Adipocytes enhance murine pancreatic cancer growth via a hepatocyte growth factor (HGF)-mediated mechanism.

INTRODUCTION: Obesity accelerates the development and progression of pancreatic cancer, though the mechanisms underlying this association are unclear. Adipocytes are biologically active, producing factors such as hepatocyte growth factor (HGF) that may influence tumor progression. We therefore sought to test the hypothesis that adipocyte-secreted factors including HGF accelerate pancreatic cancer cell proliferation. MATERIAL AND METHODS: Murine pancreatic cancer cells (Pan02 and TGP-47) were grown in a) conditioned medium (CM) from murine F442A preadipocytes, b) HGF-knockdown preadipocyte CM, c) recombinant murine HGF at increasing doses, and d) CM plus HGF-receptor (c-met) inhibitor. Cell proliferation was measured using the MTT assay. ANOVA and t-test were applied; p < 0.05 considered significant. RESULTS: Wild-type preadipocyte CM accelerated Pan02 and TGP-47 cell proliferation relative to control (59 ± 12% and 34 ± 12%, p < 0.01, respectively). Knockdown of preadipocyte HGF resulted in attenuated proliferation vs. wild type CM in Pan02 cells (35 ± 5% vs. 68 ± 14% greater than control; p < 0.05), but proliferation in TGP-47 cells remained unchanged. Recombinant HGF dose-dependently increased Pan02, but not TGP-47, proliferation (p < 0.05). Inhibition of HGF receptor, c-met, resulted in attenuated proliferation versus control in Pan02 cells, but not TGP-47 cells. CONCLUSIONS: These experiments demonstrate that adipocyte-derived factors accelerate murine pancreatic cancer proliferation. In the case of Pan02 cells, HGF is responsible, in part, for this proliferation.

Obesity, but not high-fat diet, promotes murine pancreatic cancer growth.

BACKGROUND: Obesity accelerates pancreatic cancer growth; the mechanisms underlying this association are poorly understood. This study evaluated the hypothesis that obesity, rather than high-fat diet, is responsible for accelerated pancreatic cancer growth. METHODS: Male C57BL/6J mice were studied after 19 weeks of high-fat (60 % fat; n = 20) or low-fat (10 % fat; n = 10) diet and 5 weeks of Pan02 murine pancreatic cancer growth (flank). RESULTS: By two-way ANOVA, diet did not (p = 0.58), but body weight, significantly influenced tumor weight (p = 0.01). Tumor weight correlated positively with body weight (R (2) = 0.562; p < 0.001). Tumors in overweight mice were twice as large as those growing in lean mice (1.2 ± 0.2 g vs. 0.6 ± .01 g, p < 0.01), had significantly fewer apoptotic cells than those in lean mice (0.8 ± 0.4 vs 2.4 ± 0.5; p < 0.05), and greater adipocyte volume (3.7 vs. 2.2 %, p < 0.05). Apoptosis (R (2) = 0.472; p = 0.008) and serum adiponectin correlated negatively with tumor weight (R = 0.45; p < 0.05). CONCLUSIONS: These data suggest that body weight, and not high-fat diet, is responsible for accelerated murine pancreatic cancer growth observed in this model of diet-induced obesity. Decreased tumor apoptosis appears to play an important mechanistic role in this process. The concept that decreased apoptosis is potentiated by hypoadiponectinemia (seen in obesity) deserves further investigation.

Does adiponectin upregulation attenuate the severity of acute pancreatitis in obesity?

