Food intake and meal pattern in IAPP knockout mice with and without infusion of exogenous IAPP.

OBJECTIVE: The current study used islet amyloid polypeptide (IAPP) knockout mice (KO mice) to investigate the physiological role of IAPP in the regulation of food intake (FI). MATERIAL AND METHODS: FI and body weight were measured in KO and wild-type (WT) mice for 27 weeks. In an additional short-term experiment, IAPP (25 pmol·kg(-1)min(-1)) was infused subcutaneously for 3 days in KO and WT mice, and FI, meal pattern, and body weight were analyzed. RESULTS: In the long-term experiment, no significant differences in body weight were seen between WT and KO mice at any point. FI, meal number, and meal size did not differ significantly between the groups in any of the five selected weeks that were studied. In the short-term experiment, FI decreased significantly during IAPP infusion in both WT and KO groups. FI was significantly lower in the KO mice compared with WT on days 1 and 2 (p < 0.05 and p < 0.01, respectively). CONCLUSIONS: The data showing no differences in FI and body weight were seen between KO and WT mice, indicating that FI can be controlled in the absence of IAPP. The more marked anorectic effect seen in the KO mice during IAPP infusion suggests that IAPP receptors and/or IAPP post-receptor signaling pathways are up-regulated in mice lacking endogenous IAPP.

PP56 improves energy homeostasis in a mouse model of pancreatic cancer.

In this study, we investigated whether the anti-inflammatory drug PP56 (alpha-trinositol) may improve cancer-induced metabolic disorders. We implanted human MiaPaCa2 pancreatic cancer cells in the pancreas of 14 athymic mice for 12 weeks, using six intact littermates as normal controls. During the 12 weeks, seven tumor-cell recipients were treated with PP56 by daily injection (PPT mice). The tumor-cell recipients that were otherwise untreated were used as tumor controls (TC mice). Impaired glucose tolerance and decreased body weight gain were seen in TC but not PPT mice. When an enzyme for fatty acid beta-oxidation namely medium-chain acyl-CoA dehydrogenase (MCAD) was determined in tumor grafts; tumors from PPT mice showed more MCAD than those from TC mice. This suggests that PP56 stimulated fatty acid beta-oxidation in MiaPaCa2 cells in vivo. In keeping with this notion, PPT mice had decreased plasma free fatty acids. In vitro, we demonstrated that MiaPaCa2 cells consumed more fatty acids in the presence of PP56. In another experiment, we infused PP56 or vehicle in normal mice and found that PP56 decreased circulating glucose in the animals. We also showed that PP56 increased glucose transport in L6 skeletal muscle cells in vitro. In conclusion, PP56 increases the turnover of circulating nutrients such as glucose and helps maintain energy homeostasis in mice with pancreatic cancer.

Increased lipid metabolism and cell turnover of MiaPaCa2 cells induced by high-fat diet in an orthotopic system.

In this study, we investigated whether increased dietary fat influences established pancreatic cancer cells. MiaPaCa2 human pancreatic cancer cells were grown orthotopically in athymic mice fed normal diet (ND) or high-fat diet (HF). In the resulting tumors, medium-chain acyl-coenzyme A dehydrogenase (MCAD, a regulator of fatty acid beta-oxidation) and Cu/Zn-superoxide dismutase (an antioxidant enzyme) were determined using Western blotting. The MCAD messenger RNA (mRNA) was determined by real-time polymerase chain reaction. Intracellular lipid droplets, proliferating cells (Ki67 positive), and apoptotic cells were stained in tumor sections. The HF tumors were heavier than the ND tumors (1.60 +/- 0.08 vs 1.13 +/- 0.10 g, P < .01, 6 tumors per group). The MCAD and Cu/Zn-superoxide dismutase proteins and the MCAD mRNA were increased in HF tumors compared with those seen in ND tumors. The HF tumors contained extensive central necrosis, which was surrounded with apoptotic and proliferating cells. The HF tumors also showed numerous lipid droplets. In the ND tumors, necrosis was uncommon, apoptotic cells were sporadic, and lipid droplets were few. In follow-up experiments, MiaPaCa2 cells were incubated in vitro in the presence or absence of fatty acids (oleic and linoleic acids). The fatty acid exposure increased lipid droplets, cell proliferation, and MCAD mRNA expression in MiaPaCa2 cells. In conclusion, increased dietary fat stimulates lipid metabolism and cell turnover in MiaPaCa2 human pancreatic cancer cells.

Preoperative glucocorticoid administration attenuates the systemic stress response and hyperglycemia after surgical trauma in the rat.

