[The EHEC O104:H4 outbreak in Germany 2011 - lessons learned?!]. / Der EHEC O104:H4 Ausbruch 2011 in Deutschland - Lektion gelernt?!

The EHEC O104:H4 outbreak 2011 in Germany provided numerous insights into the recognition and control of such epidemic situations. Food-borne outbreaks and their related dynamics may lead to a critical burden of disease and an eventual capacity overload of the medical care system. Possible difficulties in the microbiological diagnostics of new or significantly altered infectious agents may result in a delayed detection of the outbreak as well as the launching of interventional measures. Besides an early notification of the local public health office by the affected institutions, in which a complete electronic procedure and additional sentinel or surveillance instruments (e. g., in emergency departments of hospitals) may be of great help, an interdisciplinary cooperation of the local public health and food safety agencies is the key to an effective outbreak control. Corresponding organizations on the state and federal level should support the investigation process by microbiological diagnostics and advanced epidemiological analysis as well as examination of the food chains. Finally, successful crisis communication relies on "speaking with one voice" (not necessarily one person). Immediate, transparent, appropriate and honest information of the general public concerning the reasons, consequences and (counter-) measures of a crisis are the best means to keep the trust of the population and to counteract the otherwise inevitable speculations.

Glucagon-stimulated but not isoproterenol-stimulated glucose formation inhibition by interleukin-6 in primary cultured rat hepatocytes.

During prolonged sepsis, impairment of glucose supply by the liver leads to hypoglycemia. Our aim was to investigate whether proinflammatory cytokine interleukin-6, a major mediator of the hepatic acute phase reaction, could contribute to this impairment by inhibiting hepatic glucose production stimulated by glucagon or isoproterenol in rat hepatocytes. Interleukin-6 inhibited the stimulation of glucose formation from glycogen by glucagon but not by isoproterenol in cultured rat hepatocytes. This was confirmed in the perfused rat liver. In cultured hepatocytes, the increase in cyclic adenosine-3',5'-monophosphate formation by glucagon was inhibited by interleukin-6, which was probably due to attenuation of glucagon binding to the glucagon receptor. The increase in cyclic adenosine-3',5'-monophosphate stimulated by isoproterenol was not affected by interleukin-6. However, the cytokine inhibited both expression of the key gluconeogenic control enzyme, phosphoenolpyruvate carboxykinase, stimulated by glucagon and isoproterenol. Thus, while increased glucose demand during the acute-phase reaction might initially be accomplished by catecholamine-mediated stimulation of glucose formation from glycogen, inhibition of gluconeogenesis by interleukin-6 may contribute to the impairment of glucose homeostasis during the prolonged acute phase reaction.

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.

Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.

Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes.

Impaired stimulation of intestinal glucose absorption by portal insulin via hepatoenteral nerves in chronically ethanol-intoxicated rats.

In the isolated, jointly perfused small intestine and liver of rats insulin, infused into the portal vein, induced an increase in intestinal glucose absorption via hepatoenteral cholinergic nerves. The possible loss of function of these nerves due to ethanol-induced neuropathy was investigated with 6 weeks ethanol-fed rats. Portal insulin or arterial carbachol failed to increase intestinal glucose absorption but cAMP still did so. The intact stimulatory effect of cAMP indicated an undisturbed capacity of the enterocytes. The loss of action of portal insulin and of arterial carbachol can be explained by the impairment of the hepatoenteral nerves in line with an ethanol-induced neuropathy.

Stimulation by portal insulin of intestinal glucose absorption via hepatoenteral nerves and prostaglandin E2 in the isolated, jointly perfused small intestine and liver of the rat.

Insulin infused into the portal vein acutely enhanced intestinal glucose and galactose absorption via the sodium-dependent glucose cotransporter-1 in the isolated, jointly perfused small intestine and liver of the rat. Atropine and tetrodotoxin infused into the superior mesenteric artery completely prevented the portal insulin-dependent increase in intestinal glucose absorption, and carbachol caused an increase similar to that of portal insulin. Thus, a signal was transmitted against the bloodstream in a retrograde direction from the portal vein to the small intestine via hepatoenteral cholinergic nerves. The intracellular messenger in the enterocytes was cAMP, and the link between the muscarinic receptors, which do not increase cAMP concentrations, and adenylate cyclase was found to be prostaglandin E2.

Impaired stimulation of intestinal glucose absorption via hepatoenteral nerves in streptozotocin-diabetic rats.