INTRODUCTION: Obesity is an independent risk factor for severe acute pancreatitis, though the mechanisms underlying this association are unknown. The powerful anti-inflammatory adipokine adiponectin is decreased in obesity. We recently showed that the severity of pancreatitis in obese mice is inversely related to circulating adiponectin levels, and therefore hypothesized that adiponectin upregulation would attenuate the severity of pancreatitis in obese mice. METHODS: Forty congenitally obese mice were studied. Seven days prior to study, 20 mice received a single tail vein injection of adenovirus expressing recombinant murine adiponectin (APN; 2 × 108 plaque forming unit (pfu)), and the remainder received a control adenoviral vector expressing ß-galactosidase (ß-gal; 2 × 108 pfu). Half of the mice in each group had pancreatitis induced by cerulein injection (50 mcg/kg IP hourly for 6 h). The other half received saline on the same schedule. Serum APN concentration and pancreatic tissue concentrations of interleukin (IL)-6, IL-1ß, and MCP-1 were measured by ELISA. Histologic pancreatitis score was calculated based on the degree of inflammation (0-4), edema (0-4), and vacuolization (0-4). Data were analyzed by ANOVA and Tukey's tests; p < 0.05 was considered significant. RESULTS: No difference in body weight was observed between groups. Serum APN was significantly upregulated in the APN group compared with the ß-gal group. Pancreatic tissue concentration of IL-6 was significantly decreased in the APN group compared with the ß-gal group. No change either in pancreatic tissue concentration of IL-1ß and MCP-1 or in the severity of histologic pancreatitis were observed. CONCLUSION: Adiponectin upregulation modulates the pancreatic cytokine milieu but does not attenuate pancreatitis in this model of mild acute pancreatitis.

Validation of a novel, physiologic model of experimental acute pancreatitis in the mouse.

BACKGROUND: Many experimental models of acute pancreatitis suffer from lack of clinical relevance. We sought to validate a recently reported murine model of acute pancreatitis that more closely represents the physiology of human biliary pancreatitis. METHODS: Mice (C57BL/6J n=6 and CF-1 n=8) underwent infusion of 50µl of 5% sodium taurocholate (NaT) or 50µl of normal saline (NaCl) directly into the pancreatic duct. Twenty-four hours later, pancreatitis severity was graded histologically by three independent observers, and pancreatic tissue concentration of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were determined by ELISA. RESULTS: Twenty four hours after retrograde injection, the total pancreatitis score was significantly greater in mice infused with NaT than in those infused with NaCl (6.3 ± 1.2 vs. 1.2 ± 0.4, p<0.05). In addition, the inflammatory mediators IL-6 and MCP-1 were increased in the NaT group relative to the NaCl group. DISCUSSION: Retrograde pancreatic duct infusion of sodium taurocholate induces acute pancreatitis in the mouse. This model is likely representative of human biliary pancreatitis pathophysiology, and therefore provides a powerful tool with which to elucidate basic mechanisms underlying the pathogenesis of acute pancreatitis.

Acute pancreatitis in obesity: adipokines and dietary fish oil.

BACKGROUND: Acute pancreatitis is a substantial clinical problem accounting for 240,000 hospital admissions yearly in the United States. Obesity is epidemic and is clearly an independent risk factor for increased severity of acute pancreatitis (AP). Adipose tissue is an endocrine organ that secretes a variety of metabolically active substances termed adipokines. However, the role of adipokines in modulating acute pancreatitis severity remains incompletely understood. Dietary fish oil is rich in omega-3 free fatty acids and attenuates adipose tissue-induced inflammation. Therefore, we hypothesized that feeding obese mice diets rich in fish oil would alter the adipokine milieu and attenuate the severity of pancreatitis. METHODS: Lean (C57BL/6 J) and obese (LepDb) mice were fed either a soybean oil- or fish oil-rich diet for 4 weeks. AP was induced by six hourly intraperitoneal injections of cerulein (50 µg/kg). Serum adipokine levels were measured, and pancreatitis severity was assessed histologically and by measuring pancreatic concentrations of interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), myleoperoxidase (MPO), and monocyte chemoattractant protein-1 (MCP-1). RESULTS: Obese mice developed more severe pancreatitis than lean mice. Fish oil significantly decreased serum leptin (lean and obese) and increased serum adiponectin (lean only). Fish oil did not alter the baseline pancreatic inflammatory milieu, nor did it change histologic or biochemical pancreatitis severity. CONCLUSION: These data demonstrate that a diet rich in fish oil altered the adipokine milieu in lean and congenitally obese mice; however, fish oil did not improve pancreatitis severity induced with cerulein hyperstimulation.

Insulin, leptin, and tumoral adipocytes promote murine pancreatic cancer growth.