The stress response to surgery is characterized by activation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, and by an inflammatory response and hyperglycemia. The aim of the present study was to investigate if preoperative corticosterone could reduce the postoperative systemic stress response, without aggravating hyperglycemia or interfering with activation of the hypothalamic-pituitary-adrenal axis, in a standardized rat model of surgical trauma. We used a standardized experimental model of intestinal resection in the rat. Exogenous corticosterone (8 mg/kg body weight) or vehicle was administered 2 hours before surgery; and postoperative plasma concentrations of interleukin-6, interleukin-10, adrenaline, noradrenaline, glucose, and insulin were determined. Exogenous corticosterone decreased preoperative plasma adrenaline but did not change plasma glucose or insulin levels. Moreover, corticosterone reduced postoperative plasma interleukin-6, catecholamines, and glucose (all P < .001-.05) without any effect on the plasma corticosterone concentration compared with vehicle-treated controls. A preoperative 2-hour exposure of physiologic poststress corticosterone concentrations not only suppressed plasma IL-6 levels but also inhibited surgery-induced adrenaline release and suppressed plasma glucose levels. We hypothesize that glucocorticoids attenuated the inflammatory response in injured tissues that reduced afferent input into brain areas regulating the neuroendocrine response.

Gastric emptying in response to IAPP and CCK in rats with subdiaphragmatic afferent vagotomy.

In the subdiaphragmatic vagal deafferentation procedure (SDA), the afferent fibers of the vagus are surgically severed unilaterally where they enter the brain stem. The technique includes a subdiaphragmal truncal vagotomy performed on the contralateral side. This procedure has been used to study the control of food intake, but it has not been used previously to investigate the role of vagal afferent fibers in the control of gastric emptying (GE). The current experiment studied the effect of SDA on the inhibition of GE by islet amyloid polypeptide (IAPP or amylin) and cholecystokinin (CCK) in awake, unrestrained rats with gastric cannulas. The experimental group underwent subdiaphragmatic vagal deafferentation; the control group had sham operations. All rats received 20-min intravenous infusions of IAPP (1, 3, 9, 27, and 81 pmol/kg/min), CCK (3, 30 and 90 pmol/kg/min), and normal saline in random order. Gastric emptying of saline was measured by the phenol red method during the last 5 min of each infusion period. CCK dose-dependently inhibited gastric emptying in both the control and SDA animals. The inhibition of GE by CCK was significantly attenuated by SDA (p<0.01). IAPP also inhibited gastric emptying dose-dependently, but the difference between the SDA and control groups was not significant. The current experiment, which used a different methodology than previous studies, provides support for the hypothesis that the inhibition of gastric emptying by CCK, but not by IAPP, is mediated partly by afferent vagal fibers.

Obesity alters cytokine gene expression and promotes liver injury in rats with acute pancreatitis.

OBJECTIVE: Obesity is a negative prognostic factor in patients with critical illnesses such as acute pancreatitis (AP). The outcome of AP is determined by the severity of systemic inflammation and organ dysfunction. In a previous study, we found that AP caused more deaths in obese rats than in lean rats. In the present study, we examined whether the effect of obesity on rats with AP is associated with distinct alterations in inflammatory cytokine expression in organs involved in AP. METHODS AND PROCEDURES: AP was induced in lean and obese Zucker rats by pancreatic infusion of taurocholic acid. All survivors were killed 8 h later. Gene transcripts for two proinflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha)) and two anti-inflammatory cytokines (IL-10 and pancreatitis-associated protein (PAP)) were determined in the pancreas, liver, and lungs by quantitative real-time polymerase chain reaction. The severity of AP was assessed by means of histology and serology. RESULTS: Obese AP rats had higher TNF-alpha mRNA in all organs examined, lower IL-10 and IL-6 mRNA in the pancreas, and lower PAP mRNA in the liver, compared to lean AP rats. Lean and obese AP rats had similar pancreatic lesions as assessed by histology. However, steatohepatitis and increased serum alanine amino transferase levels, which are indications of hepatic injury, were present in obese but not lean AP rats. DISCUSSION: Our findings suggest that altered inflammatory cytokine expression and increased vulnerability in the liver underlie the detrimental influence of obesity on AP.

Comparison of the effects of chronic central administration and chronic peripheral administration of islet amyloid polypeptide on food intake and meal pattern in the rat.

Islet amyloid polypeptide (IAPP) is postulated to act as a hormonal signal from the pancreas to the brain to inhibit food intake and reduce adipose energy reserves. The present study compared the effects of chronic peripheral and chronic central administration of IAPP on food intake and meal pattern in rats. IAPP was administered subcutaneously (SC) for 7 days at doses of 0, 0.25, 2.5 and 25 pmol kg(-1) min(-1) using an osmotic minipump or administered centrally at doses of 0, 0.025, 0.25 and 2.5 pmol kg(-1) min(-1) using an osmotic minipump connected to an intracerebroventricular (ICV) catheter inserted into the third ventricle. Both SC and ICV infusion decreased total food intake dose-dependently. The minimal effective dose was 2.5 pmol IAPP kg(-1) min(-1) for SC administration and 0.25 pmol kg(-1) min(-1) for ICV infusion. The decrease in food intake produced by infusion of IAPP was mainly due to decreased meal size, although a significant decrease in meal number also occurred at the highest SC and ICV doses. SC administration produced a larger, more persistent decrease in food intake during the light period than in the dark period, while ICV infusion caused a larger, more persistent decrease during the dark period. The 10-fold difference in minimal effective doses indicates that ICV-administered IAPP acted primarily in the brain to inhibit food intake. The difference between the effects of IAPP on meal pattern with the two methods of administration suggests that IAPP does not act on the same target(s) when administered centrally as it does when it is administered peripherally.

mRNA for pancreatic uncoupling protein 2 increases in two models of acute experimental pancreatitis in rats and mice.

