Differential effects of altered patterns of movement and strain on joint cell behaviour and skeletal morphogenesis.

OBJECTIVE: There is increasing evidence that joint shape is a potent predictor of osteoarthritis (OA) risk; yet the cellular events underpinning joint morphogenesis remain unclear. We sought to develop a genetically tractable animal model to study the events controlling joint morphogenesis. DESIGN: Zebrafish larvae were subjected to periods of flaccid paralysis, rigid paralysis or hyperactivity. Immunohistochemistry and transgenic reporters were used to monitor changes to muscle and cartilage. Finite Element Models were generated to investigate the mechanical conditions of rigid paralysis. Principal component analysis was used to test variations in skeletal morphology and metrics for shape, orientation and size were applied to describe cell behaviour. RESULTS: We show that flaccid and rigid paralysis and hypermobility affect cartilage element and joint shape. We describe differences between flaccid and rigid paralysis in regions showing high principal strain upon muscle contraction. We identify that altered shape and high strain occur in regions of cell differentiation and we show statistically significant changes to cell maturity occur in these regions in paralysed and hypermobile zebrafish. CONCLUSION: While flaccid and rigid paralysis and hypermobility affect skeletal morphogenesis they do so in subtly different ways. We show that some cartilage regions are unaffected in conditions such as rigid paralysis where static force is applied, whereas joint morphogenesis is perturbed by both flaccid and rigid paralysis; suggesting that joints require dynamic movement for accurate morphogenesis. A better understanding of how biomechanics impacts skeletal cell behaviour will improve our understanding of how foetal mechanics shape the developing joint.

Within-guild dietary discrimination from 3-D textural analysis of tooth microwear in insectivorous mammals.

Resource exploitation and competition for food are important selective pressures in animal evolution. A number of recent investigations have focused on linkages between diversification, trophic morphology and diet in bats, partly because their roosting habits mean that for many bat species diet can be quantified relatively easily through faecal analysis. Dietary analysis in mammals is otherwise invasive, complicated, time consuming and expensive. Here we present evidence from insectivorous bats that analysis of three-dimensional (3-D) textures of tooth microwear using International Organization for Standardization (ISO) roughness parameters derived from sub-micron surface data provides an additional, powerful tool for investigation of trophic resource exploitation in mammals. Our approach, like scale-sensitive fractal analysis, offers considerable advantages over two-dimensional (2-D) methods of microwear analysis, including improvements in robustness, repeatability and comparability of studies. Our results constitute the first analysis of microwear textures in carnivorous mammals based on ISO roughness parameters. They demonstrate that the method is capable of dietary discrimination, even between cryptic species with subtly different diets within trophic guilds, and even when sample sizes are small. We find significant differences in microwear textures between insectivore species whose diet contains different proportions of 'hard' prey (such as beetles) and 'soft' prey (such as moths), and multivariate analyses are able to distinguish between species with different diets based solely on their tooth microwear textures. Our results show that, compared with previous 2-D analyses of microwear in bats, ISO roughness parameters provide a much more sophisticated characterization of the nature of microwear surfaces and can yield more robust and subtle dietary discrimination. ISO-based textural analysis of tooth microwear thus has a useful role to play, complementing existing approaches, in trophic analysis of mammals, both extant and extinct.

Finite element modelling of squirrel, guinea pig and rat skulls: using geometric morphometrics to assess sensitivity.

Rodents are defined by a uniquely specialized dentition and a highly complex arrangement of jaw-closing muscles. Finite element analysis (FEA) is an ideal technique to investigate the biomechanical implications of these specializations, but it is essential to understand fully the degree of influence of the different input parameters of the FE model to have confidence in the model's predictions. This study evaluates the sensitivity of FE models of rodent crania to elastic properties of the materials, loading direction, and the location and orientation of the models' constraints. Three FE models were constructed of squirrel, guinea pig and rat skulls. Each was loaded to simulate biting on the incisors, and the first and the third molars, with the angle of the incisal bite varied over a range of 45°. The Young's moduli of the bone and teeth components were varied between limits defined by findings from our own and previously published tests of material properties. Geometric morphometrics (GMM) was used to analyse the resulting skull deformations. Bone stiffness was found to have the strongest influence on the results in all three rodents, followed by bite position, and then bite angle and muscle orientation. Tooth material properties were shown to have little effect on the deformation of the skull. The effect of bite position varied between species, with the mesiodistal position of the biting tooth being most important in squirrels and guinea pigs, whereas bilateral vs. unilateral biting had the greatest influence in rats. A GMM analysis of isolated incisor deformations showed that, for all rodents, bite angle is the most important parameter, followed by elastic properties of the tooth. The results here elucidate which input parameters are most important when defining the FE models, but also provide interesting glimpses of the biomechanical differences between the three skulls, which will be fully explored in future publications.