BACKGROUND: Obesity accelerates development and growth of human pancreatic cancer. We recently reported similar findings in a novel murine model of pancreatic cancer in congenitally obese mice. The current experiments were designed to evaluate the effects of diet-induced obesity on pancreatic cancer growth. METHODS: Thirty C57BL/6J female mice were fed either control 10% fat (n = 10) or 60% fat diet (n = 20) starting at age 6 weeks. At 11 weeks, 2.5 × 10(5) PAN02 murine pancreatic cancer cells were inoculated. After 6 weeks, tumors were harvested. Serum adiponectin, leptin, insulin, and glucose concentrations were measured. Tumor proliferation, apoptosis, adipocyte content, and tumor-infiltrating lymphocytes were evaluated. RESULTS: The diet-induced obesity diet led to significant weight gain (control 21.3 ± 0.6 g; diet-induced obesity 23.1 ± 0.5 g; p = 0.03). Mice heavier than 23.1 g were considered "Overweight." Tumors grew significantly larger in overweight (1.3 ± 0.3 g) compared to lean (0.5 ± 0.2 g; p = 0.03) mice; tumor size correlated positively with body weight (R = 0.56; p < 0.02). Serum leptin (3.1 ± 0.7 vs. 1.4 ± 0.2 ng/ml) and insulin (0.5 ± 0.2 vs. 0.18 ± 0.02 ng/ml) were significantly greater in overweight mice. Tumor proliferation, apoptosis, and tumor adipocyte volume were similar. T and B lymphocytes were observed infiltrating tumors from lean and overweight mice in similar number. CONCLUSION: These data show that diet-induced obesity accelerates the growth of murine pancreatic cancer.

Adipokines and cytokines in human pancreatic juice: unraveling the local pancreatic inflammatory milieu.

BACKGROUND: Differential adipokine expression in obesity influences the inflammatory milieu, and may explain in part obesity's negative impact on pancreatic disease. Pancreatic juice analysis may provide a good means to evaluate the local pancreatic inflammatory milieu. The presence of adipokines in pancreatic juice is unknown. AIMS: This proof-of-concept study was designed to determine the presence of adipokines and cytokines in human pancreatic juice. METHODS: With institutional review board approval, pancreatic juice was obtained from ten patients with a broad range of diagnoses at the time of endoscopic retrograde cholangiopancreatography. Pancreatic juice was assayed using enzyme-linked immunosorbent assay (ELISA) for insulin, the proinflammatory adipokine leptin, the anti-inflammatory adipokine adiponectin, and the inflammatory mediators interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and monocyte chemoattractant protein-1 (MCP-1). Correlation between adipokine and inflammatory mediator expression was determined by linear regression analysis. Data are presented as mean +/- standard error of the mean (SEM); P < 0.05 was considered statistically significant. RESULTS: Leptin (0.16 +/- 0.05 ng/ml) and adiponectin (0.63 +/- 0.02 microg/ml) were both present, as were the inflammatory mediators IL-6 (112.6 +/- 28.1 pg/ml), TNF-alpha (49.0 +/- 18.8 pg/ml), and MCP-1 (32.2 +/- 0.9 pg/ml). Paradoxically, the expression of the anti-inflammatory adipokine adiponectin correlated strongly with that of the proinflammatory cytokine IL-6 (R(2) = 0.98, P < 0.001). CONCLUSIONS: This report is the first to describe adipokine expression in human pancreatic juice. Human pancreatic juice inflammatory mediators and adipokines may provide an important measurement of the local pancreatic inflammatory milieu.

Obesity potentiates the growth and dissemination of pancreatic cancer.

BACKGROUND: Obesity is an independent risk factor for pancreatic cancer development and progression, although the mechanisms underlying this association are completely unknown. The aim of the current study was to investigate the influence of obesity on pancreatic cancer growth using a novel in vivo model. METHODS: Lean (C57BL/6 J) and obese (Lep(Db) and Lep(Ob)) mice were inoculated with murine pancreatic cancer cells (PAN02), and studied after 5 weeks of tumor growth. Tumor histology was evaluated by hematoxylin and eosin staining, cellular proliferation was assessed by 5-bromodeoxyuridine, and apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Serum adiponectin, leptin, and insulin levels were assayed. RESULTS: Obese mice developed larger tumors, and a significantly greater number of mice developed metastases; mortality was also greater in obese mice. Tumor apoptosis did not differ among strains, but tumors from both obese strains had greater proliferation relative to those growing in lean animals. Serum adiponectin concentration correlated negatively and serum insulin concentration correlated positively with tumor proliferation. Intratumoral adipocyte mass in tumors from both obese strains was significantly greater than that in tumors of lean mice. CONCLUSION: Data from this novel in vivo model suggest that the altered adipokine milieu and insulin resistance observed in obesity may lead directly to changes in tumor microenvironment, thereby promoting pancreatic cancer growth and dissemination.

