Gestational hyperglycemia reprograms cardiac gene expression in rat offspring.

BackgroundRat fetuses with maternal pregestational hyperglycemia develop cardiac dysfunction, and their cardiac gene expression differs from that of healthy control fetuses near term. We hypothesized that cardiac gene expression and morphologic abnormalities of rat fetuses with maternal pregestational hyperglycemia become normal after birth.MethodsNine rats were preconceptually injected with streptozotocin to induce maternal hyperglycemia and nine rats served as controls. The hyperglycemia group comprised 82 mice and the control group 74 offspring fed by euglycemic dams. Hearts of the offspring were collected on postnatal days 0, 7, and 14, and processed for histologic and gene expression analyses.ResultsOn day 0, heart weight was increased, and expression of cardiac genes involved in contractility, growth, and metabolism was decreased in the hyperglycemia group. On day 7, although cardiomyocyte apoptosis was enhanced, most of the changes in gene expression had normalized in the hyperglycemia group. By day 14, the expression of genes important for myocardial growth, function, and metabolism was again abnormal in the hyperglycemia group.ConclusionMost cardiac gene expression abnormalities become transiently normal during the first week of life of offspring to hyperglycemic rats. However, by day 14, cardiac expressions of genes involved in growth, function, and metabolism are again abnormal in relation to control offspring.

Physical Stability of Freeze-Dried Isomalt Diastereomer Mixtures.

PURPOSE: Isomalt is a sugar alcohol used as an excipient in commercially available solid oral dosage forms. The potential of isomalt as a novel freeze-drying excipient was studied in order to increase knowledge of the behavior of isomalt when it is freeze-dried. METHODS: Isomalt was freeze-dried in four different diastereomer compositions and its physical stability was investigated with differential scanning calorimetry, Fourier-transform infrared and Raman spectroscopy, X-ray powder diffraction, Karl-Fischer titration and thermogravimetric analysis in order to verify the solid state form of isomalt after freeze-drying and observe any changes occurring during storage in three different relative humidity conditions. RESULTS: Isomalt was successfully transformed into the amorphous form with freeze-drying and three diastereomer combinations remained stable as amorphous during storage; one of the diastereomer compositions showed signs of physical instability when stored in the highest relative humidity condition. The four different crystalline diastereomer mixtures showed specific identifiable solid state properties. CONCLUSIONS: Isomalt was shown to be a suitable excipient for freeze-drying. Preferably a mixture of the diastereomers should be used, as the mixture containing only one of the isomers showed physical instability. A mixture containing a 1:1 ratio of the two diastereomers showed the best physical stability in the amorphous form.

YKL-40 as a novel factor associated with inflammation and catabolic mechanisms in osteoarthritic joints.

YKL-40 is associated with tissue injury and inflammation, and consequently to diseases in which these mechanisms lead to tissue degradation, for example, asthma and rheumatoid arthritis. The purpose of the present study was to investigate if YKL-40 is also a significant factor in osteoarthritis (OA) by assessing associations of YKL-40 with mediators related to the pathogenesis of OA: cartilage destructing matrix metalloproteinases (MMPs) and proinflammatory cytokines interleukin-6 (IL-6) and interleukin-17 (IL-17). Cartilage, synovial fluid (SF), and plasma samples were obtained from 100 OA patients undergoing total knee replacement surgery. SF levels of YKL-40 (1027.9 ± 78.3 ng/mL) were considerably higher than plasma levels (67.2 ± 4.5 ng/mL) and correlated with YKL-40 released from cartilage samples obtained from the same patients (r = 0.37, P = 0.010), indicating that YKL-40 is produced by OA cartilage. Interestingly, YKL-40 concentrations in OA SF correlated positively with MMP-1 (r = 0.36, P = 0.014), MMP-3 (r = 0.46, P = 0.001), IL-6 (r = 0.57, P < 0.001), and IL-17 (r = 0.52, P = 0.010) levels. Moreover, IL-6 and IL-17 enhanced YKL-40 production in human primary chondrocyte cultures. The present study introduces YKL-40 as a cartilage-derived factor associated with mediators of inflammation and cartilage destruction involved in the pathogenesis of OA.

