Expression studies of six human obesity-related genes in seven tissues from divergent pig breeds.

Obesity has reached epidemic proportions globally and has become the cause of several major health risks worldwide. Presently, more than 100 loci have been related to obesity and metabolic traits in humans by genome-wide association studies. The complex genetic architecture behind obesity has triggered a need for the development of better animal models than rodents. The pig has emerged as a very promising biomedical model to study human obesity traits. In this study, we have characterized the expression patterns of six obesity-related genes, leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor (MC4R), fat mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin (ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production pigs and Göttingen minipigs) that deviate phenotypically and genetically from each other with respect to obesity traits. We observe significant differential expression for LEP, LEPR and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP expression is only detected in the fat minipigs. FTO shows significant differential expression in all tissues analyzed, and NEGR1 shows significant differential expression in muscle, pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be detected only in hypothalamus. In general, the expression profiles of the investigated genes are in accordance with those observed in human studies. Our study shows that both the differences between the investigated breeds and the phenotypic state with respect to obesity/leanness play a large role for differential expression of the obesity-related genes.

Mammary remodeling in primiparous and multiparous dairy goats during lactation.

Milk production is generally lower but lactation persistency higher in primiparous (PP) than in multiparous (MP) goats. This may be related to differences in development and maintenance of mammary gland function, but the underlying mechanisms are not well understood. The present study aimed to elucidate whether differences in lactational performance between PP and MP mammary glands are related to the time course of development and maintenance, not only of the mammary epithelial cell (MEC) population, but also of the mammary vasculature that sustains synthetic activity. Mammary biopsies were obtained from both mammary glands of 3 PP and 6 MP (>or=2 parity) dairy goats at parturition (d 1), d 10, 60, and 180 of lactation. Gene transcription relating to MEC turnover and vascular function was quantified by real-time reverse transcription-PCR, mammary morphology was characterized (quantitative histology), and cell turnover was determined (terminal deoxynucleotidyl transferase dUTP nick end labeling assay and Ki-67). Primiparous glands showed higher expression for the genes involved in angiogenesis; namely, vascular endothelial growth factor receptor 2, and angiopoietin 1 and 2 and their receptor, a few days after parturition (d 10). Primiparous glands also had higher rates of MEC proliferation in early lactation. It therefore appears that initiation of lactation is associated with development and growth of the mammary gland into early lactation, which continues for a longer period in PP compared with MP glands. In addition, MEC survival was found to be higher in PP glands throughout lactation, and MEC in PP glands underwent more extensive differentiation. This could explain the reported flatter lactation curve and higher lactation persistency in PP glands. Although some of the genes included in this study were differentially expressed in PP and MP glands during the course of lactation, it was not possible to identify any specific genomic factor(s) that could account for the differences between PP and MP glands with respect to mammary development and MEC survival during lactation. It remains to be established why parity number affects MEC and vascular development and survival during lactation, and, in particular, which regulatory mechanisms are involved.

Ultrastructure and electrolyte transport of the epithelium of coprodeum, colon and the proctodeal diverticulum of Rhea americana.

The structure and function of the lower intestinal tract of Rhea americana were characterized to evaluate the evolutionary relationship to other struthioniform and avian species. In 5 rheas the gross anatomy and the light and transmission electron microscopy were studied in parallel to in vitro electrophysiological measurements of ion transport. The mucosa in the colon was amplified with villi, often branched, and in the coprodeum with folds. In both tissues the epithelium was a monolayer composed of columnar absorptive cells, goblet cells and mitochondria-rich cells. Colon and coprodeum appeared to produce large amounts of mucus. The proctodeal diverticulum was rich in lymphoid tissue arranged into lobuli bursales, and it was concluded that this structure is a modified bursa of Fabricius. The sparse interlobular epithelium of the diverticulum resembled that of colon and coprodeum. Baseline short circuit currents (I(SC)) averaged 114.5+/-13.8 microA/cm(2) in colon, 193.1+/-30.3 microA/cm(2) in coprodeum and 60.4+/-9.1 microA/cm(2) in the diverticulum. Amiloride sensitive Na+-transport amounted to 31, 88 and 38% of the baseline I(SC) in these three tissues, respectively. In all tissues, there was also a modest, theophylline activated chloride secretion response, and ouabain, the Na+/K+-ATPase inhibitor, abolished most of the I(SC). The transepithelial resistance (TER) of the diverticulum was much higher than the other tissues. Upon dissection, urate from ureteral urine was observed in the lower third of the colon and to a lesser extent in the proctodeal diverticulum, indicating retrograde peristalsis of the urine. Thus, unlike the ostrich, there is no sphincter separating colon and coprodeum. On the other hand, a thick mucus layer was seen overlying the mucosa in both colon and coprodeum, as in the ostrich. This may help to prevent osmotic water loss, despite the presence of hyperosmotic urine (up to 800 mOsm) in the lower intestine. Both morphological and electrophysiological data from the rhea support the hypothesis that the rhea lower intestine contributes to post-renal modification of ureteral urine and to the regulation of osmotic balance, as also seen in domestic fowl and other avian species. The proctodeal diverticulum functions mainly as an immune organ, with only limited transport capability.