Nuclear magnetic resonance spectroscopy-based metabolomics of the fatty pancreas: implicating fat in pancreatic pathology.

BACKGROUND: Obesity is a worldwide epidemic and a significant risk factor for pancreatic diseases including pancreatitis and pancreatic cancer; the mechanisms underlying this association are unknown. Metabolomics is a powerful new analytical approach for describing the metabolome (compliment of small molecules) of cells, tissue or biofluids at any given time. Our aim was to analyze pancreatic fat content in lean and congenitally obese mice using both metabolomic analysis and conventional chromatography. METHODS: The pancreatic fat content of 12 lean (C57BL/6J), 12 obese leptin-deficient (Lep(ob)) and 12 obese hyperleptinemic (Lep(db)) mice was evaluated by metabolomic analysis, thin-layer and gas chromatography. RESULTS: Pancreata of congenitally obese mice had significantly more total pancreatic fat, triglycerides and free fatty acids, but significantly less phospholipids and cholesterol than those of lean mice. Metabolomic analysis showed excellent correlation with thin-layer and gas chromatography in measuring total fat, triglycerides and phospholipids. CONCLUSIONS: Differences in pancreatic fat content and character may have important implications when considering the local pancreatic proinflammatory milieu in obesity. Metabolomic analysis is a valid, powerful tool with which to further define the mechanisms by which fat impacts pancreatic disease.

Cannabinoid receptor-1 blockade attenuates acute pancreatitis in obesity by an adiponectin mediated mechanism.

BACKGROUND: Obesity is a risk factor for increased severity of acute pancreatitis. Adipocytes produce adiponectin, an anti-inflammatory molecule that is paradoxically decreased in the setting of obesity. We have shown that adiponectin concentration inversely mirrors the severity of pancreatitis in obese mice. Cannabinoid receptor CB-1 blockade increases circulating adiponectin concentration. We, therefore, hypothesize that blockade of CB-1 would increase adiponectin and attenuate pancreatitis severity. METHODS: Forty lean (C57BL/6J) and 40 obese (Lep(Db)) mice were studied. Half of the mice in each strain received intraperitoneal injection of the CB-1 antagonist rimonabant (10 mg/kg daily for 7 days); the others received vehicle. Pancreatitis was induced by intraperitoneal injection of cerulein (50 microg/g hourly x 6). Pancreatitis severity was determined by histology. Pancreatic chemokine and proinflammatory cytokine concentrations were measured by ELISA. RESULTS: Rimonabant treatment significantly increased circulating adiponectin concentration in obese mice (p < 0.03 vs. vehicle). After induction of pancreatitis, obese mice treated with rimonabant had significantly decreased histologic pancreatitis (p < 0.001), significantly lower pancreatic tissue levels of monocyte chemoattractant protein-1 (p = 0.03), tumor necrosis factor-alpha (p < 0.001), interleukin-6 (p < 0.001), and myeloperoxidase (p = 0.006) relative to vehicle-treated animals. CONCLUSIONS: In obese mice, cannabinoid receptor CB-1 blockade with rimonabant attenuates the severity of acute pancreatitis by an adiponectin-mediated mechanism.

Adiponectin receptor-1 expression is decreased in the pancreas of obese mice.

