Diet-Dependent Changes of the DNA Methylome Using a Göttingen Minipig Model for Obesity.

Objective: Environmental factors can influence obesity by epigenetic mechanisms. The aim of this study was to investigate obesity-related epigenetic changes and the potential for reversal of these changes in the liver of Göttingen minipigs subjected to diet interventions. Methods: High-throughput liquid hybridization capture-based bisulfite sequencing (LHC-BS) was used to quantify the methylation status of gene promotor regions in liver tissue in three groups of male castrated Göttingen minipigs: a standard chow group (SD, N = 7); a group fed high fat/fructose/cholesterol diet (FFC, N = 10) and a group fed high fat/fructose/cholesterol diet during 7 months and reversed to standard diet for 6 months (FFC/SD, N = 12). Expression profiling by qPCR of selected metabolically relevant genes was performed in liver tissue from all pigs. Results: The pigs in the FFC diet group became morbidly obese. The FFC/SD diet did not result in a complete reversal of the body weight to the same weight as in the SD group, but it resulted in reversal of all lipid related metabolic parameters. Here we identified widespread differences in the patterning of cytosine methylation of promoters between the different feeding groups. By combining detection of differentially methylated genes with a rank-based hypergeometric overlap algorithm, we identified 160 genes showing differential methylation in corresponding promoter regions in the FFC diet group when comparing with both the SD and FFC/SD groups. As expected, this differential methylation under FFC diet intervention induced de-regulation of several metabolically-related genes involved in lipid/cholesterol metabolism, inflammatory response and fibrosis generation. Moreover, five genes, of which one is a fibrosis-related gene (MMP9), were still perturbed after diet reversion. Conclusion: Our findings highlight the potential of exploring diet-epigenome interactions for treatment of obesity.

Extensive changes in innate immune gene expression in obese Göttingen minipigs do not lead to changes in concentrations of circulating cytokines and acute phase proteins.

The usefulness of Göttingen minipigs as models for obesity and obesity-related pathologies is well established. The low-grade inflammation associated with obesity involves a range of innate immune factors; however, to our knowledge, the impact of obesity on innate immune factor expression has not been studied in Göttingen minipigs. Therefore, we studied the expression of innate immune genes in liver and adipose tissues as well as serum concentrations of cytokines and acute phase proteins in obese vs. lean Göttingen minipigs. In the liver, of 35 investigated genes, the expression of nine was significantly different in obese pigs (three up-regulated, six down-regulated). Of 33 genes in adipose tissues, obesity was associated with changed expression of 12 genes in the visceral adipose tissue (VAT) (three up-regulated), 11 in the abdominal retroperitoneal adipose tissue (RPAT) (seven of these up-regulated) and eight in the subcutaneous adipose tissue (SAT) from the neck (five of which were up-regulated). Obesity-associated expression changes were observed for three genes in all adipose tissues, namely chemokine (C-C motif) ligand 3-like 1 (up-regulated), CD200 molecule (down-regulated) and interleukin 1 receptor antagonist (up-regulated) with interleukin 1 receptor antagonist being the most highly regulated gene in both VAT and RPAT. Looking at patterns of expression across the three types of adipose tissues, obesity was associated with an increased number of acute phase proteins differentially expressed between adipose tissues and a decreased tissue-specific expression of cytokines and chemokines. In contrast to obese humans, no changes in serum concentrations of haptoglobin, C-reactive protein, serum amyloid A, tumor necrosis factor-α and interleukin 6 were found in obese Göttingen minipigs.

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.

Evalution of the castrated male Sprague-Dawley rat as a model of the metabolic syndrome and type 2 diabetes.

