Gallbladder myocytes are short and cholecystokinin-resistant in obese diabetic mice.

BACKGROUND: Obesity is associated with diabetes and gallstone formation. Obese leptin-deficient (Lepob) and leptin-resistant (Lepdb) mice are hyperglycemic and have enlarged gallbladders with diminished response in vitro to cholecystokinin (CCK) and acetylcholine (ACh). Whether this phenomenon is secondary to hyperosmolar myocytes and/or decreased neuromuscular transmission remains unclear. We hypothesize that myocytes from Lepob and Lepdb obese mice would not respond normally to neurotransmitters. METHODS: Cholecystectomy was performed on 39 lean, 19 Lepob, and 20 Lepdb 12-week-old female mice. The gallbladder was divided and enzymatically digested. Half of each gallbladder's myocytes had contraction induced by CCK (10(-8) mol/L, n = 38) or ACh (10(-5) mol/L, n = 40). RESULTS: Body weights, gallbladder volumes, and serum glucoses were greater for Lep(ob) and Lepdb mice compared to controls (P < .001). Resting myocyte lengths from Lepob and Lepdb mice were 93% and 91% of the length of controls (P < .001). In response to CCK, lean myocytes shortened 6% (P < .01), while myocytes from obese mice demonstrated no shortening. None of the myocytes demonstrated significant shortening with ACh. CONCLUSIONS: These data suggest that gallbladder myocytes from obese mice are (1) foreshortened and (2) have a diminished response to cholecystokinin. We conclude that altered leptin and/or increased glucose may foreshorten myocytes and decrease response to cholecystokinin.

Leptin-resistant obese mice have paradoxically low biliary cholesterol saturation.

BACKGROUND: Human obesity is associated with leptin resistance, elevated serum glucose and lipids, hepatic steatosis, and cholesterol gallstone formation. These gallstones are thought to result from hypersecretion of biliary cholesterol as well as biliary stasis. Leptin-resistant Lep(db) obese mice, which are known to have elevated serum leptin, glucose, and lipids, as well as hepatic steatosis, should be an appropriate model for human gallstone formation. Therefore, we tested the hypothesis that leptin-resistant mice would have increased gallbladder volume, biliary cholesterol saturation, and cholesterol crystal formation. METHODS: Sixty lean control mice and 60 Lep(db) obese mice on a low cholesterol chow diet were studied. Gallbladder volumes were measured and bile was pooled to calculate cholesterol saturation index. Serum cholesterol, glucose, and leptin levels were determined from pooled serum. Hepatic fat vacuoles were counted. Bile from a second group of 90 lean control and 59 obese mice was observed microscopically for cholesterol crystal formation. RESULTS: Leptin-resistant obese mice have significantly higher serum cholesterol, glucose, and leptin levels, hepatic fat vacuoles, and gallbladder volume than lean control mice. However, biliary cholesterol saturation index and cholesterol crystal formation were significantly diminished in the obese mice. CONCLUSIONS: These data suggest that leptin-resistant Lep(db) obese mice have (1) increased gallbladder volume, (2) decreased biliary cholesterol saturation despite elevated serum cholesterol and hepatic steatosis, and (3) decreased in vitro cholesterol crystal formation. We conclude that the link between obesity and gallstone formation does not require hypersecretion of biliary cholesterol.

Decreased gallbladder response in leptin-deficient obese mice.