BACKGROUND: Obesity is epidemic in the 21st century and has been shown to be a risk factor for developing severe acute pancreatitis. Adipose tissue produces small molecules called adipokines, which are important in modulating metabolism and inflammation. The anti-inflammatory adipokine adiponectin is decreased in obesity and inversely mirrors the severity of pancreatitis in a murine experimental model. Adiponectin acts through two receptors, AdipoR1 and AdipoR2; no data are currently available regarding adiponection receptor expression in the obese murine pancreas. MATERIALS AND METHODS: Immunohistochemical and reverse transcription-polymerase chain reaction analysis were undertaken to determine expression of adiponectin receptors AdipoR1 and AdipoR2 in the pancreas and liver of lean (C57BL/6J) and congenitally obese (Lep(Ob) and Lep(Db)) mice. RESULTS: Immunohistochemistry confirmed expression of both AdipoR1 and AdipoR2 in the pancreas of all three murine strains. Staining was positive in acinar cells and to a lesser extent in islet cells. Pancreatic gene expression of AdipoR2 was similar among lean and obese mice. AdipoR1 gene expression, however, was significantly (P < 0.001) decreased in the pancreas of both Lep(Ob) and Lep(Db) mice compared to wild-type lean animals. Gene expression of both AdipoR1 and AdipoR2 was significantly less in the liver of obese (Lep(Ob) and Lep(Db)) mice compared to wild-type lean animals (P < 0.001). CONCLUSIONS: These data show for the first time that the adiponectin receptors AdipoR1 and AdipoR2 are expressed in the obese murine pancreas. The paucity of AdipoR1 receptors may be important when considering the role played by adipokines in the genesis of severe pancreatitis in obesity.

A murine model of obesity implicates the adipokine milieu in the pathogenesis of severe acute pancreatitis.

Obesity is clearly an independent risk factor for increased severity of acute pancreatitis (AP), although the mechanisms underlying this association are unknown. Adipokines (including leptin and adiponectin) are pleiotropic molecules produced by adipocytes that are important regulators of the inflammatory response. We hypothesized that the altered adipokine milieu observed in obesity contributes to the increased severity of pancreatitis. Lean (C57BL/6J), obese leptin-deficient (LepOb), and obese hyperleptinemic (LepDb) mice were subjected to AP by six hourly intraperitoneal injections of cerulein (50 microg/kg). Severity of AP was assessed by histology and by measuring pancreatic concentration of the proinflammatory cytokines IL-1beta and IL-6, the chemokine MCP-1, and the marker of neutrophil activation MPO. Both congenitally obese strains of mice developed significantly more severe AP than wild-type lean animals. Severity of AP was not solely related to adipose tissue volume: LepOb mice were heaviest; however, LepDb mice developed the most severe AP both histologically and biochemically. Circulating adiponectin concentrations inversely mirrored the severity of pancreatitis. These data demonstrate that congenitally obese mice develop more severe AP than lean animals when challenged by cerulein hyperstimulation and suggest that alteration of the adipokine milieu exacerbates the severity of AP in obesity.

Leptin regulates gallbladder genes related to gallstone pathogenesis in leptin-deficient mice.

BACKGROUND: Little is known about the genetic factors that cause alterations in gallbladder motility, cholesterol crystal nucleation, biliary lipids, and, ultimately, cholesterol gallstones. Obese, leptin-deficient (Lep(ob)) mice have large gallbladder volumes with decreased contraction in vitro and are predisposed to cholesterol crystal formation. Leptin administration to these mice causes weight loss and restores gallbladder function. We hypothesize that administration of leptin to Lep(ob) mice would cause weight loss, decrease gallbladder volume, and change gallbladder genes related to gallbladder motility, nucleating factors, and lipid metabolism. STUDY DESIGN: Twenty-four 8-week-old Lep(ob) mice were fed a nonlithogenic diet for 4 weeks. Twelve mice received daily IP saline injections, and 12 received 5 mug/g recombinant leptin. Gallbladder mRNA was pooled and analyzed on murine genome microarray chips. Selected genes were confirmed by real-time polymerase chain reaction (PCR) in a second group of mice treated by the same protocol. RESULTS: Leptin-deficient mice given leptin had significant weight loss and reductions in gallbladder volume. These mice had upregulation of the leptin receptor (p = 0.007; PCR = 1.1-fold increase) but downregulation of leptin (p = 0.003; PCR = 13.5-fold decrease). Leptin upregulated the cholecystokinin A receptor (p < 0.001; PCR = 3.1-fold increase), acetylcholine beta2 receptor (p = 0.005), and the Ca+-calmodulin-dependent protein kinase (p = 0.002) genes. Leptin also altered immunoglobulin heavy chain 4 (p = 0.005; PCR = 17.7-fold increase), mucin 3 (p = 0.006), and carboxylesterase (p = 0.016; PCR = 2.5-fold decrease) genes. Leptin downregulated 3-hydroxy 3-methylglutaryl coenzyme A reductase (p = 0.006; PCR = 2.5-fold decrease) and LDL receptor (p = 0.003). CONCLUSIONS: Leptin modulates obesity and regulates gallbladder genes related to cholesterol gallstone pathogenesis.

