Altered intestinal motility in leptin-deficient obese mice

INTRODUCTION: Leptin is produced by adipocytes and causes satiety by regulating hypothalamic neurotransmission and energy expenditure. Leptin functions through the active long form of its receptor, which is expressed throughout the gastrointestinal tract, including the vagal neurons concerned with small intestinal motility. However, the role of leptin in small intestinal motility is poorly understood. Therefore, we hypothesized that leptin-deficient (Lepob) obese mice would have altered small intestinal response to neurotransmitters and transit time. MATERIALS AND METHODS: Responses of jejunal and ileal segments from lean control and leptin-deficient obese animals to acetylcholine (ACh) and cholecystokinin (CCK) were determined in an organ bath. In addition, gastric emptying was determined as the amount of gavaged liquid diet remaining in the stomach after 1 h, and intestinal transit time was determined by calculating the geometric center (GC) of passage of a fluorescent-labeled marker. RESULTS: Leptin deficiency resulted in increased jejunal responses to CCK (P <0.05) and a similar response to ACh compared to lean controls. Also, gastric emptying (97% versus 91%, P <0.001) in obese mice was greater. Overall small intestinal transit (GC) in obese mice was decreased (7.3 versus 8.4, P <0.05) even though proximal transit was increased (5.3 versus 1.5, P <0.06). CONCLUSIONS: These studies indicate that leptin-deficient (Lepob) obese mice have an increased jejunal response to CCK as well as an increased proximal intestinal transit, but an overall decrease in small intestinal transit.     

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.     

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.     

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.     

Gallbladder motility in agouti-yellow and leptin-resistant obese mice

BACKGROUND: Obesity is a polygenic disorder that is associated with gallstone disease. We have previously shown that leptin deficiency in obese mice correlates with decreased gallbladder motility, suggesting that leptin plays a role in the link between gallstone disease and obesity. However, most obese humans are leptin-resistant, and relatively few are leptin-deficient. To confirm that leptin dysfunction is responsible for impaired gallbladder motility in obese mice, we hypothesized that leptin-resistant obese mice (Lep(db)) would have abnormal gallbladder motility while obese mice with intact leptin function (Agouti Yellow, A(y)) would have normal gallbladder motility. MATERIALS AND METHODS: Eighteen lean control (C57BL/6J), 10 A(y) and 12 Lep(db) female mice were fasted overnight, weighed, and livers and gallbladders were harvested. Liver weights and gallbladder volumes were measured. Gallbladder contractile responses (N/cm(2)) to acetylcholine (10(-5)M), neuropeptide Y (10(-8,-7,-6) M) and cholecystokinin (10(-10,-9,-8,-7)M) were determined in muscle bath chambers. Results were analyzed by analysis of various (ANOVA) and with the Mann-Whitney Rank Sum Test. RESULTS: Both Agouti yellow (A(y)) and leptin-resistant (Lep(db)) obese mice had body weights, liver weights and gallbladder volumes that were significantly greater (P < 0.01) than lean control mice. Leptin-resistant obese mice had gallbladder responses to acetylcholine, neuropeptide Y and cholecystokinin that were significantly less (P < 0.01) than both lean control and Agouti yellow obese mice. CONCLUSIONS: These data suggest that (1). leptin-resistant obese mice (Lep(db)) have abnormal gallbladder motility and (2). obese mice with normal leptin metabolism (A(y)) have normal gallbladder response to neurotransmitters. We conclude that leptin represents a link between obesity, gallbladder motility and gallstone formation.     

Diabetes and hyperlipidemia correlate with gallbladder contractility in leptin-related murine obesity

Obesity is associated with many comorbid conditions including diabetes, hyperlipidemia, and gallstones. However, the interaction among these modalities remains unclear. We recently demonstrated that both leptin-deficient and leptin-resistant obese mice have impaired biliary motility. These obese mice also are diabetic and hyperlipidemic. Therefore, we tested the hypothesis that serum glucose, insulin, cholesterol, and triglyceride levels would correlate with gallbladder contractility. Thirty-four lean control, 10 lean heterozygous leptin-deficient, 18 obese homozygous leptin-deficient, and 12 obese homozygous leptin-resistant mice were fed a nonlithogenic chow diet while nine lean control and nine obese homozygous leptin-deficient mice were fed a high-cholesterol diet for 4 weeks. In vitro gallbladder responses to cholecystokinin (CCK; 10^-8 mol/L), acetylcholine (ACh; 10^-5 mol/L), and neuropeptide Y (NPY; 10^-6 mol/L) were measured. Serum glucose, insulin, cholesterol, and triglyceride levels were measured from pooled serum from an additional 704 animals. Gallbladder responses were greatest for CCK, intermediate for ACh, and least for NPY. Serum glucose, insulin, cholesterol, and triglyceride levels and body weight all correlated similarly, negatively, and significantly (P < 0.001) with gallbladder contractility. Hyperglycemia, insulin-resistance, hyperlipidemia, and body weight in obese mice with leptin dysfunction are associated with poor gallbladder contractility, which in turn may contribute to the association between obesity and gallstone formation. Presented in part at Digestive Disease Week 2003, SSAT Plenary Session and Residents’ Conference, Orlando, Florida, May 17–22, 2003 (oral presentation); and at the Association for Academic Surgery, Poster Session, Boston, Massachusetts, November 7–9, 2002. Supported by grant NIH R-01 DK44279 from the National Institutes of Health.     

