A morphometric study of human submandibular gland in type 2 diabetic status

Diabetes Mellitus Type 2 represents one of the principal diseases that afflict the world population. It is well documented that diabetes affects both morphology and function of several organs. In diabetic rats significant structural changes have been demonstrated in salivary glands, such as accumulation of secretory material and lipid droplets within secretory cells, parenchymal degeneration and its replacement with fibrous connective tissue (1). With regard to human salivary glands, the data are scanty and conflicting. Our work, carried out by light and electron microscopy, is based on the evaluation of the morphological changes which occur in human submandibular glands of diabetic with respect to non diabetic patients. Surgical fragments of glandular tissue were fixed, dehydrated, and processed for light and electron microscopy. Randomly chosen images were analyzed with Image Pro Plus software to record the dimension of acini, serous cells, secretory granules and other variables. Data were analyzed by Student’s t-test and Mann Whitney test. In diabetic glands statistically significant morphological changes were observed, such as enlargement of serous acini and increase of secretory granules area. These results suggest that the secretory activity of human submandibular gland is severely affected by the diabetic status. Obviously these data need to be confirmed with further measurements in order to explain better how diabetes affects human salivary glands

Dynamics of the melatonin MT1 receptor in the rat parotid gland upon melatonin administration

Our recent ultrastructural study of human parotid glands revealed that the melatonin receptors, MT1 and MT2, are localised in the plasma cell membranes of acinar and ductal cells but also, and intriguingly, predominantly in acinar secretory granules, giving rise to the working hypothesis that secretory granules are a part of a transcytotic transport system for melatonin. To put this hypothesis to the test in rat parotid glands, anaesthetised animals were exposed to a high melatonin dose (3 mg/kg per hour), infused intravenously over two hours and aiming to stimulate a glandular melatonin-receptor-dependent intracellular transport system, if any. Thirty minutes later, the right parotids were removed. Pre-stimulation, left parotid gland tissue was removed to serve as (untreated) controls. Gland tissues were processed for the gold post-embedding technique and for western blot analysis. In untreated glands, on transmission electron microscope images, melatonin receptors displayed a distribution pattern similar to that in human parotids, i.e. here, too, the receptors were principally associated with the acinar secretory granules. In melatonin- treated glands, the number of granules associated with the MT1 receptor was twice that in untreated glands, despite the same total granule number in the two glands. Moreover, the density of gold particles showing MT1-receptor immunoreactivity associated with granules in melatonin-treated glands was 2.5 times that in untreated glands. The number of MT1 receptors associated with the granule membrane was about three times higher in melatonin-treated glands than in untreated glands, while the number of MT1 receptors inside the granules was about twice that in untreated glands. The immunoblotting of membrane-enriched samples showed that the MT1-receptor expression was about three times that of untreated glands. When it came to the MT2 receptor, no changes were observed. Melatonin itself thus exerts dynamic effects on its MT1 receptor, which may reflect an adaptive receptor-linked carrier system for melatonin, delivering - upon gland stimulation - melatonin to the saliva by exocytosis

Melatonin ultrastructural localization in mitochondria of human salivary glands

The hormone melatonin was initially believed to be synthesized exclusively by the pineal gland and the enterochromaffin cells, but nowadays its production and distribution were observed in several other tissues and organs. Among others, the ultrastructural localization of melatonin and its receptors has been reported in human salivary glands. In these glands, the fine localization of melatonin in intracellular organelles, above all in mitochondria, remains to be explored comprehensively. Bioptic samples of parotid and submandibular glands were treated to search for melatonin using the immunogold staining method by transmission electron microscopy. Morphometric analysis was applied to micrographs. The results indicated that, both in parotid and submandibular glands mitochondria, a certain melatonin positivity was present. Within glandular cells, melatonin was less retrieved in mitochondria than in secretory granules; however, its presence in this organelle was clearly evident. Inside striated duct cells, melatonin staining in mitochondria was more prominent than in glandular cells. Our data provide an ultrastructural report on the presence of melatonin in mitochondria of human major salivary glands and represent a fundamental prerequisite for a better understanding of the melatonin role in this organelle

Ultrastructural evidence of a secretory role for melatonin in the human parotid gland