Steatocholecystitis: the influence of obesity and dietary carbohydrates.

INTRODUCTION: We have recently demonstrated that obese and lean mice fed a high fat diet have increased gallbladder wall fat and decreased gallbladder contractility, cholecystosteatosis. Animal and human data also suggest that diets high in refined carbohydrates lead to gallstone formation. However, no data are available on the role of dietary carbohydrates on gallbladder wall fat and inflammation. Therefore, we tested the hypothesis that both obesity and dietary carbohydrates would increase gallbladder fat and cytokines, steatocholecystitis. METHODS: At 8 wk of age, 47 lean and 22 obese female mice were fed a 45% carbohydrate (CHO) diet while an equal number of lean and obese mice were fed a 75% CHO diet for 4 wk. All mice underwent cholecystectomy, and the gallbladders were snap-frozen. Individual and total lipids were measured by gas chromatography. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6 were measured by enzyme-linked immunosorbent assay. Data were analyzed by analysis of variance and Tukey test. RESULTS: Gallbladder total fat, triglycerides, and cholesterol were maximum (P < 0.001) in obese mice on the 75% CHO diet. Gallbladder TNF-alpha and IL-1beta as well as serum cholesterol levels showed a similar pattern (P < 0.001). Gallbladder saturated free fatty acids and IL-6 levels were highest (P < 0.001) in obese mice on the 45% CHO diet. CONCLUSIONS: These data suggest that (1) both obesity and dietary carbohydrates increase gallbladder total fat, triglycerides, cholesterol, TNF-alpha, and IL-1beta and (2) obesity also increases gallbladder free fatty acids and IL-6. Therefore, we conclude that obesity is associated with steatocholecystitis and that a high carbohydrate diet exacerbates this phenomenon.

Ezetimibe ameliorates cholecystosteatosis.

BACKGROUND: Cholecystosteatosis is the accumulation of gallbladder wall fats leading to decreased gallbladder emptying. Ezetimibe inhibits intestinal fat absorption and prevents murine gallstone formation. However, the influence of ezetimibe on gallbladder emptying and cholecystosteatosis has not been studied. Therefore, we tested the hypothesis that ezetimibe would improve gallbladder motility by preventing the buildup of fats in the gallbladder wall. METHODS: Forty lean female mice were fed either a control diet or a lithogenic diet for 6 weeks. Half of the mice on each diet received ezetimibe. At 11 weeks of age, all mice were fasted overnight and underwent gallbladder ultrasonography to determine ejection fraction. One week later, the mice were fasted and underwent cholecystectomy. Bile was examined for cholesterol crystals. The gallbladders were snap-frozen for lipid analysis. RESULTS: The lithogenic diet significantly (P < 0.05) increased serum cholesterol, biliary crystals, gallbladder wall cholesterol and cholesterol/phospholipid ratio, and decreased gallbladder ejection fraction. All of these abnormalities were reversed (P < 0.05) by the addition of ezetimibe to the diet. CONCLUSIONS: These data suggest that ezetimibe lowers serum cholesterol, prevents biliary crystals, and normalizes gallbladder wall fat and function. We conclude that ezetimibe ameliorates cholecystosteatosis and may be an effective agent for gallstone prevention.

High dietary carbohydrates decrease gallbladder volume and enhance cholesterol crystal formation.