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.     

Luminal leptin induces rapid inhibition of active intestinal absorption of glucose mediated by sodium-glucose cotransporter 1

The effect of leptin on glucose transport was studied in rat jejunal mucosa in Ussing chambers. Leptin was added in the luminal or the serosal compartment before the tissues were challenged with 1, 10, or 50 mmol/l glucose. In response to 10 mmol/l glucose, the increase in short-circuit current (DeltaIsc) reached 26.8 +/- 2.1 microA/cm(2). Luminal addition of leptin dramatically decreased glucose-induced Isc (90.5% for 10 nmol/l leptin). Inhibition was maximal after 5 min and dose dependent (IC(50) = 0.13 nM). Western blot analysis showed that rapid inhibition of glucose-induced Isc by leptin was associated with a parallel decrease in the abundance of sodium-glucose transporter-1 in brush border membranes. Inhibition by luminal leptin of DeltaIsc was prevented by inhibitor of conventional protein kinase C isoforms. Serosal addition of leptin did not decrease glucose-induced Isc within 5 min and reached maximum after 10 min. The effect of leptin from serosal side was blocked by cholecystokinin (CCK) receptor-2 receptor antagonist YM022. Altogether, these data demonstrate that luminal leptin induces rapid inhibition of glucose entry into enterocyte. The slower action of leptin on the serosal side of mucosa seems indirect and is likely mediated by endogenous CCK. They demonstrate that gut leptin is a major regulator of rapid intestinal glucose transport.     

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. Presented at the Forty-Second Annual Meeting of The Society for Surgery of the Alimentary Tract, Atlanta, Georgia, May 20–23, 2001 (oral presentation). Supported by grant RO1-DK442 79–07 from the National Institutes of Health.     

Role of selective leptin resistance in diet-induced obesity hypertension

Leptin is an adipocyte-derived hormone that plays a key role in the regulation of body weight through its actions on appetite and metabolism. Leptin also increases sympathetic nerve activity (SNA) and blood pressure. We tested the hypothesis that diet-induced obesity is associated with resistance to the metabolic actions of leptin but preservation of its renal SNA and arterial pressure effects, leading to hypertension. Mice were fed a high-fat diet for 10 weeks to induce moderate obesity. The decrease in food intake and body weight induced by intraperitoneal or intracerebroventricular leptin was significantly attenuated in the obese mice. Regional SNA responses to leptin were differentially altered in diet-induced obese mice. Renal SNA response to leptin was preserved, whereas lumbar and brown adipose tissue SNA responses were attenuated in obese mice. Radiotelemetric arterial pressure was approximately 10 mmHg higher in obese mice. Furthermore, the increase in arterial pressure in response to long-term (12 days) leptin treatment was preserved in obese mice. Thus, mice with diet-induced obesity exhibit circulating hyperleptinemia and resistance to the metabolic actions of leptin. However, there is preservation of the renal sympathetic and arterial pressure responses to leptin, which represent a potential mechanism for the adverse cardiovascular consequences of obesity.     

Duodenal leptin stimulates cholecystokinin secretion: evidence of a positive leptin-cholecystokinin feedback loop

Some of the actions of leptin depend on cholecystokinin (CCK). However, it is unknown whether leptin modulates the release of CCK. Here, we demonstrate in vitro that leptin induces the phosphorylation of extracellular signal-related kinase (ERK)-1/2 proteins and increases CCK release (EC(50) = 0.23 nmol/l) in CCK-secreting STC-1 cells. We showed that rat duodenal juice contains leptin that circulates free and bound to macromolecules, suggesting that leptin has a lumenal action on the intestine. In vivo in the rat, duodenal infusion of leptin increased plasma CCK at levels comparable to those induced by feeding. Moreover, meal-induced increases in plasma CCK were markedly reduced in obese fa/fa rats, whereas the mobilization of the gastric leptin pool was similar in lean and obese Zucker rats. The release of CCK by leptin presumably generates a positive feedback loop. Indeed, the blockade of CCK receptors reversed the meal reduction of the stomach leptin pool and the meal-increased plasma insulin, consistent with the previous concept of an entero-insular axis. Collectively, these data support a novel mode of action of leptin where leptin and CCK may potentiate their own effects by cross-stimulating their secretion. The impairment of this leptin-CCK loop may have pathological implications related to obesity and diabetes.     