Obesity is a major risk factor for gallstone formation, but the pathogenesis of this phenomenon remains unclear. Human data on gallbladder emptying are conflicting, and no animal data exist on the effect of obesity on gallbladder motility. Leptin, a hormone produced by adipocytes, is known to have central effects on neuropeptide Y and cholecystokinin, but the influence of leptin on the biliary effects of these hormones is unknown. Therefore we tested the hypothesis that leptin-deficient C57BL/6J-lep(ob) obese mice would have decreased gallbladder responses to excitatory stimuli. Twelve-week-old lean control (C57BL/6J) (n = 22) and C57BL/6J-lep(ob) obese (n = 20) female mice were fed a nonlithogenic diet. The mice were fasted overnight and underwent cholecystectomy. Whole gallbladders were placed in 3 ml muscle baths. After optimal length was determined with acetylcholine (10(-5) mol/L, responses to increasing doses of neuropeptide Y (10(-8) to 10(-6) mol/L) and cholecystokinin-8 (10(-10) to 10(-7) mol/L) were measured. Student's t test and two-way analysis of variance were used where appropriate. Results were expressed as Newtons per cross-sectional area. The lean control mice had significantly greater excitatory responses to acetylcholine than the obese mice (0.37 +/- 0.05 vs. 0.16 +/- 0.02, P < 0.01). The gallbladder responses were also greater when mice were treated with neuropeptide Y (10(-8) mol/L: 0.00 +/- 0.00 vs. 0.00 +/- 0.00, NS; 10(-7) mol/L: 0.12 +/- 0.02 vs. 0.05 +/- 0.01, P < 0.01; 10(-6) mol/L: 0.26 +/- 0.08 vs. 0.06 +/- 0.01, P < 0.01) and cholecystokinin (10(-10) mol/L: 0.27 +/- 0.04 vs. 0.13 +/- 0.02, P < 0.01; 10(-9) mol/L: 0.59 +/- 0.08 vs. 0.27 +/- 0.04, P < 0.01; 10(-8) mol/L: 0.80 +/- 0.11 vs. 0.37 +/- 0.05, P < 0.01; 10(-7) mol/L: 0.86 +/- 0.11 vs. 0.44 +/- 0.06, P < 0.01). These data suggest that genetically obese, leptin-deficient mice have decreased responses to acetylcholine, neuropeptide Y, and cholecystokinin. We conclude that decreased gallbladder motility contributes to the increased incidence of gallstones associated with obesity.

Nonobese diabetic mice have diminished gallbladder motility and shortened crystal observation time.

Diabetes and obesity are strongly associated and are risk factors for cholesterol gallstone disease. Leptin-deficient and leptin-resistant diabetic obese mice have enlarged, hypomotile gallbladders. In addition, bile from gallbladders of leptin-deficient mice has enhanced cholesterol crystal formation, whereas bile from gallbladders of leptin-resistant mice has delayed crystal observation time. To determine the effect of diabetes alone, we hypothesized that leptin-normal, nonobese diabetic (NOD) mice would have reduced biliary motility and rapid crystal formation. Twenty control and 9 prediabetic and 11 diabetic NOD, 12- to 26-week-old mice underwent glucose measurement and cholecystectomy for muscle bath stimulation with neurotransmitters. An additional group of 200 control and 78 NOD 12-week-old mice underwent microscopic bile examination for cholesterol crystal formation. Compared with control mice, prediabetic NOD mice had similar glucose levels and gallbladder volumes. Diabetic NOD mice had higher sugar levels and larger gallbladder volumes (P < 0.001) than control mice. Prediabetic NOD gallbladders had less contractility (P < 0.01) than control gallbladders, and contractility worsened (P < 0.01) in diabetic NOD mice. NOD mice formed cholesterol crystals earlier than did control mice (P < 0.05). Nonobese diabetic NOD mice have (1) decreased gallbladder contraction to neurotransmitters, which worsens with development of diabetes, and (2) rapid crystal formation. We conclude that diabetes alone alters gallbladder motility and cholesterol crystal formation.

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.

Ciliary neurotrophic factor restores gallbladder contractility in leptin-resistant obese diabetic mice.

BACKGROUND: Obesity and diabetes are major risk factors for cholesterol gallstones, and the majority of obese people are leptin-resistant. Our previous work has shown that both leptin-deficient (Lepob) and leptin-resistant (Lepdb) obese diabetic mice have decreased in vitro gallbladder motility. Leptin administration to leptin-deficient (Lepob) animals restores gallbladder motility and reverses obesity and hyperinsulinemia. However, additional leptin in leptin-resistant obesity would not be expected to improve obesity-related parameters. Recent studies demonstrate that ciliary neurotrophic factor (CNTF) reduces weight and hyperinsulinemia in leptin-resistant obesity. Our hypothesis is that CNFT would cause weight loss, lower blood sugars, and restore gallbladder contractility in leptin-resistant (Lepdb) mice. MATERIALS AND METHODS: 20 C57b/6J and 20 Lepdb 8-week-old female mice were injected daily with either intraperitoneal saline or 0.3 microg/g CNTFAx15 for 17 days. Gallbladders were mounted in muscle baths and stimulated with acetylcholine, neuropeptide Y, and cholecystokinin. Gallbladder volume, serum glucose, insulin, liver weight, liver fat, and gallbladder responses were measured. Data were analyzed by ANOVA. RESULTS: Saline treated obese mice had greater body weight and obesity parameters, but decreased gallbladder contractility to neurotransmitters compared to saline treated lean mice. CNTF administration to obese mice decreased body weight and obesity parameters, and restored gallbladder contractility. CNTF treated lean animals had weight loss and decreased gallbladder contraction to acetylcholine and cholecystokinin compared to saline treated lean animals. CONCLUSIONS: Ciliary neurotrophic factor (CNTF) causes 1) weight loss, 2) improvement of diabetes, and 3) alterations in gallbladder motility that is improved in obese mice but decreased in lean mice. We conclude that CNTF may improve gallbladder contractility in leptin-resistant obesity with diabetes.