Effects of antisecretory factor-derived peptides on contractions in guinea pig colon.

Two antisecretory factor (AF)-derived peptides have been studied in relation to effects on motility of guinea pig colon. Colon segments were isolated from adult guinea pigs and incubated in Tyrode Ringer. Motility was measured as the force and frequency of contractions upon addition of the derived peptides AF 1 (8 amino acids (aa)) and AF 3 (10 amino acids). At the lowest concentration (5 pM), peptide AF 1 induced a negative effect on the force of contraction in colon segments; an effect that was abolished by the cholinergic agonist carbachol. Peptide AF 3 induced a significant increase in the force of colon contractions at all concentrations (5-180 pM), with carbachol only reducing the effect of peptide AF 3 at a concentration of 15 pM. Both peptides increased contractile frequency, although the overall response was lower for peptide AF 3 than for peptide AF 1. It is concluded that antisecretory factor-derived peptides may play a role in regulating colon motility such that under pathophysiological conditions, they may serve to hasten the evacuation of noxious agents from the large intestine.

Gastrointestinal-tract models and techniques for use in safety pharmacology.

The gastrointestinal tract (GI tract), which extracts nutrients, electrolytes, minerals, and water, is prone to injury as a result of oral drug administration. Clinical assessment of the GI tract is often limited to measurements of transit time and observations of vomiting or diarrhoea, despite the existence of methods and techniques capable of assessing specific changes in GI function at the membrane, cell, and whole animal levels. Membrane studies, record the uptake of solutes, and electrolyte transport, assessing the affects of compounds on transepithelial GI transport and flux. Such methods lend themselves to permeability, immunohistochemistry, morphology, and molecular biology techniques. Isolated cells from the GI tract or cultured cell lines provide knowledge of regulation and function at a cellular level, whilst motility patterns, taken in vivo or from biopsies, provide information at a more integrated level. In anesthetised animals, ligated segments of the intestine can be infused with test compounds, providing information about absorptive and secretory processes important for the treatment of diarrhoea. Computer simulations and modelling are used to predict the disposition of a chemical and its metabolite and can, to some extent, replace animal testing, thereby reducing development costs. Indeed, software programs can be used to simulate the dissolution, absorption, distribution, metabolism, and excretion (ADME) properties of potential drugs in the human GI tract. Finally, advances in the field of imaging, combined with endoscopy, have resulted in a wireless capsule, allowing the inspection of the GI tract anatomy and pathology without surgical intervention. It is concluded that the field of safety pharmacology could rapidly, cheaply, and routinely incorporate membrane, isolated tissue, and endoscopy techniques for GI tract testing of drugs.

Does the ostrich (Struthio camelus) coprodeum have the electrophysiological properties and microstructure of other birds?