Shape and mechanics in thalattosuchian (Crocodylomorpha) skulls: implications for feeding behaviour and niche partitioning.

Variation in modern crocodilian and extinct thalattosuchian crocodylomorph skull morphology is only weakly correlated with phylogeny, implying that factors other than evolutionary proximity play important roles in determining crocodile skull shape. To further explore factors potentially influencing morphological differentiation within the Thalattosuchia, we examine teleosaurid and metriorhynchid skull shape variation within a mechanical and dietary context using a combination of finite element modelling and multivariate statistics. Patterns of stress distribution through the skull were found to be very similar in teleosaurid and metriorhynchid species, with stress peaking at the posterior constriction of the snout and around the enlarged supratemporal fenestrae. However, the magnitudes of stresses differ, with metriorhynchids having generally stronger skulls. As with modern crocodilians, a strong linear relationship between skull length and skull strength exists, with short-snouted morphotypes experiencing less stress through the skull than long-snouted morphotypes under equivalent loads. Selection on snout shape related to dietary preference was found to work in orthogonal directions in the two families: diet is associated with snout length in teleosaurids and with snout width in metriorhynchids, suggesting that teleosaurid skulls were adapted for speed of attack and metriorhynchid skulls for force production. Evidence also indicates that morphological and functional differentiation of the skull occurred as a result of dietary preference, allowing closely related sympatric species to exploit a limited environment. Comparisons of the mechanical performance of the thalattosuchian skull with extant crocodilians show that teleosaurids and long-snouted metriorhynchids exhibit stress magnitudes similar to or greater than those of long-snouted modern forms, whereas short-snouted metriorhynchids display stress magnitudes converging on those found in short-snouted modern species. As a result, teleosaurids and long-snouted metriorhynchids were probably restricted to lateral attacks of the head and neck, but short-snouted metriorhynchids may have been able to employ the grasp and shake and/or 'death roll' feeding and foraging behaviours.

Virus-induced alterations in cyclic adenosine monophosphate generation in hamster islets of Langerhans.

Inoculation of golden Syrian hamsters with Venezuelan encephalitis (VE) virus results in a sustained diminution in glucose-stimulated insulin release that is correctable by cyclic (c) AMP analogs and phosphodiesterase inhibitors. This suggested the importance of directly measuring cAMP content in VE-infected and control islets in response to insulin secretagogues. The basal cAMP content of VE-infected islets (0.14 +/- 0.02 pmol/micrograms islet DNA) was approximately half that of control islets (0.27 +/- 0.02 pmol/micrograms islet DNA) (P less than 0.05). In the presence of 10 microM glucagon (and 3 mM glucose), the rate of cAMP generation in VE-infected islets was only half that of control islets. With 10 mM alpha-ketoisocaproic acid, the rates of cAMP generation were indistinguishable between control and experimental groups. In response to 20 mM glucose and 3-isobutyl-1-methylxanthine (IBMX) (a phosphodiesterase inhibitor), cAMP generation in VE-infected islets was 81% (NS) of the control rate. When a more specific phosphodiesterase inhibitor, RO 20-1724, was used with 20 mM glucose, cAMP generation in the infected islets was only 44% (P less than 0.001) of the control value. Insulin secretion over the perifusion period paralleled the cAMP levels. In the presence of 10 mM alpha-ketoisocaproic acid, there was no difference in insulin secretion between VE-infected and control islets, while there was a statistically significant (P less than 0.05) difference with 10 microM glucagon or 20 mM glucose (in 1 mM RO 20-1724). These data point to a defect in the cAMP generation system of VE-infected islets, although additional factors involved in insulin secretion may also be impaired by the virus.

Virus-induced alterations in insulin release in hamster islets of Langerhans.

After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.

Rubella virus-induced diabetes in the hamster.