Uncoupling-protein 2 (UCP2) is a mitochondrial protein that appears to be involved in cellular oxidant defense and in the regulation of oncotic cell death, both of which are important features of acute pancreatitis. However, UCP2 expression in acute pancreatitis has not been previously reported. In the current experiments, pancreatic gene expression was studied by real-time reverse-transcription/polymerase chain reaction and Northern blots. Two models of acute experimental pancreatitis were investigated: cerulein-induced pancreatitis in mice at two different time points and taurocholate-induced pancreatitis in rats at two degrees of severity. After cerulein administration, acinar injury and leukocyte infiltration was significantly higher at 24 h compared with 12 h after the first injection of cerulein (P<0.05, P<0.005, respectively). UCP2 mRNA was unchanged at 12 h but was nearly 12-fold greater than control levels after 24 h (P<0.001). UCP2 gene expression correlated with acinar injury (r=0.69; P<0.001). By 72 h after taurocholate administration, the severe group had more necrosis than the mild group (P<0.005). Pancreatic UCP2 mRNA was increased fourfold in the severe group compared with controls (P<0.01). UCP2 expression correlated with parenchymal necrosis (r=0.61; P<0.01). Thus, pancreatic UCP2 mRNA increased in two models of acute pancreatitis. The increase in UCP2 gene expression was correlated with the severity of the disease. Up-regulation of UCP2 in the pancreas may be a protective response to oxidative stress, but this increase may also have a negative influence on cellular energy metabolism. Therefore, acinar UCP2 may be an important modifier of the severity of acute pancreatitis.

Impaired insulin action on phosphatidylinositol 3-kinase activity and glucose transport in skeletal muscle of pancreatic cancer patients.

INTRODUCTION: Glucose intolerance or overt diabetes occurs in 80% of patients with pancreatic cancer (PC). This associated metabolic disorder includes peripheral insulin resistance, which may be caused by factors produced by the PC. The mechanism underlying PC-associated insulin resistance has not been clearly defined. AIM: To characterize basal and insulin-stimulated glucose transport, phosphatidylinositol (PI) 3-kinase activity, and glucose transporter 4 (GLUT4) in skeletal muscles of PC patients. METHODOLOGY: Skeletal muscle samples were obtained from the abdominal wall of 17 PC patients during surgery. Control muscles were sampled in the same way from 11 donors undergoing abdominal surgery for benign diseases. PI 3-kinase activity, glucose transport, and GLUT4 were assessed in vitro in these muscles. RESULTS: In the presence of physiologic concentrations of insulin, glucose transport and PI 3-kinase activity were significantly decreased in the PC group compared with controls. At supraphysiologic insulin concentrations, glucose transport was significantly decreased but PI 3-kinase activity was normalized. In the absence of insulin, these parameters were not significantly different between PC and control groups. Muscle GLUT4 contents were similar between PC and control groups. CONCLUSION: Defects in insulin-mediated PI 3-kinase activity and glucose transport contribute to the insulin resistance in patients with PC.

Early impairment of insulin secretion in rats after surgical trauma.

OBJECTIVE: Hyperglycaemia associated with insulin resistance is common after trauma and surgical procedures. Both reduced insulin sensitivity and altered insulin secretion may contribute to the impaired glucose homeostasis. We have demonstrated that skeletal muscle insulin resistance is present 2 h after small intestinal resection in rats. In this study, the aim was to investigate insulin secretion in the same experimental model. DESIGN: Small intestinal resection (5 cm) was performed in adult rats. The control animals underwent anaesthesia only. METHODS: The intravenous glucose tolerance test (IVGTT), the hyperglycaemic clamp and in vitro studies in isolated pancreatic islets were performed after surgery. Concentrations of blood glucose, plasma insulin, corticosterone and interleukin-6 (IL-6) were determined 0-5 h postoperatively. RESULTS: The insulin response in the IVGTT was attenuated 2 h (P<0.05) but not 4 h or during the hyperglycaemic clamp (3.5-4.5 h) postoperatively. Insulin secretion in response to glucose in vitro was decreased 2 h after the surgery (P<0.05), but no change was seen in arginine-stimulated secretion. Plasma levels of corticosterone were increased 3.5-5 h postoperatively (P<0.001-0.05). Increases in IL-6 were also seen postoperatively. CONCLUSION: We demonstrate that glucose-induced, but not arginine-induced, insulin secretion is temporarily impaired after intestinal resection in rats. The later appearance of elevated corticosterone and IL-6 levels, as well as the preservation of the beta-cell inhibition in vitro, argues against the possibility that these two circulating factors are causally responsible for reduced insulin release seen after surgery in this model.