The ostrich is unique among birds in having complete separation of urine and faeces. The coprodeal epithelium is thus during dehydration exposed to a fluid 500 mOsm hyperosmotic to plasma. We have investigated whether the coprodeum is adapted like a mammalian bladder. The coprodeal epithelium was studied by electrophysiology in the Ussing chamber, and the anatomy by light microscopy and scanning electron microscopy. ELECTROPHYSIOLOGY: The short-circuit current (SCC) and open circuit electrical potential difference were recorded. The change induced by 0.1 mmol mucosal amiloride was recorded. An average basal SCC of 162+/-29 microA/cm(2) was observed, and a resistance of 297+/-34 Omega cm(2) calculated. These values are as observed in other avian coprodea. The resistance is much lower than in mammalian bladders (10000 Omega cm(2)). The amiloride-sensitive SCC, equal to net sodium absorption, was approximately 5 micromol/cm(2)h as observed in other avian species. ANATOMY: The mucosal membrane is composed of broad irregular folds with very short intestinal glands containing an unusually high proportion of goblet cells. CONCLUSION: The ostrich coprodeum is not adapted like a mammalian bladder. The abundance of goblet cells results in a copious secretion of mucus that establishes a thick unstirred layer giving effective osmotic protection.

Aldosterone suppresses expression of an avian colonic sodium-glucose cotransporter.

Transport in the colon of the domestic fowl switches from sodium-linked hexose and amino acid cotransport on high-salt intake to amiloride-sensitive sodium channel expression on low-salt (LS) diets. The present experiments were designed to investigate the role of aldosterone in suppression of the colonic sodium-glucose luminal cotransporter (SGLT). LS-adapted hens were resalinated with or without simultaneous aldosterone treatment. Changes in the electrophysiological responses and SGLT protein expression levels were examined at 1, 3, and 7 days of treatment. Serum aldosterone levels fell from approximately 400 pmol/l in LS-adapted hens to values below the detection limit (<44 pmol/l) after 1 day of resalination. At the same time, glucose-stimulated short circuit current (I(SC)) increased from 20.9 +/- 8.7 to 56.3 +/- 15.5 microA/cm(2), whereas amiloride-sensitive I(SC) decreased from -68.9 +/- 12.7 microA/cm(2) on LS to +0.6 +/- 12.0 microA/cm(2). Glucose-stimulated I(SC) increased further at 3 and 7 days of resalination, whereas amiloride-sensitive I(SC) remained suppressed. When resalinated birds were simultaneously treated with aldosterone, the LS pattern of high amiloride-sensitive I(SC) and low glucose-stimulated I(SC) was maintained. Immunoblotting results from the same tissues demonstrated that SGLT-like protein expression increased following resalination. Aldosterone treatment completely blocked this effect. These results demonstrate that aldosterone suppresses both activity and protein expression of hen colonic SGLT. Resalination either through decreased aldosterone or other factors may be able to activate SGLT activity independently of increases in protein expression.

Differences in epithelial morphology correlate to Na(+)-transport: a study of the proximal, mid, and distal regions of the coprodeum from hens on high and low NaCl diet.

A study was performed to correlate regional morphology and amiloride inhibitable Na(+)-transport in the coprodeal epithelium in hens, Gallus domesticus, on low-NaCl diet and in controls. Proximal (close to colon), mid and distal (close to urodeum) regions were examined using light microscopy, transmission- and scanning electron microscopy. Na(+)-transport was measured electrophysiologically in Ussing-chambers in the proximal and distal regions. The epithelium, simple and columnar, is composed of absorptive intestinal epithelial cells, goblet cells, brush cells, migrating lymphoid cells, and enteroendocrine cells. Brush cells, identified in avians for the first time, occur in highest number in the proximal part of the coprodeum in low-NaCl hens. Na(+)-transport is high in the low-NaCl hens, ranging from 347 microA/cm2 (proximal) to 187 microA/cm2 (distal). In control hens, which correspond to hens on high-NaCl diet, it is low in all regions (0-4 microA/cm2). Absorptive intestinal epithelial cells as well as brush cells adapt to variations in transepithelial Na(+)-transport by regulating height and packing density of their microvilli, number, size, and localization of apical vesicles, and the width of the intercellular space. Regional differences in the epithelial cell composition and ultrastructure are closely correlated to transepithelial Na(+)-transport but only in low-NaCl hens, as controls do not show these variations.

Morphological adaptations to induced changes in transepithelial sodium transport in chicken lower intestine (coprodeum): a study of resalination, aldosterone stimulation, and epithelial turnover.