BACKGROUND: Animal and human data suggest that a diet high in refined carbohydrates leads to gallstone formation. However, no data are available on the role of dietary carbohydrates on gallbladder volume or on cholesterol crystal formation. Therefore, we tested the hypothesis that a high carbohydrate diet would alter gallbladder volume and enhance cholesterol crystal formation. METHODS: At 8 weeks of age, 60 lean and 36 obese leptin-deficient female mice were fed a 45% carbohydrate diet while an equal number of lean and obese mice were fed a 75% carbohydrate diet for 4 weeks. All animals then underwent cholecystectomy, and gallbladder bile volume was recorded. Bile was pooled, filtered, and maintained in a water bath at 37 degrees C for 14 days. Birefringent cholesterol crystals in bile were counted daily; crystal observation time and crystal mass were determined. RESULTS: The crystal observation time was significantly shortened in both lean and obese mice on the 75% diet compared with their counterparts on the 45% diet. The crystal mass was significantly increased in the lean mice on the 75% diet compared with the 45% diet. Gallbladder volumes were significantly reduced in both lean and obese mice on the 75% diet compared with their counterparts on the 45% diet. CONCLUSIONS: These data suggest that a high carbohydrate diet decreases gallbladder volume, shortens cholesterol crystal observation time, and increases crystal mass. We conclude that dietary carbohydrates may play a role in cholesterol gallstone formation by altering biliary motility and by enhancing crystal formation.

Leptin regulates gallbladder genes related to absorption and secretion.

Dysregulation of gallbladder ion and water absorption and/or secretion has been linked to cholesterol crystal and gallstone formation. We have recently demonstrated that obese, leptin-deficient (Lep(ob)) mice have enlarged gallbladder volumes and decreased gallbladder contractility and that leptin administration to these mice normalizes gallbladder function. However, the effect of leptin on gallbladder absorption/secretion is not known. Therefore, we sought to determine whether leptin would alter the expression of genes involved in water and ion transport across the gallbladder epithelium. Affymetrix oligonucleotide microarrays representing 39,000 transcripts were used to compare gallbladder gene-expression profiles from 12-wk-old control saline-treated Lep(ob) and from leptin-treated Lep(ob) female mice. Leptin administration to Lep(ob) mice decreased gallbladder volume, bile sodium concentration, and pH. Leptin repletion upregulated the expression of aquaporin 1 water channel by 1.3-fold and downregulated aquaporin 4 by 2.3-fold. A number of genes involved in sodium transport were also influenced by leptin replacement. Epithelial sodium channel-alpha and sodium hydrogen exchangers 1 and 3 were moderately downregulated by 2.0-, 1.6-, and 1.3-fold, respectively. Carbonic anhydrase-IV, which plays a role in the acidification of bile, was upregulated 3.7-fold. In addition, a number of inflammatory cytokines that are known to influence gallbladder epithelial cell absorption and secretion were upregulated. Thus leptin, an adipocyte-derived cytokine involved with satiety and energy balance, influences gallbladder bile volume, sodium, and pH as well as multiple inflammatory cytokine genes and genes related to water, sodium, chloride, and bicarbonate transport.

Resistin-like molecule alpha reduces gallbladder optimal tension.

INTRODUCTION: Insulin resistance is associated with increased gallbladder volume and impaired gallbladder emptying. Resistin and resistin-like molecule alpha (RELM-alpha) are adipose-derived hormones that are believed to mediate insulin resistance. Therefore, we tested the hypothesis that administration of resistin or RELM-alpha would cause insulin resistance and diminish gallbladder contractility. METHODS: In two sequential studies 40 eight-week-old nondiabetic lean mice were fed a chow diet for 4 weeks. In Study A, 10 mice received 20 microg of resistin IP, while in Study B 10 mice received 20 microg of RELM-alpha IP for seven days. In each study, 10 control mice received an equal volume of saline IP for seven days. At 12 weeks animals were fasted and underwent cholecystectomy, and in vitro gallbladder response to neurotransmitters was determined. Serum resistin, RELM-alpha, glucose, and insulin levels were measured. HOMA index, a measure of insulin resistance, was calculated. RESULTS: RELM-alpha significantly increased HOMA index. RELM-alpha decreased gallbladder optimal tension, but did not alter responses to neurotransmitters. Resistin had no effect on HOMA index or on gallbladder optimal tension or response. CONCLUSION: These data suggest that in nondiabetic lean mice: 1) resistin does not alter insulin resistance or gallbladder optimal tension, but 2) RELM-alpha increases insulin resistance and reduces gallbladder optimal tension. Therefore, we concluded that RELM-alpha may play a role in insulin-resistance mediated gallbladder dysmotility.