OBJECTIVE: Low testosterone levels have been shown to be predictive for the development of the metabolic syndrome in men. The aim of this study was to describe effects of testosterone deficiency on metabolic syndrome-related parameters in male rats in order to evaluate the rat as a model for the human metabolic syndrome related to low testosterone levels. METHODS: Male Sprague-Dawley rats were castrated or sham operated at 16 weeks of age and fed either a standard or a high energy diet. Measured parameters were: food intake, body weight, fat distribution, energy expenditure, physical activity and blood/plasma parameters related to glucose and lipid metabolism. RESULTS: Castration led to an increase in the amount of subcutaneous fat, but did not result in any changes in the visceral fat. Fasting blood glucose levels were increased and free fatty acids concentration decreased in the castrated rats from 2 weeks after castration and throughout the study, whereas no significant differences between the groups were found in any of the other parameters measured. A high-energy diet did not change the response to castration in male Sprague-Dawley rats. CONCLUSION: Compared to humans rats respond differently to testosterone deficiency. Only few of the features typical for the human metabolic syndrome were observed in castrated male Sprague-Dawley rats. Therefore, we conclude that with the present experimental setup the castrated rat is not an optimal model for studies on the influence of testosterone deficiency on body fat distribution and the development of other central components of the metabolic syndrome.

[Creutzfeldt-Jakob syndrome in the county of Storstrom during the period 1990-1999. Was there a significant accumulation of cases?]. / Creutzfeldt-Jacobs sygdom i Storstrøms Amt i perioden 1990-1999. Har der vaeret tale om en overhyppighed af tilfaelde?

INTRODUCTION: In the county of Storstrøm with about 250,000 inhabitants, ten cases of Creutzfeldt-Jacob disease (CJD) were diagnosed at the Department of Neurology, Naestved County Hospital during the decade 1990-1999. Only one case is to be expected every four years in the county. Therefore we decided to investigate if a significant accumulation had occurred. METHODS: Case records were examined to see whether the patients complied with the WHO criteria for possible, probable, or certain sporadic CJD. Poisson distribution with correction for age was used to compare the observed incidence with the expected incidence of 1 case/million/year. RESULTS: Eight cases complied with the diagnostic criteria: two certain, three probable, two possible, and one familial. The incidence of certain and probable cases was 1.9 per million per year during the period, which does not constitute a significant increase (p = 0.18). CONCLUSION: The result is consistent with the fact that all time or space clustering of CJD ever reported has proved ascribable to familial cases, and with the fact that case-control studies have never identified environmental risk factors for sporadic CJD.

Secretin stimulates HCO3(-) and acetate efflux but not Na+/HCO3(-) uptake in rat pancreatic ducts.

Pancreatic ducts secrete HCO3(-), but transport mechanisms are unresolved and possibly vary between species. Our aim was to study the intracellular pH (pHi) regulation and thus H+/HCO3- transport in rat pancreatic ducts. Of particular interest was the Na+/HCO3(-) cotransporter, thought to be important in HCO3(-) -transporting epithelia. pHi was measured with BCECF in freshly isolated intralobular ducts. A reduction in extracellular Na+ concentration or application of HOE 694 (1 microM) decreased pHi by 0.1 to 0.6 pH units, demonstrating Na+/H+ exchanger activity. A reduction in extracellular Cl- concentration or addition of H2DIDS (10 microM) increased pHi by 0.1 to 0.5 pH units, demonstrating Cl-/ HCO(3)- (OH ) exchanger activity. In experimental acidosis, extracellular HCO3(-)/CO2 buffer did not increase the rate of pHi recovery, indicating that provision of HCO3(-) by the Na+/HCO3(-) cotransporter was not apparent. Most importantly, Na+/HCO3(-) cotransport was not stimulated by secretin (1 nM). In contrast, in experimental alkalosis the pHi recovery was increased in HCO3(-)/CO2 buffer, possibly due to Na+/HCO3(-) cotransport in the efflux mode. Secretin (1 nM) and carbachol (1 microM) stimulated HCO3(-) efflux, which can account for the observed HCO3(-) concentrations in rat pancreatic juice. Acetate and HCO3(-) buffers were handled similarly, indicating similar transport mechanisms in pancreatic ducts.

Purinoceptors evoke different electrophysiological responses in pancreatic ducts. P2Y inhibits K(+) conductance, and P2X stimulates cation conductance.