Transepithelial sodium transport and epithelial morphology during short-term adaptation to resalination or aldosterone stimulation were studied in the chicken coprodeum. Coprodeum was sampled for light and electron microscopy after 0-3 days of resalination in hens on a low-NaCl diet and after 0-6 days of aldosterone stimulation in hens on a high-NaCl diet. Sodium transport was measured in vitro with Ussing chambers. Plasma osmolality and electrolyte concentrations were measured in aldosterone-stimulated hens. Epithelial proliferation and migration between 1 h and 16 days were investigated in chickens on high-NaCl and low-NaCl diets using a bromodeoxyuridine technique. Resalination abolished the otherwise high sodium transport within 1 day, while the height and number of microvilli, as well as the number of brush cells, decreased over 3 days. Aldosterone stimulation increased sodium transport, the height and number of microvilli, and the brush-cell number. Bromodeoxyuridine studies indicated an epithelial cell turnover of more than 16 days. The results thus demonstrate that epithelial cells have an unusual capacity to adjust rap- idly to variations in sodium intake. A strong correlation between structure and function was apparent.

The effect of alpha-trinositol on cholera toxin-induced fluid accumulation in pig (Sus scrofa domesticus) jejunum in vivo.

The effect of alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate) on cholera toxin-induced fluid accumulation (i.e., net fluid secretion) was studied in the pig jejunum in vivo. Cholera toxin caused a dose-dependent fluid accumulation in control experiments. Intravenous injection of alpha-trinositol produced a reduction of the response to cholera toxin with a significant maximal inhibition of 36%. However, in high concentrations of alpha-trinositol this inhibition was absent.

The effect of alpha-trinositol on cholera toxin-induced hypersecretion and morphological changes in pig jejunum.

alpha-Trinositol (D-myo-inositol 1,2,6-trisphosphate, PP56) is a novel antiinflammatory drug. This study elucidates the effect of intravenous alpha-trinositol on basal and acute fluid transport and morphological changes following cholera toxin administration in pig jejunum in vivo. Using isolated jejunal tied-off loops, the fluid hypersecretory (accumulation) effect of different doses of cholera toxin was studied in pigs treated intravenously with saline added different doses (0, 4, 8, 16 and 32 mg x kg-1 x hr-1) of alpha-trinositol. Levels of alpha-trinositol, as well as stereomicroscopical, light microscopical and scanning electron microscopical morphological studies were performed. Cholera toxin evoked a dose-dependent fluid hypersecretion. Treatment with alpha-trinositol caused a dose-dependent inhibition of the cholera toxin-induced fluid hypersecretion and did not affect basal fluid absorption. The 16 mg x kg-1 x hr-1 alpha-trinositol dose gave a maximal inhibition of 36%. Morphological studies showed only minor changes following 6 hr of exposure to 20 micrograms x loop-1 cholera toxin. These changes consisted of dilation of the villus capillaries, an increase of apical membrane blebbing and a reduction of the intercellular space. Treatment with 16 mg x kg-1 x hr-1 alpha-trinositol alone did not induce any morphological changes, and did not alter the morphological changes induced by cholera toxin, which caused fluid hypersecretion and only minor acute morphological changes. In conclusion, alpha-trinositol treatment reduced cholera toxin-induced fluid hypersecretion without altering basal fluid absorption, basal morphology, or cholera toxin-induced morphological changes in pig jejunum in vivo.

Mucosal acidification and an acid microclimate in the hen colon in vitro.

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63 +/- 0.25 microEq.cm-2.h-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10(-3) mol.l-1), and serosal ouabain (5 x 10(-4) mol.l-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10(-3) mol.l-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59 +/- 0.13 microEq.cm-2.h-1, P < 0.002 versus controls) and addition of glucose (15 mmol.l-1), but not galactose, partially restored acidification to control levels. Anoxia (N2 gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1-7.4, was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and enzymatic studies on the plasma membrane domains and sodium pump enzymes of absorptive epithelial cells in the avian lower intestine.