The congenital rubella syndrome provides the best documentation in humans that a viral infection is associated with the subsequent development of insulin-dependent diabetes mellitus. We have developed an animal model in neonatal golden Syrian hamsters infected with rubella virus passaged in beta-cells that closely parallels the diabetes observed with congenital rubella. The hamsters develop hyperglycemia and hypoinsulinemia, which are sustained throughout the 15-wk study period. A mononuclear infiltration of the islets, isolation of rubella virus from whole pancreas, the presence of viral antigen in beta-cells by immunofluorescent localization, and cytoplasmic islet cell antibodies (40%) are demonstrated. These data suggest that an autoimmune process and diabetes develop after rubella virus infection in neonatal hamsters. This model may uncover the precise mechanism by which rubella virus induces similar disease in humans.

Virus-induced pancreatic disease by Venezuelan encephalitis virus. Alterations in glucose tolerance and insulin release.

Viral infections have been implicated in the induction of diabetes mellitus in man and laboratory animals. Since virus-specific immunofluorescence (FA) is detectable in hamster pancreas during the acute phase of Venezuelan encephalitis (VE), experiments were designed to correlate pathologic and virologic events with metabolic studies in VE-infected hamsters. Golden Syrian hamsters were inoculated s.c. in groups of four to 12 with 100,000 plaque-forming units (PFU) of the vaccine strain (TC-83) of VE or 1,000 PFU of the virulent Trinidad strain of VE. Ultrastructurally, during Trinidad infection, mature virions were associated with the cell surfaces and within pancreatic beta cells in contrast to absence of virus-related changes in TC-83-infected hamsters. Virus-specific-FA was noted in islet cells and acinar cells of Trinidad-infected hamsters. VE growth curves demonstrated viral replication in pancreas with both strains. Although ultrastructural and FA changes were much more prominent in Trinidad-infected hamsters in contrast to TC-83-infected hamsters during the first few days of illness, the rapid lethality of the Trinidad-infected group necessitated performing all metabolic studies in TC-83-strain-infected hamsters. Accordingly, for the metabolic studies, glucose tolerance tests (GTT) using 2 mg. or 5 gm./kg. glucose i.p. were performed in groups of hamsters acutely infected two days earlier with the TC-83 vaccine strain and in 24-day and 90-day convalescent hamsters after TC-83 vaccine strain. Samples were obtained for glucose and immunoreactive insulin (IRI) determinations. Glucose intolerance occurred in hamsters in each of the infected groups given 5 gm./kg. glucose except for the 90-day convalescent TC-83 group. Severely decreased IRI responses occurred in the 24-day and 90-day convalescent TC-83 hamsters following both 2- and 5-gm./kg. glucose. Pancreatic IRI content in 24-day convalescent TC-83 hamsters was within normal limits, suggesting a defect in IRI release from the beta cells at this stage of convalescence.

Relationship between serum chromium concentrations and glucose utilization in normal and infected subjects.

Studies in healthy individuals demonstrate that serum chromium concentrations fall precipitiously following the intravenous administration of a 30-gm. glucose load. Significant decreases from baseline control fasting serum Cr concentrations were also observed when intravenous glucose was given during sandfly fever. Glucose disappearance rates also decreased significantly to approximately one half of pre-illness control values while serum Cr values declined still further. In addition, serum Cr disappearance rates could be calculated. When individual preexposure and postexposure serum glucose and Cr disappearance rates were compared, significant linear correlation was found (P smaller than 0.05). Acute infection appears to reduce the availability of circulating Cr, which may contribute to the altered glucose metabolism characteristic of acute infections even in the presence of elevated insulin levels and other hormonal changes.

HLA antigens, cytoplasmic islet cell antibodies, and carbohydrate tolerance in families of children with insulin-dependent diabetes mellitus.

Cytoplasmic pancreatic islet cell antibodies were found in 21% of 244 unaffected first degree relatives of type I diabetic patients. Twenty-five percent of HLA-identical, 35% of HLA-haploidentical, 16% of HLA-nonidentical siblings, and 14% of parents were ICA-positive. In the HLA-identical sibs, irrespective of ICA, and in the 18 ICA-positive parents but not the other groups, increased plasma glucose levels were observed after the administration of glucose. In most children, these were associated with reduced insulin levels, while in the adults elevated insulin responses were noted. In 48% of the ICA-positive children and 84% of the ICA-positive parents, other evidence of "autoimmunity" was obtained either by history or by testing for specific autoantibodies. Two of the originally unaffected HLA-identical and ICA-positive siblings developed diabetes during the course of the study. These findings, plus previously reported data in families with two diabetic sibs demonstrating that the empiric risk for developing IDDM is of the order of 30% for HLA-identical sibs but less than 5% for those that are HLA-haploidentical, suggest that HLA-identity may be a useful predictor of potential type I diabetes. The presence of ICA may, at times, portend the need for future antidiabetic therapy but prospective studies must be continued to fully elucidate this relationship.