In an ex situ organ perfusion system, that of the isolated nonrecirculating joint perfusion of rat small intestine and liver, insulin infused into the portal vein increased intestinal glucose absorption. This insulin action against the bloodstream can be blocked by TTX, indicating a propagation of the insulin signal via hepatoenteral nerves, which conforms with previous studies with atropine and carbachol. Insulin action could also be mimicked by dibutyryl cAMP (DBcAMP) acting directly on the absorptive enterocytes. Because autonomic neuropathy is a common late complication of diabetes mellitus, the possible impairment of these nerves in the diabetic state was studied in streptozotocin-diabetic rats. In the isolated joint intestine-liver perfusion, glucose was applied as a bolus into the lumen; its absorption was measured in the portal vein. In 5-day diabetic as well as in control rats, portal insulin, arterial carbachol, and arterial DBcAMP increased intestinal glucose absorption. In 3-mo diabetic rats portal insulin and arterial carbachol failed to stimulate glucose absorption, whereas arterial DBcAMP still did so, indicating an undisturbed function of the absorptive enterocytes. The lack of an effect of portal insulin and arterial carbachol and the unchanged action of DBcAMP in the chronically diabetic rats indicated that the signaling chain via the hepatoenteral nerves was impaired, which is in line with a diabetic neuropathy.

Role of hepatic, intrahepatic and hepatoenteral nerves in the regulation of carbohydrate metabolism and hemodynamics of the liver and intestine.

The liver as an effector organ is the major glucose reservoir, the utilization of which is controlled by hormones but also by hepatic sympathetic nerves. The liver as a sensory organ detects a glucose concentration gradient between the hepatic artery and the portal vein by intrahepatic sensory-effector nerves, generating a cholinergic signal for an insulin-dependent net hepatic glucose uptake. The liver senses the insulin concentration by hepatoenteral sensory-effector nerves, generating a cholinergic signal to increase glucose absorption in the intestine and thus its coordinated utilization in liver, muscle and adipose tissue.

Acute increase, stimulated by prostaglandin E2, in glucose absorption via the sodium dependent glucose transporter-1 in rat intestine.

BACKGROUND/AIMS: Acute stimulation by cAMP of the sodium dependent glucose cotransporter SGLT1 has previously been shown. As prostaglandin E2 (PGE2) increases intracellular cAMP concentrations via its receptor subtypes EP2R and EP4R, it was investigated whether PGE2 could enhance intestinal glucose absorption. METHODS: The action of PGE2 on carbohydrate absorption in the ex situ perfused rat small intestine and on 3-O-[14C]methylglucose uptake in isolated villus tip enterocytes was determined. Expression of mRNA for the PGE2 receptor subtypes 1-4 was assayed in enterocytes by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In the perfused small intestine, PGE2 acutely increased absorption of glucose and galactose, but not fructose (which is not a substrate for SGLT1); in isolated enterocytes it stimulated 3-O-[14C]methylglucose uptake. The 3-O-[14C]methylglucose uptake could be inhibited by the cAMP antagonist RpcAMPS and the specific inhibitor of SGLT1, phlorizin. High levels of EP2R mRNA and EP4R mRNA were detected in villus tip enterocytes. CONCLUSION: PGE2 acutely increased glucose and galactose absorption by the small intestine via the SGLT1, with cAMP serving as the second messenger. PGE2 acted directly on the enterocytes, as the stimulation was still observed in isolated enterocytes and RT-PCR detected mRNA for the cAMP-increasing PGE2 receptors EP2R and EP4R.

Connexin 32 gap junctions enhance stimulation of glucose output by glucagon and noradrenaline in mouse liver.

Gap junctions connect neighboring cells via intercellular channels composed of connexins (Cx). Connexin 32 (Cx32) is the main connexin in hepatocytes. Gap junctions propagate a signal from periportal to perivenous hepatocytes generated by electrical stimulation of sympathetic liver nerves. Therefore, it was the aim of this study to examine the involvement of hepatocellular gap junctions in hormonal regulation. In perfused livers from wild-type mice and Cx32-deficient mice, the stimulation of glucose release by varying noradrenaline and glucagon concentrations was investigated. At saturating hormone concentrations, glucose release was the same in wild-type and Cx32-deficient livers. However, glucose output was significantly smaller in Cx32-deficient than wild-type livers at half-maximally effective hormone concentrations. Because the two hormones circulate at less than half-saturating concentrations and because they are degraded during passage of blood through the liver, they lose efficiency from the periportal to the perivenous zone. In wild-type livers, this decrease in efficiency can be partially compensated by intercellular signal propagation through gap junctions, resulting in higher hormone actions than in Cx32-deficient livers. It is concluded that gap junctions are not only involved in intercellular propagation of nervous, but also of hormonal signals from periportal to perivenous hepatocytes.