In epithelia, extracellular nucleotides are often associated with regulation of ion transporters, especially Cl(-) channels. In this study, we investigated which purinoceptors are present in native pancreatic ducts and how they regulate ion transport. We applied whole-cell patch-clamp recordings, intracellular Ca(2+) and pH measurements, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. The data show two types of purinoceptors and cellular responses. UTP and ATP produced large Ca(2+) transients, a decrease in intracellular pH, 8-10-mV depolarization of the membrane voltage, and a decrease in the whole-cell conductance. The membrane effects were due to closure of K(+) channels, as confirmed by dependence on extracellular K(+). UTP/ATP effects could be associated with P2Y(2) purinoceptors, and RT-PCR revealed mRNAs for P2Y(2) and P2Y(4) receptors. On the other hand, 2', 3'-O-4-benzoylbenzoyl-ATP induced Ca(2+) influx and approximately 20-mV depolarization of the membrane voltage with a concomitant increase in the whole-cell conductance. These effects were dependent on extracellular Na(+), not Cl(-), indicating opening of cation channels associated with P2X(7) purinoceptors. RT-PCR showed mRNAs for P2X(7) and P2X(4) receptors. In microperfused ducts, luminal (but not basolateral) ATP caused large depolarizations of membrane voltages recorded with microelectrodes, consistent with luminal localization of P2X(7) receptors. Thus, P2Y(2) (and possibly P2Y(4)) purinoceptors inhibit K(+) channels and may not support secretion in native ducts. P2X(7) (and possibly P2X(4)) receptors are associated with cation channels and may contribute to regulation of secretion.

Different purinergic receptors lead to intracellular calcium increases in pancreatic ducts.

Extracellular adenosine 5'-triphosphate (ATP) has been described to act as a regulator in many cells and tissues, including epithelia, and in the gastrointestinal tract ATP is one of the substances involved in non-cholinergic non-adrenergic control. However, very little is known about the effect of ATP on pancreatic ducts, which normally secrete bicarbonate-rich fluid in response to secretin. Hence, the aim of our present study was to test the effect of ATP and other nucleotides on intracellular Ca2+ activity ([Ca2+]i) of pancreatic ducts, and thereby get information about purinergic receptors that might play a role in the regulation of pancreatic bicarbonate transport. Native intralobular ducts were obtained from rat pancreas and [Ca2+]i in 10-20 cells was measured using the fura-2 method. ATP (10(-4) mol/l) evoked a characteristic biphasic Ca2+ transient in duct cells. Nucleotides, used to classify the P2 receptors, acted with the following potency on the peak Ca2+ in many ducts: uridine 5'-triphosphate (UTP) >/= ATP >inosine 5'-triphosphate >/= 2-methylthio-ATP > beta,gamma-methyl-ATP > adenosine. However, although the peak [Ca2+]i responses to ATP and UTP were similar, the plateau [Ca2+]i was nearly doubled with UTP. Moreover, in about one-third of the ducts studied, UTP had no effect on cell Ca2+, while the response to ATP was normal. In further experiments we found that removal of extracellular Mg2+ increased the peak [Ca2+]i evoked in response to ATP. 2'&3'-O-(4-benzoylbenzoyl) ATP (BzATP) evoked a monophasic and slower increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+, or by addition of 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). Taken together, our data indicate that there are two types of purinergic receptors on pancreatic ducts through which ATP can act. These are pharmacologically known as P2U and P2Z receptors and may correspond to P2Y2 and P2X7 receptors.

Role of mitochondria-rich cells in epithelial chloride uptake.