The coprodaeum of the domestic hen maintained on a low-NaCl diet adapts by enhanced sodium transport. This study examines the adaptive response at the single cell and whole organ levels. Surface areas of apical (microvillous) and basolateral plasma membranes of columnar absorptive epithelial cells were estimated by use of ultrastructural stereology. The activities of succinic dehydrogenase (a mitochondrial enzyme) and ouabain-sensitive, potassium-dependent paranitrophenyl phosphatase (a sodium pump enzyme) were determined in tissue homogenates. Sodium, potassium-ATPase (pump enzyme) activity in cell membranes was localized by ultrastructural cytochemistry. Apical and basolateral membranes responded differently. In high-NaCl hens, the membrane signature of the average cell was 32 microns 2 (apical), 932 microns 2 (lateral) and 17 microns 2 (basal). Cells from low-NaCl hens had more apical membrane (49 microns 2 per cell) but essentially the same area of basolateral membrane. However, total surfaces per organ were greater for all membranes. Sodium pump enzymes were localized in basolateral membranes. Enzyme activities per unit mitochondrial volume and per unit basolateral membrane surface were higher in low-NaCl birds. These findings are discussed in the context of known mechanisms of transcellular sodium transport via apical ion channels and basolateral pumps.

Quantitative analysis of factors contributing to expansion of microvillous surface area in the coprodaeum of hens transferred to a low NaCl diet.

A stereological study of the lower intestine (coprodaeum) of the domestic hen was undertaken using combined light and electron microscopy. Numbers of columnar absorptive epithelial cells and the dimensions and numbers of microvilli were estimated. The aim was to identify the main factors contributing to an increase in microvillous surface area following transfer of hens from a high to a low NaCl diet on which they were kept for at least 3 wk. The principal contributor to observed changes between organs was cell number. Birds adapted to the low NaCl diet had 57% more cells than high NaCl controls. The average cell had a larger microvillous surface (55%) and this could be explained by the presence of longer (34%) and more densely packed (38%) microvilli. The total number of microvilli per coprodaeum doubled (from 35 x 10(9) on the high NaCl to 71 x 10(9) on the low NaCl diet). The increase in cell number accounted for 67% of the change in surface area and 78% of the change in number of microvilli per organ. These findings emphasise that, when assessing the form and function of a whole organ, it is important to monitor cell populations as well as single cells. This is especially true when studying renewing and expanding (rather than static) populations.

Na-transport during long-term incubation of the hen lower intestine: no aldosterone effect.

1. In order to test the aldosterone effect in vitro, Na-transport of the coprodeal epithelium from hens on low-NaCl diet was measured in the Ussing chamber for up to 8 hr. Short-circuit current (SCC, near equal to the amiloride inhibitable Na-transport) was recorded. 2. Incubation media were either Krebs-phosphate or bicarbonate buffer with and without addition of beta-hydroxybutyrate, glutamine and mannose as "metabolic fuels". The media were replaced every hour. The Krebs-phosphate buffer was further tested with and without indomethacin and media replacement. Na-transport was best maintained in this buffer with replacement: SCC at 4 hr: 156 +/- 21 microA/cm2, 8 hr: 73 +/- 14 microA/cm2. 3. The aldosterone experiments were carried out on tissues from hens resalinated for 24 hr. No effects were demonstrated at concentrations up to 10(-5) M. The SCC showed an unexpected raise within 2-4 hr to a very high level (4 hr: 221 +/- 61 microA/cm2) both in the control and in all aldosterone-treated tissues. This SCC decreased slowly to 210 +/- 29 microA/cm2 at 8 hr. It was abolished by amiloride. 4. No increase in SCC was observed in tissues from hens after 48 and 72 hr of resalination either after aldosterone or on chronic high-NaCl diet.

A sampling scheme intended for tandem measurements of sodium transport and microvillous surface area in the coprodaeal epithelium of hens on high- and low-salt diets.

A tissue sampling protocol for combined morphometric and physiological studies on the mucosa of the avian coprodaeum is presented. The morphometric goal is to estimate the surface area due to microvilli at the epithelial cell apex and the proposed scheme is illustrated using material from three White Plymouth Rock hens. The scheme is designed to satisfy sampling requirements for the unbiased estimation of surface areas by vertical sectioning coupled with cycloid test lines and it incorporates a number of useful internal checks. It relies on multi-level sampling with four levels of stereological estimation. At Level I, macroscopic estimates of coprodaeal volume are obtained. Light microscopy is employed at Level II to calculate epithelial volume density. Levels III and IV require low and high power electron microscopy to estimate the surface density of the epithelial apical border and the amplification factor due to microvilli. Worked examples of the calculation steps are provided.