Maternal hyperglycemia leads to fetal cardiac hyperplasia and dysfunction in a rat model.

Accelerated fetal myocardial growth with altered cardiac function is a well-documented complication of human diabetic pregnancy, but its pathophysiology is still largely unknown. Our aim was to explore the mechanisms of fetal cardiac remodeling and cardiovascular hemodynamics in a rat model of maternal pregestational streptozotocin-induced hyperglycemia. The hyperglycemic group comprised 107 fetuses (10 dams) and the control group 219 fetuses (20 dams). Fetal cardiac function was assessed serially by Doppler ultrasonography. Fetal cardiac to thoracic area ratio, newborn heart weight, myocardial cell proliferative and apoptotic activities, and cardiac gene expression patterns were determined. Maternal hyperglycemia was associated with increased cardiac size, proliferative, apoptotic and mitotic activities, upregulation of genes encoding A- and B-type natriuretic peptides, myosin heavy chain types 2 and 3, uncoupling proteins 2 and 3, and the angiogenetic tumor necrosis factor receptor superfamily member 12A. The genes encoding Kv channel-interacting protein 2, a regulator of electrical cardiac phenotype, and the insulin-regulated glucose transporter 4 were downregulated. The heart rate was lower in fetuses of hyperglycemic dams. At 13-14 gestational days, 98% of fetuses of hyperglycemic dams had holosystolic atrioventricular valve regurgitation and decreased outflow mean velocity, indicating diminished cardiac output. Maternal hyperglycemia may lead to accelerated fetal myocardial growth by cardiomyocyte hyperplasia. In fetuses of hyperglycemic dams, expression of key genes that control and regulate cardiomyocyte electrophysiological properties, contractility, and metabolism are altered and may lead to major functional and clinical implications on the fetal heart.

Leptin enhances MMP-1, MMP-3 and MMP-13 production in human osteoarthritic cartilage and correlates with MMP-1 and MMP-3 in synovial fluid from OA patients.

OBJECTIVES: In the present study, we investigated the role of adipocytokine leptin in the pathogenesis of osteoarthritis (OA) by measuring its effects on matrix metalloproteinase (MMP) production in human OA cartilage. In addition, the correlations between leptin and MMP concentrations in synovial fluid from OA patients were studied. METHODS: Cartilage tissue obtained from leftover pieces of total knee replacement surgery from patients with OA was used in the experiments. Production of collagenases MMP-1, MMP-8 and MMP-13, and stromelysin-1 (MMP-3) in the cartilage was measured by immunoassay and the signalling pathways were explored by pharmacological means. In addition, synovial fluid samples were collected from 84 OA patients undergoing knee replacement surgery. The concentrations of leptin and MMPs in synovial fluid were measured by immunoassay. RESULTS: Leptin alone and in combination with IL-1ß enhanced production of MMP-1, MMP-3, and MMP-13 in human OA cartilage, while MMP-8 concentrations remained undetectable. The effects of leptin on MMP-1, MMP-3 and MMP-13 production were mediated through transcription factor NF-κß, and through protein kinase C and MAP kinase pathways. Interestingly, leptin concentrations in synovial fluid from OA patients correlated positively with MMP-3 (r=0.51, p<0.001) and MMP-1 (r=0.41, p<0.001) levels. CONCLUSIONS: To our knowledge, this is the first study to show that leptin up-regulates MMP-1 and MMP-3 production in human OA cartilage and correlates positively to MMP-1 and MMP-3 in synovial fluid from OA patients. The findings suggest that leptin has catabolic effects in OA joints by increasing MMP production in cartilage.

