be useful in quantitative evaluation of anti-angiogenic agents.

The role of phosphoenolpyruvate in insulin secretion: the effect of L-phenylalanine.

Incubation of rat islets with phenylalanine increased the tissue content of phosophoenolpyruvate, both in the presence and in the absence of glucose. At the same time, L-phenylalanine neither stimulated nor inhibited insulin release. It is unlikely that insulin secretion is tightly coupled to the availability of phosphoenolpyruvate in rat islets.

The internalisation of [125I] insulin by isolated rat hepatocytes.

This work was undertaken to examine the relationship between the binding, internalisation and degradation of insulin by isolated rat hepatocytes. Internalisation of hormone reached a maximum after 5-7 minutes at 37 degrees C; at an insulin concentration of 0.1 nM, 25% of the specifically bound hormone was internalised. Internalisation and the degradation of internalised insulin were inhibited by the intralysosomal protease inhibitors chloroquine and methylamine but not by the thiol oxidant diamide. It is concluded that internalisation of insulin by rat hepatocytes is not a necessary step in the degradation of the hormone molecule.

125I-labelled insulin degradation by isolated rat hepatocytes: the roles of glutathione-insulin transhydrogenase and insulin-specific protease.

Isolated rat hepatocytes degraded 125I-insulin with a Km of 150 nmol/l. Degradation was stimulated by the addition of glutathione and dithiothreitol. In cells incubated with diamide, glutathione was oxidised to the disulphide. Regeneration of reduced glutathione commenced after a further 30 min incubation at 37 degrees C. Diamide (1 mmol/l) significantly inhibited insulin degradation by hepatocytes (p less than 0.001). The 'apparent Vmax' for insulin degradation was decreased tenfold and the Km decreased to 25 nmol/l. The diamide-insensitive degrading activity was cell-associated and produced an intermediate of hormone degradation that was apparently of a higher molecular weight than insulin A chain. The biological activity of the intermediate was 0.03% of that of insulin. The diamide-insensitive activity was not due to release of protease into the medium by cell lysis. We conclude that there are at least two pathways capable of degrading insulin existing in rat hepatocytes.

Immunological and kinetic properties of pyruvate kinase in rat pancreatic islets.

Pyruvate kinase in rat pancreatic islets was characterized immunologically and kinetically. It is concluded that this activity is predominantly if not totally of the M(2) type.

A neuropeptide of the blowfly Calliphora vomitoria with an amino acid composition homologous with vertebrae pancreatic polypeptide.

A neuropeptide purified from the brain of the blowfly (Calliphora vomitoria) that cross-reacts in a bovine pancreatic polypeptide radioimmunoassay has been subjected to amino acid analysis. The amino acid composition of the peptide shows homology with vertebrate pancreatic polypeptide species. Amounts of the neuropeptide calculated from amino acid analysis record with those measured by the pancreatic polypeptide radioimmunoassay. These results suggest that the primary structure of the Calliphora neuropeptide is very similar to that of mammalian pancreatic polypeptides.

The metabolism of 125I-labelled insulin by isolated Zucker rat hepatocytes.

The metabolism of 125I-labelled insulin by hepatocytes isolated from 48-h-starved Zucker lean and obese rats was studied. Hepatocytes from the lean animals bound significantly more 125I-labelled insulin and had a greater receptor number per cell than did cells from obese littermates. Hepatocytes from the lean animals degraded and internalized more hormone than did those from obese ones. Increased degradation and internalization correlated with the increased receptor number.

Isolation and partial characterization of pancreatic polypeptide-like material in the brain of the blowfly Calliphora vomitoria.

Using 10(6) flies (5 kg of heads) a pancreatic polypeptide-like material has been partially purified from the blowfly Calliphora vomitoria. The isolated material was eluted on Sephadex G-50 similarly to bovine pancreatic polypeptide and had an RF on polyacrylamide-gel electrophoresis that was identical with that of the bovine hormone. The material diluted linearly and showed parallelism with bovine standards in a bovine pancreatic polypeptide immunoassay. In specificity controls the immunoreactivity was not abolished by trasylol and no cross-reactivity was discerned in assay for glucagon, proangiotensin and cyclic AMP. These data suggest that the pancreatic polypeptide material in the brain of the blowfly has close structural similarity to the mammalian hormone.

A monomeric insulin from the porcupine (Hystrix cristata), an Old World hystricomorph.

The insulins of New World hystricomorph rodents exhibit many novel amino acid changes in primary structure when compared with other mammalian insulins. These changes give rise to unusual properties (low potency, failure to self-associate) not shared by other naturally-occurring insulins. We report here on the primary structure, zinc-binding properties and circular dichroism (CD) of porcupine-insulin (Hystrix cristata), the first Old World hystricomorph insulin to be investigated, and discuss the changes in primary structure of the hormone in relation to its properties. Residue B22 is strongly implicated as being responsible for the unusual properties of porcupine insulin.