In vivo animal studies show that pentagastrin, cholecystokinin and melatonin cause the secretion and synthesis of salivary proteins. Melatonin occurs in large amounts in the gut and is released into the blood on food intake. In vitro experiments suggest that pentagastrin exerts secretory activity in human salivary glands, as judged by ultrastructural changes, reflecting secretion, and an actual protein output. Currently, it is hypothesised that melatonin induces secretory exocytotic events in the human parotid gland. Human parotid tissues were exposed to a high single concentration of melatonin in vitro, processed for high resolution scanning electron microscopy and then assessed morphometrically with the emphasis on the membrane of the intercellular canaliculi, a site of protein secretion. Compared with controls and in terms of density, the melatonin-exposed parotid tissues displayed increases in protrusions (signalling anchored granules) and microbuds (signalling membrane recycling and/or vesicle secretion) and decreases in microvilli (signalling cytoskeletal re-arrangement related to exocytosis), phenomena abolished or very largely reduced by the melatonin receptor blocker, luzindole. In conclusion, acinar serous cells of parotid tissue displayed in vitro exocytotic activity to melatonin, signalling protein secretion. Whether, under physiological conditions, melatonin influences the secretion of human parotid glands remains to be explored, however

Diabetes induces changes in salivary gland melatonin reactivity

The fine localization of melatonin and its receptors in the human salivary glands were reported in our previous works revealing, by transmission electron microscopy (TEM), that serous cells are able to store melatonin and to secrete it by regulated pathways (1, 2). Moreover, changing in morphology during secretion was observed after melatonin treatment by high resolution scanning electron microscopy (3). As in saliva of patients suffering from type 2 diabetes melatonin was reduced, we focused our study on salivary glands removed from diabetic subjects, in order to add diabetic data to our survey on melatonin and salivary glands. Aim of this investigation was to establish if diabetic status may affect subcellular melatonin distribution and traffic. Bioptic samples of parotid and submandibular glands, removed from diabetic patients, were fixed, dehydrated, embedded in Epon Resin and processed to search for melatonin reactivity by the immunogold staining method. The labelling density (expressed as number of gold particles per μm2/granule) and the percentage of melatonin-positive granules were estimated in diabetic samples. The resulting values were compared with those of non-diabetic ones and the differences were statistically evaluated. In diabetic samples the pattern of melatonin staining was unchanged with respect to non-diabetic ones, as the gold particles were specifically localized within secretory granules and vesicles of serous cells. The quantitative evaluation of gold particles showed that the labeling density changed in parotid diabetic samples with respect to those measured in non-diabetics, as the percentage of melatonin positive granules showed a tendency to decrease in the diabetic status in both glands.This work was supported by grant from RAS, L7/2007, Progetti di Ricerca Fondamentale o di Base, Bando 201

Mitochondrial metabolism and morphology in a rat model of NAFLD

NAFLD (non-alcoholic fatty liver disease) is a condition, characterized by fatty liver, that, if untreated, can progress to NASH (non-alcoholic steatohepatitis) and, finally, to cirrhosis. Mitochondrial dysfunction is a crucial element for the progression of NAFLD to NASH. Our objective was studying both morphological and physiological mitochondrial alterations in a rat model of NAFLD and NASH. Sprague Dawley rats were fed either by a standard diet (35% of energy from fat) or by a high fat diet (HFD) (71% of energy from fat). Diets were given ad libitum for a maximum of four weeks and then rats were killed by decapitation after 1, 2, 3, and 4 weeks. A portion of liver samples was processed for light and transmission electron microscopy (TEM) for steatosis assessment, a part for high resolution scanning electron microscopy (HRSEM) to evaluate 3D mitochondria morphology, the remaining part for oxidative phosphorylation experiments (OXPHOS). Measurements of OXPHOS were performed on freshly isolated mitochondria in a Clark-type oxygen electrode. Several substrates were added to mitochondria to test the efficiency of electron transport chain complexes (ETC) and to test the efficiency of several enzymes involved in fatty acid oxidation (FAO). At three and four weeks of HFD, signs of hepatic steatosis were evident (as an increased amount of intracellular lipid droplets) on histological sections and TEM images. OXPHOS measurements indicated that, compared to controls, in the first week, HFD rats had an increased oxygen consumption with each substrate, in the second, third and fourth week OXPHOS efficiency had a tendency to decline with substrates inherent to glycolitic metabolism, but it was higher with substrates stimulating FAO. There were not notable differences in Respiratory Control Ratio and ADP/O ratio between HFD and controls, proving that the integrity of mitochondria was preserved and coupled to phosphorylation. HRSEM observations indicated that variations on inner mitochondrial morphology in HFD rats, appeared in the third and fourth week, when mitochondrial cristae were less, and cristal shape was often lamellar rather than tubular. Concluding, following HFD mitochondria exhibited first an increase in oxidative metabolism (with normal ultrastructural morphology) and later an alteration in the oxidative phosphorylation apparatus associated to mitochondrial cristae injury. These preliminary results suggest that in order to develop a consistent mitochondrial impairment associated to NASH is necessary to extend HFD treatment for longer. This study was supported by a grant by RAS (Regione Autonoma della Sardegna)

Male and Female Mitochondria Respond Differently after Exercising in Acute Hypoxia