Leptin enhances synthesis of proinflammatory mediators in human osteoarthritic cartilage--mediator role of NO in leptin-induced PGE2, IL-6, and IL-8 production.

Obesity is an important risk factor for osteoarthritis (OA) in weight-bearing joints, but also in hand joints, pointing to an obesity-related metabolic factor that influences on the pathogenesis of OA. Leptin is an adipokine regulating energy balance, and it has recently been related also to arthritis and inflammation as a proinflammatory factor. In the present paper, the effects of leptin on human OA cartilage were studied. Leptin alone or in combination with IL-1 enhanced the expression of iNOS and COX-2, and production of NO, PGE(2), IL-6, and IL-8. The results suggest that the effects of leptin are mediated through activation of transcription factor nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathway c-Jun NH(2)-terminal kinase (JNK). Interestingly, inhibition of leptin-induced NO production with a selective iNOS inhibitor 1400 W inhibited also the production of IL-6, IL-8, and PGE(2), and this was reversed by exogenously added NO-donor SNAP, suggesting that the effects of leptin on IL-6, IL-8, and PGE(2) production are dependent on NO. These findings support the idea of leptin as a factor enhancing the production of proinflammatory factors in OA cartilage and as an agent contributing to the obesity-associated increased risk for osteoarthritis.

Central injection of oxytocin reduces food intake and affects hypothalamic and adipose tissue gene expression in chickens.

Oxytocin (OT) is a well-characterized neurotransmitter that participates in a wide range of physiological processes including the inhibition of food intake. The avian ortholog, mesotocin (MT), differs from OT by a single amino acid. Little is known regarding the function of OT in regulating energy balance in birds; thus, this study was designed to determine the effects of central OT injection on food intake and adipose tissue physiology in chicks. At 4-d post-hatch, broiler chicks were fasted for 3 h and injected intracerebroventricularly with 0 (vehicle), 0.63, 2.5, 5.0, or 10 nmol OT. Oxytocin decreased food and water intake during the entire 180-min observation period. The reduction in water intake was likely not prandial because chicks that were food restricted after OT injection also drank less. There was increased c-Fos immunoreactivity in several appetite-associated hypothalamic nuclei in OT-injected chicks at 1 h, including the arcuate (ARC), dorsomedial nucleus (DMN), lateral hypothalamus (LH), paraventricular nucleus (PVN), and ventromedial hypothalamus (VMH). OT treatment was associated with reduced hypothalamic corticotropin-releasing factor (CRF) mRNA and increased cloacal temperature at 1 h post-injection. We then investigated appetite- and adipose tissue-associated effects of OT in chicks from lines that have undergone long-term selection for either low (LWS) or high (HWS) juvenile body weight. Central injection of OT decreased food intake in both lines with the magnitude of response greater in the HWS than LWS chicks. Adipose tissue abundance of fatty acid-binding protein 4, monoglyceride lipase (MGLL), MT, and perilipin-1 mRNA was greater in LWS than HWS chicks. Lipoprotein lipase, MGLL, and MT mRNAs increased in response to OT injection in LWS but not HWS chicks. In conclusion, central injection of OT induced anorexia, reduced water intake, increased body temperature, and was associated with activation of the ARC, DMN, LH, PVN, and VMH in the hypothalamus. The effects on appetite and body temperature may involve CRF signaling in the hypothalamus and lipolysis in the adipose tissue, respectively. There were differences in the appetite, and adipose tissue response to OT in body weight-selected lines of chicks supports that MT plays a role in energy balance regulation in chickens.

Leptin - a link between obesity and osteoarthritis. applications for prevention and treatment.