Potential mechanisms promoting restenosis in diabetic patients.

Diabetes is associated with greater restenosis rates after successful balloon angioplasty. The metabolic alterations that occur as a result of hyperglycemia or hyperinsulinemia can accelerate many of the pathophysiologic processes that lead to restenosis. Diabetes results in endothelial dysfunction and accelerated platelet deposition, which increase the propensity to thrombosis. Several growth factors known to promote the restenosis process are overexpressed in the presence of hyperglycemia. Advanced glycosylation promotes inflammatory cell recruitment and smooth muscle cell proliferation. Many of the potential mechanisms promoting restenosis in diabetic patients can be ameliorated by improved metabolic control.

Studies of carbohydrate metabolism after hemodialysis and hemofiltration in uremic patients.

To assess the effects of dialysis or hemofiltration on carbohydrate metabolism in uremia, we performed intravenous (IV) glucose tolerance tests (IV GTTs) after an overnight fast 48 hours following the last treatment in ten patients with chronic renal failure. Samples were obtained for plasma glucose, insulin, glucagon, and growth hormone levels throughout the GTTs in addition to basal samples for levels of plasma potassium and bicarbonate. The IV GTTs were performed at the end of a four-month period of standard hemodialysis (period 1) and then at the end of a four-month period of hemofiltration (period 2). Patients had mild glucose intolerance that did not change after hemofiltration, although the exaggerated insulin responses to glucose administration did significantly decrease in period 2. The fasting hyperglucagonemia did not decrease after hemofiltration but exhibited normal suppression with IV glucose. Levels of basal plasma bicarbonate and basal plasma potassium did not change significantly in period 2. Further studies investigating the beneficial metabolic effect of hemofiltration would seem to be indicated based on the data reported herein.

Localizaiton of insulinomas by radioimmunoassay of blood obtained by the transport route.

Transhepatic catheterization of the portal system was performed to obtain blood for RIA of insulin. This technique localized insulinomas in two patients after negative celiac, superior mesenteric, and subselective gastroduodenal arteriograms. The tumors were found in the predicted locations at surgery.

Nonautonomous function of a pancreatic insulinoma.

A 56-year-old woman with symptoms of weakness, visual blurring, and sweating underwent diagnostic studies to evaluate the etiology of her hypoglycemia. Fasting hypoglycemia was never documented; in diagnostic studies performed during her two hospitalizations and several outpatient glucose tolerance tests, the lowest fasting plasma glucose recorded was 56 mg/dl. The patient displayed exaggerated plasma insulin responses following oral glucose (peak response: 447 muU/ml at 30 min) and following 1 gm of iv tolbutamide (peak response: 719 muU/ml at 5 min) with symptomatic profound hypoglyceria during both tests. Basal per cent proinsulin was elevated at 49% (normal range 5-22%). Throughout a 72 h fast, values for plasma glucose, insulin, and glucose/insulin ratios were all within the normal range. During the infusion of exogenous insulin (0.1 U/kg for 60 min) serum C-peptide reactivity suppressed to less than 1.3 ng/ml when the plasma glucose fell below 40 mg/dl representing normal suppression. At surgery, a pancreatic beta cell adenoma was found and removed. This patient represents the uncommon circumstances in which stimulation tests with tolbutamide and glucose were more helpful in establishing a preoperative diagnosis than were the suppression tests.

Diabetes, sleep disorders, and male sexual function.

The prevalence and role of sleep disorders in the sexual problems of diabetic patients remain unexplored. This study was conducted on 40 diabetic men carefully screened to exclude the confounding effects of other medical illnesses or drugs likely to impair sexual function and 40 age-matched healthy volunteers. They underwent an extensive psychosexual interview, medical and psychiatric evaluations, and three recorded nights in a sleep laboratory. Electroencephalogram, eye movements, muscle tone, and nocturnal penile tumescence were monitored continuously. Respiratory airflow and bilateral anterior tibialis recordings were obtained during the first sleep session. Diabetic men had significantly higher prevalences of respiratory and periodic leg movement disturbances during sleep. There was clinical, although not nocturnal penile tumescence, evidence suggesting that respiratory abnormalities during sleep are associated with erectile difficulties in diabetic men. Future studies should include blood oxygenation and respiratory effort measures to clarify the significance of sleep-related airflow disturbances in diabetic patients.