Altered small intestinal absorptive enzyme activities in leptin-deficient obese mice: influence of bowel resection.

BACKGROUND: Residual bowel increases absorption after massive small bowel resection. Leptin affects intestinal adaptation, carbohydrate, peptide, and lipid handling. Sucrase, peptidase, and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) are involved in carbohydrate, protein, and lipid absorption. We hypothesized that leptin-deficient obese mice would have altered absorptive enzymes compared with controls before and after small bowel resection. METHODS: Sucrase, peptidase (aminopeptidase N [ApN], dipeptidyl peptidase IV [DPPIV]), and MGAT activities were determined from lean control (C57BL/6J, n = 16) and leptin-deficient (Lep(ob), n = 16) mice small bowel before and after 50% resection. RESULTS: Ileal sucrase activity was greater in obese mice before and after resection. Jejunal ApN and DPPIV activities were lower for obese mice before resection; ileal ApN activity was unaltered after resection for both strains. Resection increased DPPIV activity in both strains. Jejunal MGAT in obese mice decreased postresection. In both strains, ileal MGAT activity decreased after resection, and obese mice had greater activity in remnant ileum. CONCLUSIONS: After small bowel resection, leptin-deficient mice have increased sucrase activity and diminished ileal ApN, DPPIV, and MGAT activity compared with controls. Therefore, we conclude that leptin deficiency alters intestinal enzyme activity in unresected animals and after small bowel resection. Altered handling of carbohydrate, protein, and lipid may contribute to obesity and diabetes in leptin-deficient mice.

Leptin-resistant obese mice do not form biliary crystals on a high cholesterol diet.

INTRODUCTION: Human obesity is associated with leptin resistance and cholesterol gallstone formation. Previously, we demonstrated that leptin-resistant (Lep(db)) obese mice fed a low cholesterol diet have enlarged gallbladders, but a decreased cholesterol saturation index, despite elevated serum cholesterol. Obese humans, however, consume a high cholesterol diet. Therefore, we hypothesized that on a high cholesterol diet, leptin-resistant mice would have cholesterol saturated bile and would form biliary crystals. METHODS: Eight-week old female lean control (n = 70) and leptin-resistant (n = 72) mice were fed a 1% cholesterol diet for 4 weeks. All animals then had cholecystectomies. Bile was collected, grouped into pools to determine cholesterol saturation index (CSI), and examined for cholesterol crystals. Serum cholesterol and leptin were also measured. RESULTS: Gallbladder volumes for Lep(db) mice were enlarged compared with the lean mice (35.8 microl versus 19.1 microl, P < 0.001), but the CSI for the Lep(db) mice was lower than for the lean animals (0.91 versus 1.15, P < 0.03). The obese animals did not form cholesterol crystals, whereas the lean animals averaged 2.2 crystals per high-powered field (hpf) (P < 0.001). Serum cholesterol and leptin were also elevated (P < 0.001) in the obese animals. CONCLUSIONS: These data suggest that Lep(db) obese mice fed a high cholesterol diet have increased gallbladder volume and decreased biliary cholesterol saturation and crystal formation despite elevated serum cholesterol compared with lean control mice. We conclude that the link among obesity, diet, and gallstone formation may not require hypersecretion of biliary cholesterol and may be related to the effects of diabetes, hyperlipidemia, or both on gallbladder motility.