Osteoarthritis (OA) is the most common cause of musculoskeletal disability and pain in the world. The current drug treatment for OA is symptom relieving, and there is an urgent need for treatments that could retard, prevent or repair cartilage destruction in OA. Obesity is a major risk factor for OA. Traditionally, it has been thought to contribute to the development of OA by increasing the load on weight-bearing joints. However, this appears to be an over-simplification, because obesity is also linked to OA in the hand and finger joints. Recent studies have shown that adipocytokine leptin is a possible link between obesity and OA: Leptin levels in synovial fluid are increased in obese patients, leptin receptor (Ob-R) is expressed in cartilage, and leptin induces the production of matrix metalloproteinases (MMPs), pro-inflammatory mediators and nitric oxide (NO) in chondrocytes. Furthermore, according to the very recent findings, not only leptin levels in the joint but also leptin sensitivity in the cartilage are enhanced in obese OA patients. The findings supporting leptin as a causative link between obesity and OA offer leptin as a potential target to the development of disease-modifying drugs for osteoarthritis (DMOAD), especially for obese patients.

Delay in rat lung alveolarization after the combined exposure of maternal hyperglycemia and postnatal hyperoxia.

BACKGROUND: Maternal diabetes interferes with fetal lung development and postnatal treatments may further disturb pulmonary growth. Therefore, we investigated the effect of postnatal oxygen exposure on alveolar development in neonatal rat lungs pre-exposed to intrauterine hyperglycemia. METHODS: Diabetes was induced in Sprague-Dawley rats with streptozotocin injection before pregnancy. Hyperglycemia-exposed and control litters were randomized to breath room air or 85% oxygen for 7 days after birth. Lungs were analyzed on postnatal d7 for weight, morphology, apoptosis, proliferation, and biomarkers of oxidative stress. RESULTS: Maternal hyperglycemia accelerated lung development as demonstrated by thinner alveolar walls and slightly increased secondary septation when compared to room air bred rats. Hyperoxia alone caused thin-walled and enlarged alveoli with few secondary septa. Interestingly, the dual exposure inhibited the thinning of alveolar walls and the disappearance of mesenchymal cells from the alveolar walls together with the delay in the formation of alveoli and secondary crests. While the lungs' oxidative stress was similar in all groups, pulmonary apoptosis and proliferation were altered. CONCLUSION: Our results thus indicate that the hyperglycemic priming of the fetal lung modifies the deleterious effect of hyperoxia on alveolarization in neonatal rats.

Adipokines as drug targets in joint and bone disease.

White adipose tissue is now recognized to be a multifactorial organ secreting several adipose-derived factors that have been collectively termed 'adipokines'. Adipokines are pleiotropic molecules that contribute to the so-called 'low-grade inflammatory state' of obese subjects creating a cluster of metabolic aberrations including autoimmune and inflammatory diseases that affect joints and bone. The aim of this review is to present knowledge about the role of adipokines in bone and cartilage function, as well as in inflammatory and degenerative joint disease. We discuss clinical implications and then survey attempts to exploit this role for therapeutic gain, which holds potential as a novel approach for drug development in bone and joint disease.

Fetal hyperglycemia alters lung structural development in neonatal rat.

Maternal diabetes is associated with increased risk for abnormal fetal organogenesis, but its effects on the developing lungs are still insufficiently known. To determine the effect of maternal hyperglycemia on postnatal lung development, we studied lung structural and cellular changes in newborn rats exposed to intrauterine hyperglycemia. We induced hyperglycemia in Sprague-Dawley rats with i.p. streptozotocin before pregnancy and allowed the hyperglycemic and control dams deliver at term. Lungs were obtained on postnatal day (d) 0, d7, and d14 and analyzed for lung weight and morphology, as well as cellular apoptosis (TUNEL staining) and proliferation (PCNA staining). Quantitative micro-CT analysis of the lung vasculature was additionally performed at d14. At birth, maternal hyperglycemia resulted in decreased relative lung weight, thinner alveolar septa and increased cellular apoptosis and proliferation, when compared to controls. At 1 and 2 weeks of age pulmonary cell apoptosis and alveolar chord length remained unchanged, but cell proliferation and number of secondary crests were increased in the hyperglycemia-exposed neonatal lungs in comparison with the controls. Density of small arterioles on histological examination and the structure of pulmonary arterial vasculature in micro-CT analysis of the neonatal lungs were not influenced by maternal hyperglycemia. Our results suggest, that maternal hyperglycemia is related to developmental structural alterations in postnatal rat lungs. These early changes may reflect aberrant maturational adaptation in response to the hyperglycemic fetal environment.