Enteric glucagon 37 rather than pancreatic glucagon 29 stimulates glucose absorption in rat intestine.

BACKGROUND & AIMS: Glucose and galactose are absorbed by the small intestine via the sodium-dependent glucose transporter 1 (SGLT1) and fructose via the facilitated glucose transporter 5. A stimulatory effect of enteric glucagon 37 and pancreatic glucagon 29 on intestinal carbohydrate absorption has been shown. However, only glucagon 37 is released after nutrient uptake and would thus fit into a regulatory circuit of nutrient-dependent hormone release that enhances carbohydrate absorption. Therefore, the aim of the present study was to evaluate whether glucagon 37 rather than glucagon 29 is the physiological stimulus of intestinal glucose absorption. METHODS: We examined the effects of glucagon 37, glucagon 29, and dibutyryl adenosine 3',5'-cyclic monophosphate on intestinal carbohydrate absorption and hepatic glucose output in the isolated perfused small intestine, isolated enterocytes, and isolated perfused liver of the rat. RESULTS: Reciprocal dose-response curves for the effects of the two hormones in intestine and liver were demonstrated: glucagon 37 was one order of magnitude more potent than glucagon 29 in increasing intestinal absorption of glucose via the SGLT1. In contrast, glucagon 29 more efficiently stimulated hepatic glucose release. The intracellular messenger was shown to be adenosine 3',5'-cyclic monophosphate. CONCLUSIONS: Glucagon 37 rather than glucagon 29 is the physiological stimulus of intestinal glucose absorption and exerts its effect via a specific glucagon 37 receptor.

Impaired glucose sensing by intrahepatic, muscarinic nerves for an insulin-stimulated hepatic glucose uptake in streptozotocin-diabetic rats.

Insulin-induced net hepatic glucose uptake depends on the sensing by muscarinic, intrahepatic nerves of a glucose concentration gradient between portal vein and hepatic artery. The function of these intrahepatic nerves was examined in streptozotocin-diabetic rats. In the presence of the glucose gradient insulin induced net glucose uptake in isolated perfused livers from control and acutely diabetic but not from chronically diabetic animals. The neurotransmitter acetylcholine still mimicked the existence of the gradient, excluding a metabolic impairment of livers of chronically diabetic animals. The impairment of the intrahepatic nerves due to diabetic neuropathy could contribute to postprandial hyperglycemia in diabetes mellitus.

A new role for enteric glucagon-37: acute stimulation of glucose absorption in rat small intestine.

Glucagon-37 is secreted by intestinal L-cells following carbohydrate uptake. It is known to inhibit gastric acid secretion (hence also named oxyntomodulin) and appears to increase intracellular cyclic AMP concentrations. Since cyclic AMP could enhance intestinal glucose absorption, a possible stimulatory effect of glucagon-37 on glucose transport was examined. Glucagon-37 acutely increased glucose absorption in the isolated, vascularly perfused small intestine and in isolated enterocytes of the rat. In these cells the stimulation by glucagon-37 could be completely blocked by the cAMP antagonist Rp-cAMPS and was therefore mediated by cAMP. The stimulation of intestinal glucose absorption by glucagon-37 appears to be a major new physiological function.

Sensing by intrahepatic muscarinic nerves of a portal-arterial glucose concentration gradient as a signal for insulin-dependent glucose uptake in the perfused rat liver.

In vivo, insulin increases net hepatic glucose uptake efficiently only in the presence of a portal-arterial glucose gradient. In isolated perfused rat livers supplied with a glucose gradient (portal 10 mM/arterial 5 mM) insulin-induced glucose uptake was blocked by atropine; in livers not supplied with the gradient (portal = arterial 5 mM) insulin-dependent glucose uptake was elicited by acetylcholine. Apparently, the gradient was sensed and transformed into a metabolic signal by intrahepatic nerves, releasing acetylcholine to muscarinic receptors.

Impairment of metabolic hepatic nerve action by chronic but not acute ethanol intoxication studied in isolated perfused rat liver.