Effect of glucose infusion on the concentration of individual serum free amino acids during sandfly fever in man.

After an intravenous glucose load in man, total serum amino acid concentrations are rapidly depressed and remain below baseline values for at least 2 to 3 hr after serum glucose and insulin have returned to preload concentrations. Despite the presence of basal hypoaminoacidemia, a decreased glucose disappearance rate, and hyperinsulinemia in volunteers who were ill with sandfly fever, an intravenous glucose load resulted in a further depression of serum amino acids which was equal to or slightly greater than that observed in the same individuals before exposure to the virus. Although the infectious process may have some effect on insulin-stimulated hepatic disposal of a glucose load, it does not appear to influence the ability on insulin to decrease the rate of release of certain amino acids from skeletal muscle.

Disopyramide (Norpace)-induced hypoglycemia.

Disopyramide (Norpace) is a recently released antiarrhythmic agent with quinidine-like actions, but structurally unique. We describe a patient in whom impressive hypoglycemia developed following treatment with this agent. Blood glucose levels returned to normal after cessation of therapy, but dropped again following rechallenge with the drug. The pathogenesis of the hypoglycemia was investigated by assessment of serum insulin, plasma glucagon and serum alanine levels during disopyramide rechallenge. Clinicians should be aware of fasting hypoglycemia as an unusual but potentially serious complication of disopyramide therapy.

Patterns of foot examination in a diabetes clinic.

Peripheral vascular disease is a well-known source of morbidity and potential mortality in diabetic patients. Sixty-five subjects with diabetes were studied in order to describe the contributions of the patient, the physician, and the health care system to the performance of a preventive foot examination. The incidences of foot examination on the day of study (12.3 percent) and during the previous year (49.2 percent) were similar to those found by other investigators. The feet of patients at higher risk for limb amputation were not examined with greater frequency at the clinic, although such patients were more often referred to a podiatrist. The most significant determinants of physician foot examination were patient recall of foot-related education received at the clinic and inter-physician variability. These data suggest that the patient, physician, and clinic routine all play an important role in the success of a foot screening program for patients with diabetes.

Infection and diabetes: the case for glucose control.

This review summarizes data concerning the host resistance to infection in diabetes and the influence of an acute infection upon the endocrinologic-metabolite status of the diabetic patient. While it is well known that acute infections lead to difficulty in controlling blood sugar levels and the infection is the most frequently documented cause of ketoacidosis, controversy persists as to whether or not patients with diabetes mellitus are more susceptible to infection than age- and sex-matched nondiabetic control subjects. Our data obtained from the charts of 241 diabetic patients who were being followed as outpatients show a striking direct correlation between the overall prevalence of infection (p less than 0.001) and the mean plasma glucose levels (representing three or more fasting glucose determinations taken at times when no evidence of infection existed). There is a significant diminution in intracellular bactericidal activity of leukocytes with Staphylococcus aureus and Escherichia coli in subjects with poorly controlled diabetes in comparison with the control group. Serum opsonic activity for both Staph. Aureus and E. coli were significantly lower than in the control subjects. Taken together, the results from published reports as well as our data suggest to us that good control of blood sugar in diabetic patients is a desirable goal in the prevention of certain infections (Candida vaginitis, for example) and to ensure maintenance of normal host defense mechanisms that determine resistance and response to infection.

Elevated hemoglobin A1c and low-density lipoprotein cholesterol levels in thiazide-treated diabetic patients.

Despite the well-known hyperglycemic effect of thiazide diuretics, these agents are often administered to diabetic patients. This study compared 89 insulin-treated diabetic patients receiving hydrochlorothiazide, 57 receiving furosemide, and 255 receiving no diuretic. Hemoglobin A1c level was 7.2 +/- 1.8 percent (mean +/- SD) with hydrochlorothiazide, significantly higher than the levels of 5.9 +/- 2.3 percent with furosemide and 6.4 +/- 2.0 percent with no diuretic. Low-density lipoprotein cholesterol level was 154 +/- 43 mg/dl with hydrochlorothiazide, but 134 +/- 42 mg/dl with furosemide and 130 +/- 42 mg/dl with no diuretic. Multivariate analysis showed that the associations remained significant after adjustment for age, sex, race, type and duration of diabetes, body mass index, blood pressure, serum potassium level, insulin dose, and treatment with other medications. These findings suggest that treatment with thiazide diuretics in the diabetic population may increase low-density lipoprotein cholesterol and hemoglobin A1c levels.