Adiponectin associates with markers of cartilage degradation in osteoarthritis and induces production of proinflammatory and catabolic factors through mitogen-activated protein kinase pathways.

INTRODUCTION: Adiponectin is an adipokine that regulates energy metabolism and insulin sensitivity, but recent studies have pointed also to a role in inflammation and arthritis. The purpose of the present study was to investigate the association and effects of adiponectin on inflammation and cartilage destruction in osteoarthritis (OA). METHODS: Cartilage and blood samples were collected from 35 male OA patients undergoing total knee replacement surgery. Preoperative radiographs were evaluated using Ahlbäck classification criteria for knee OA. Circulating concentrations of adiponectin and biomarkers of OA, that is, cartilage oligomeric matrix protein (COMP) and matrix metalloproteinase 3 (MMP-3), were measured. Cartilage samples obtained at the time of surgery were cultured ex vivo, and the levels of adiponectin, nitric oxide (NO), IL-6, MMP-1 and MMP-3 were determined in the culture media. In addition, the effects of adiponectin on the production of NO, IL-6, MMP-1 and MMP-3 were studied in cartilage and in primary chondrocyte cultures. RESULTS: Plasma adiponectin levels and adiponectin released from OA cartilage were higher in patients with the radiologically most severe OA (Ahlbäck grades 4 and 5) than in patients with less severe disease (Ahlbäck grades 1 to 3). Plasma adiponectin concentrations correlated positively with biomarkers of OA, that is, COMP (r = 0.55, P = 0.001) and MMP-3 (r = 0.34, P = 0.046). Adiponectin was released by OA cartilage ex vivo, and it correlated positively with production of NO (r = 0.43, P = 0.012), IL-6 (r = 0.42, P = 0.018) and MMP-3 (r = 0.34, P = 0.051). Furthermore, adiponectin enhanced production of NO, IL-6, MMP-1 and MMP-3 in OA cartilage and in primary chondrocytes in vitro in a mitogen-activated protein kinase (MAPK)-dependent manner. CONCLUSIONS: The findings of this study show that adiponectin is associated with, and possibly mediates, cartilage destruction in OA.

Maternal hyperglycemia modifies extracellular matrix signaling pathways in neonatal rat lung.

BACKGROUND: Maternal diabetes is associated with numerous adverse effects in fetal and neonatal organs, including the lungs. OBJECTIVE: To investigate the effects of intrauterine hyperglycemia on neonatal lung biological signaling, we performed a microarray analysis in the lungs of four 14-day-old rat pups born to a hyperglycemic dam and in four age mate control pup lungs. METHODS: Total RNA was isolated and cDNA was hybridized to the Illumina Sentrix® RatRef-12 BeadChip. A total of 22,000 genes were analyzed for expression profiles and functional gene clustering. Ten selected genes differentially expressed in microarray were additionally analyzed by the real-time polymerase chain reaction. RESULTS: Two hundred twenty-seven genes were differentially expressed in neonatal rat lungs exposed to intrauterine hyperglycemia when compared to normoglycemic controls (fold change > 1.2, p < 0.001). Functional clustering analysis revealed increased expression in signaling pathways involved with extracellular matrix regulation. The most significantly downregulated functions were cell proliferation, extracellular region, cell adhesion and reactive oxygen species metabolism. CONCLUSION: We found significant hyperglycemia-induced gene expression alterations in neonatal rat pulmonary tissue which may interfere with lung growth and biological signaling pathways.