BACKGROUND/AIMS: Liver carbohydrate metabolism and blood flow are regulated by hepatic nerves and hormones such as glucagon, insulin or catecholamines. Acute and chronic application of alcohol are known to depress the function of central and peripheral nerves. The extent of inhibition of the autonomic nervous system is not well characterized; thus, the possible impairment of hepatic nerve function by acute and chronic application of ethanol was investigated. METHODS: Rat livers were perfused simultaneously via both the portal vein and hepatic artery. Hepatic nerves were stimulated electrically for 2 min (20 Hz, 20 V, 2 ms). As a control, noradrenaline (1 microM) was infused into the portal vein for 2 minutes. RESULTS: During acute application of ethanol in portal concentrations of 50, 150 and 300 mM, which elevated basal glucose release, stimulation of hepatic nerves as well as portal noradrenaline infusion caused the same increase in glucose output and decrease in portal and arterial flow as in controls. Following chronic application of ethanol by feeding rats the Lieber-DeCarli liquid diet containing 5% (v/v) ethanol for 4 and 6 weeks, only nerve stimulation caused a significantly reduced enhancement of glucose output (50%, p < 0.025), whereas portal noradrenaline was as effective as in controls. Noradrenaline overflow was significantly reduced following nerve stimulation. CONCLUSION: The decrease in nerve stimulation-dependent glucose output and noradrenaline overflow in chronically ethanol fed rats indicates an impaired function of hepatic nerves.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Acute increase by portal insulin in intestinal glucose absorption via hepatoenteral nerves in the rat.

BACKGROUND & AIMS: Insulin exerts a strict short-term control of glucose disappearance by glucose storage as well as degradation in the liver and peripheral insulin target tissues, but an acute control of glucose appearance by glucose absorption in the intestine is as yet unknown. The aim of the present study was to evaluate, whether insulin acutely modulates intestinal glucose absorption. METHODS: In the isolated, nonrecirculating joint perfusion of the small bowel and liver of the rat via the celiac trunc and the superior mesenteric artery, glucose absorption was examined without and with infusion of insulin via the portal vein. RESULTS: Portal insulin enhanced acutely intestinal glucose absorption. This thus far unknown stimulatory effect of portal insulin was dose-dependent and detectable at physiological insulin concentrations. Atropine infused into the superior mesenteric artery completely prevented the insulin-dependent increase in intestinal glucose absorption, and carbachol caused a similar increase as portal insulin. CONCLUSIONS: Portal insulin dose-dependently generated a signal in the liver or portal vein. This signal was transmitted in a retrograde direction against the blood stream in the portal vein to the small intestine via hepatoenteral muscarinic nerves. This signal markedly increased intestinal glucose absorption.

Loss of regulation by sympathetic hepatic nerves of liver metabolism and haemodynamics in chronically streptozotocin-diabetic rats.

The consequences of autonomic diabetic neuropathy, a common complication of chronic diabetes mellitus, have been studied mainly with regard to heart and stomach function. Since the autonomic nervous system also regulates liver carbohydrate metabolism and haemodynamics via hepatic nerves, it was the purpose of this study to examine the function of hepatic nerves in chronically diabetic rats. Diabetes was induced by i.p. injection of streptozotocin. Rat livers were perfused via both portal vein and hepatic artery. Hepatic nerves were stimulated for 2 min using a platinum electrode placed around the portal vein and the hepatic artery; in an additional stimulation phase noradrenaline was infused into the portal vein. Stimulation of hepatic nerves as well as portal noradrenaline infusion increased hepatic glucose output and reduced flow in control and in acutely (48-h) diabetic animals, which still had almost normal glycogen content. In addition stimulation also caused an overflow of noradrenaline into the caval vein. However, nerve stimulation neither increased glucose output nor decreased flow in 4-month diabetic rats. In these rats noradrenaline overflow was nearly completely abolished and hepatic glycogen content was markedly depleted. Portal noradrenaline infusion in chronically diabetic rats reduced flow to a similar extent as in controls, yet the increase in glucose output was diminished. The lack of nerve stimulation-dependent glucose output, flow reduction and noradrenaline overflow is indicative of a profound loss of function of hepatic autonomic nerves in chronically diabetic rats.

Acute actions of insulin-like growth factor II on glucose metabolism in adult rats.

The metabolic potency of recombinant human insulin-like growth factor II was studied in anaesthetized adult rats by obtaining dose-response curves for the hypoglycaemic action and for the stimulation of glucose metabolism during euglycaemic clamping. Compared to insulin, about 50 times higher doses of insulin-like growth factor II were required to result in identical in vivo responses, with half-maximally effective serum concentrations for the stimulation of glucose disposal during clamp studies of about 0.8 and 50 pmol/ml, respectively. A similar difference in potency was observed for the dose-dependent stimulatory actions on glucose metabolism in individual target tissues. Half-maximally effective serum concentrations in the range of 0.8 to 3.0 pmol/ml for insulin and of 40 to 70 pmol/ml for insulin-like growth factor II were seen to be required for 2-deoxyglucose uptake, glycogen formation in skeletal muscle and lipogenesis in epididymal fat. Maximal responses were identical with both peptides. These data suggest that in vivo acute metabolic actions of insulin-like growth factor II on carbohydrate metabolism occurred through insulin receptors.