The use of hypoxic devices among athletes who train in normobaric hypoxia has become increasingly popular; however, the acute effects on heart and brain metabolism are not yet fully understood. This study aimed to investigate the mitochondrial bioenergetics in trained male and female Wistar rats after acute hypoxia training. The experimental plan included exercising for 30 min on a treadmill in a Plexiglas cage connected to a hypoxic generator set at 12.5% O2 or in normoxia. After the exercise, the rats were sacrificed, and their mitochondria were isolated from their brains and hearts. The bioenergetics for each complex of the electron transport chain was tested using a Clarktype electrode. The results showed that following hypoxia training, females experienced impaired oxidative phosphorylation through complex II in heart subsarcolemmal mitochondria, while males had an altered ADP/O in heart interfibrillar mitochondria, without any change in oxidative capacity. No differences from controls were evident in the brain, but an increased electron transport system efficiency was observed with complex I and IV substrates in males. Therefore, the study’s findings suggest that hypoxia training affects the heart mitochondria of females more than males. This raises a cautionary flag for female athletes who use hypoxic devices

Cardiovascular remodelling in female diabetic rats

Diabetic cardiomyopathy involves both cardiac and large vessels alterations in their biochemical and biomechanical properties. Part of these dysfunctions is due to ROS overproduction and advanced glycated end-products (AGEs) synthesis caused by high blood glucose concentrations (1). Epidemiological studies usually ignore sexgender outcomes of diabetes that has higher cardiovascular risk in women than in men (2). The aim of the present study was to assess the effects of diabetes on aorta, portal vein and myocardium morphology in females Wistar rats. Diabetes was induced by a single dose of streptozotocin 65 mg/kg, and, after 4 and half months, we evaluated the cardiovascular remodelling by light and transmission electron microscopy (TEM). Paraformaldehyde fixed samples of aorta and portal vein were stained with Masson Trichrome method (for collagen fibers), Weigert’s stain (for elastic fibers), Hematoxylin and Eosin (for nuclei), and underwent to morphometric analysis. TEM samples were prepared accordingly to common protocols. Morphometric analysis performed on diabetic aortas showed a reduction of tunica media thickness, but the internal diameter width or the lumen cross-area was unchanged compared to controls. The number of smooth muscle cells increased in tunica media of diabetic aortas. The main change observed in diabetic portal veins was a reduction of the area occupied by elastic fibers in tunica adventitia. TEM observations of papillary muscles did not reveal any changes in the sarcomere lengths across the two experimental groups. These results display slight differences on what was reported in male rats (3) and account for a different development of diabetes in female subjects

Immunolocalization of AQP5 in human parotid salivary glands

Background. Aquaporins (AQPs) are channel protein essential in the transport of water across the biological cell membranes. Thirteen different AQPs have been identified in different tissues of mammals. In rat parotid, the AQP-5 is principally localized in the luminal membrane of the acinar serous cells (1). In mouse parotid a weak but distinct labeling has also observed in lateral, facing the neighboring cells, and basal plasma membranes (2). The AQP seems to be directly involved in transepithelial pathway for osmotic water flow, and in salivary fluid secretion. To date, in human only a report (3) testifies the presence of AQP-5 in luminal surface of secretory cells of parotid glands. So, in order to extend the knowledge regard to aquaporins, our intention was to define the sublocalization of AQP-5 in human parotid glands by immunogold post embedding method. Methods. Surgical samples of human parotid glands were cut into small fragments, fixed in a mixture of paraformaldehyde and glutaraldehyde and embedded in Epon resin without previous osmication. Ultrathin sections were incubated overnight at 4°C with primary antibody (1:25) against human AQP-5. Labeled sections were examined by transmission electron microscope (TEM). Results. The AQP-5 labeled was observed to the apical membrane of the serous cells. Gold particles were, occasionaly, found in the surface of intercellular canaliculi. Few small vesicles exhibited reactivity for AQP-5. Conclusions. Our preliminary studies show that the apical membrane of serous cells of human parotid glands is a specific site of AQP-5. Because the water-rich fluid is secreted in the luminal membrane where aqp5 was present, it is reasonable to speculate that aqp5 plays an important role in the secretion in the human parotid salivary gland. The reactivity of labeled vesicles could mean that in serous cells of human parotid gland occurs a translocation of aqp5 from the intracytoplasmatic compartment to the apical membrane (4). This study is supported by a grant by Regione autonoma della Sardegna. 1. Nielsen et al., Am J Physiol Cell Physiol, 1997. 2. Larsen et all., J Mol Hist, 2001. 3. Gresz et al., Am J Physiol Gastrointest Liver Physiol, 2001. 4. Hishikawa et all., biochemical and biophysical research communications, 1998