Uptake of NaCl by amphibian tight epithelia, such as skin, urinary bladder and collecting duct, requires considerable thermodynamic work. By calculation it is demonstrated that NaCl absorption from dilute external solutions ([NaCl] approximately 1 mM) demands more energy than can be provided by the Na(+)-K(+)-ATPase alone. Thus, in addition to the Na+ pump, another transport ATPase must be involved. Previously, we have suggested that the other transport ATPase is an apical proton pump in mitochondria-rich (MR) cells. By driving an apical Cl(-)-HCO3- exchange, a rheogenic H+ pump would energize entrance of Cl- across the apical membrane. Since Cl- channels are present in the basolateral membrane, the net result would be a transcellular active rheogenic uptake of Cl-, and secretion of H+ and HCO3-. At higher external concentrations, the Cl- uptake by MR cells is driven by the Na+ pump-generated transepithelial electrical potential difference, which also requires the presence of apical Cl- channels. Electrophysiological methods have been developed by which we have been able to study the polarity of single MR cells and identify apical and basolateral transport systems. We have verified the existence of rheogenic H+ pumps in the apical membrane and Cl- channels in both membranes.

Tissue kinetics, ion transport, and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water.

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm2 (DMRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO3 (Ag) or methylene blue (MB). Part of the increased DMRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (Kb) decreased and cell loss by moulting (Kd) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in DMRC was accompanied by a 3-fold increase in Cl- current (ICl). At day-2 there was a transient reduction in the ICl per MRC. H+ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased ICl and H+ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl- (but not H+) transport capacity, was restored at day-7. We conclude that increased DMRC following exposure to low external Cl-, rather than being due to an increased Kb, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where 'morphological differentiation' precedes 'functional differentiation'.

Role of proton pump of mitochondria-rich cells for active transport of chloride ions in toad skin epithelium.

1. Active Cl- currents were studied in short-circuited toad skin epithelium in which the passive voltage-activated Cl- current is zero. Under visual control double-barrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border. 2. The net inward (active) 36Cl- flux of 27 +/- 8 pmol s-1 cm-2 (16) (mean +/- S.E.M (number of observation)) was abolished by 2 mM-CN- (6.3 +/- 3.5 pmol s-1 cm-2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 microM-mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM-serosal ouabain. 3. In Ringer solution buffered by 24 mM-HCO3- -5% CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISC was maintained when gluconate replaced mucosal Cl-, and it was reversibly reduced in CO2-free 5 mM-Tris-buffered Ringer solution (pH = 7.40) or by the proton pump inhibitor oligomycin. These observations indicate that the source of the outward ISC is an apical proton pump. 4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of -73 +/- 3 (22) to -93 +/- 1 mV (26), and superfusion with CO2-free Tris-buffered Ringer solution induced a further hyperpolarization (Vb = -101 +/- 1 mV (26)) which could be blocked by Ba2+. The CO2-sensitive current changes were null at Vb = EK (potassium reversal potential, -106 +/- 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISC which by default seems to be located to mitochondria-rich (MR) cells. 5. In the absence of mucosal Cl- a pH gradient was built up above MR cells with pH = 7.02 +/- 0.04 (42) and pH increasing to 7.37 +/- 0.02 (10) above principal cells (pH = 7.40 in bulk solution buffered by 0.1 mM-Tris). This observation localizes a proton pump to the apical membrane of MR cells. Using the integrated diffusion equation it was shown that the measured external pH gradient would account within an order of magnitude for measured currents. 6. Standing gradients of protons were eliminated in the presence of mucosal Cl- suggesting that active uptake of Cl- is associated with the exit of base equivalents across the apical membrane of MR cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic paroxysmal hemicrania (CPH). The clinical manifestations. A review.

Eight definite and 10 possible cases of CPH are known to the authors. Decisive diagnostic features in the differential diagnosis versus ordinary cluster headache (Horton's headache) seem to be: the presence of headache every day, a high maximum daily attack frequency (greater than or equal to attacks/24 hours) and an absolute indomethacin effect. There is increasing evidence for a female preponderance in CPH. It emerges from this study that there frequently (or invariably?) is a pre-CPH stage with atypical attack pattern, usually lasting several years. Pregnancy seems to have a rather clear ameliorating effect on attack frequency and severity. In other patients, the very onset of headache is immediately after delivery. The importance of recognizing this special headache from a clinical point of view is clear since this disabling disorder can be completely abolished by drug therapy.