Nonalcoholic fatty pancreas disease.

BACKGROUND: Obesity leads to fat infiltration of multiple organs including the heart, kidneys, and liver. Under conditions of oxidative stress, fat-derived cytokines are released locally and result in an inflammatory process and organ dysfunction. In the liver, fat infiltration has been termed nonalcoholic fatty liver disease, which may lead to nonalcoholic steatohepatitis. No data are available, however, on the influence of obesity on pancreatic fat and cytokines, and nonalcoholic fatty pancreas disease (NAFPD) has not been described. Therefore, we designed a study to determine whether obesity is associated with increased pancreatic fat and cytokines. MATERIALS AND METHODS: Thirty C57BL/6J lean control and 30 leptin-deficient obese female mice were fed a 15% fat diet for 4 weeks. At 12 weeks of age all animals underwent total pancreatectomy. Pancreata from each strain were pooled for measurement of a) wet and dry weight, b) histologic presence of fat, c) triglycerides, free fatty acids (FFAs), cholesterol, phospholipids, and total fat, and d) interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha). Data were analyzed by Student's t test and Fisher's exact test. RESULTS: Pancreata from obese mice were heavier (p<0.05) and had more fat histologically (p<0.05). Pancreata from obese mice had more triglycerides, FFAs, cholesterol, and total fat (p<0.05). Triglycerides represented 11% of pancreatic fat in lean mice compared with 67% of pancreatic fat in obese mice (p<0.01). Cytokines IL-1beta and TNF-alpha also were elevated in the pancreata of obese mice (p<0.05). CONCLUSIONS: These data suggest that obese mice have 1) heavier pancreata, 2) more pancreatic fat, especially triglycerides and FFAs, and 3) increased cytokines. We conclude that obesity leads to nonalcoholic fatty pancreatic disease.

Pioglitazone increases gallbladder volume in insulin-resistant obese mice.

BACKGROUND: Both obesity and diabetes are associated with an increased incidence of gallstones. Recent animal and human data from our laboratory suggest that insulin resistance is associated with increased gallbladder volume and/or impaired gallbladder emptying. Pioglitazone is a thiazolidinedione that has been shown to improve insulin resistance. Therefore, we tested the hypothesis that pioglitazone would improve insulin resistance, decrease resting gallbladder volume and improve gallbladder response to neurotransmitters in insulin-resistant obese mice fed a 25% carbohydrate diet. MATERIALS AND METHODS: Twenty eight-week-old insulin-resistant obese (Lep(ob)) mice fed a 25% carbohydrate diet for 4 weeks. Half of the animals had 0.3 g/kg pioglitazone added to their diet. At 12 weeks all animals were fasted and underwent cholecystectomy. Gallbladder volume and weight were measured, and fresh gallbladders were placed in a muscle bath to assess response to acetylcholine (ACh 10(-5)M), neuropeptide Y (NPY 10(-8,-7,-6)M) and cholecystokinin (CCK 10(-10,-9,-8,-7)M). Serum glucose and insulin were measured, and HOMA Index, a measure of insulin resistance, was calculated. RESULTS: Fasting serum insulin and HOMA Index were significantly decreased (P < 0.03), but gallbladder volume was significantly increased (P < 0.03) in the pioglitazone treated group. Pioglitazone did not alter gallbladder weight or response to ACh, NPY, or CCK. CONCLUSION: These data suggest that in insulin-resistant obese mice pioglitazone 1) lowers insulin-resistance, 2) increases resting gallbladder volume, and 3) does not alter gallbladder response to neurotransmitters. Therefore, we conclude that pioglitazone, while improving insulin resistance, paradoxically increases gallbladder volume and, thereby, may increase the propensity for gallstone formation by enhancing gallbladder stasis.