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.     

Role of leptin in the regulation of glucagon-like peptide-1 secretion

Glucagon-like peptide-1 (GLP-1), released from intestinal endocrine L cells, is a potent insulinotropic hormone. GLP-1 secretion is diminished in obese patients. Because obesity is linked to abnormal leptin signaling, we hypothesized that leptin may modulate GLP-1 secretion. Leptin significantly stimulated GLP-1 secretion (by up to 250% of control) from fetal rat intestinal cells, a mouse L cell line (GLUTag), and a human L cell line (NCI-H716) in a dose-dependent manner (P < 0.05-0.001). The long form of the leptin receptor was shown to be expressed, and leptin induced the phosphorylation of STAT3 in the three cell types. The leptin receptor was also expressed by rodent and human intestinal L cells, and leptin (1 mg/kg i.p.) significantly stimulated GLP-1 secretion in rats and ob/ob mice. To determine the effect of leptin resistance on GLP-1 secretion, C57BL/6 mice were fed a high-fat (45%) or low-fat (10%) diet for 8 weeks. Mice on the high-fat diet became obese; developed glucose intolerance, hyperinsulinemia, and hyperleptinemia; and were leptin resistant. Mice on the high-fat diet also had twofold lower basal plasma GLP-1 and a diminished GLP-1 response to oral glucose, by 28.5 +/- 5.0% (P < 0.05). These results show for the first time that leptin stimulates GLP-1 secretion from rodent and human intestinal L cells, and they suggest that leptin resistance may account for the decreased levels of GLP-1 found in obese humans.     

Leptin stimulates esophageal adenocarcinoma growth by nonapoptotic mechanisms

BACKGROUND: Leptin is a hormone primarily produced by adipocytes and serum leptin is elevated in obese persons. One risk factor associated with adenocarcinoma of the esophagus is obesity. We hypothesized that leptin would have stimulatory effects on esophageal adenocarcinoma and alter apoptosis in vitro. METHODS: Barrett's esophageal adenocarcinoma cells (BIC-1 and SEG-1) were cultured with human recombinant leptin (80 ng/mL) for 24 hours. Cell growth was determined by MTT assay. Apoptosis and necrosis was measured after 16 hours of treatment with leptin using a Cell Death Kit. RESULTS: Exogenous leptin stimulated cell proliferation in both cell lines. No changes in apoptosis or necrosis resulted between control and leptin-treated groups. CONCLUSIONS: We have shown that leptin increases the proliferation of human esophageal adenocarcinoma, but does not alter cell apoptosis or necrosis. The data suggest that leptin stimulates esophageal adenocarcinoma growth by nonapoptotic mechanisms. Leptin antagonism may have potential efficacy in esophageal cancer therapy.     

Cholecystokinin and leptin: their influence upon the eating behaviour and nutrient intake of dialysis patients.

BACKGROUND: We have used serial visual analogue scores to demonstrate disturbances of the appetite profile in dialysis patients. This is potentially important as dialysis patients are prone to malnutrition yet have a lower nutrient intake than controls. Appetite disturbance may be influenced by accumulation of appetite inhibitors such as leptin and cholecystokinin (CCK) in dialysis patients. METHODS: Fasting blood samples were drawn from 43 controls, 50 haemodialysis (HD) and 39 peritoneal dialysis (PD) patients to measure leptin and CCK. Hunger and fullness scores were derived from profiles compiled using hourly visual analogue scores. Nutrient intake was derived from 3 day dietary records. RESULTS: Fasting CCK was elevated for PD (6.73 +/- 4.42 ng/l vs control 4.99 +/- 2.23 ng/l, P < 0.05; vs HD 4.43 +/- 2.15 ng/l, P < 0.01). Fasting CCK correlated with the variability of the hunger (r = 0.426, P = 0.01) and fullness (r = 0.52, P = 0.002) scores for PD. There was a notable relationship with the increase in fullness after lunch for PD (r = 0.455, P = 0.006). When well nourished PD patients were compared with their malnourished counterparts, CCK was higher in the malnourished group (P = 0.004). Leptin levels were higher for the dialysis patients than controls (HD and PD, P < 0.001) with pronounced hyperleptinaemia evident in some PD patients. Control leptin levels demonstrated correlation with fullness scores (e.g. peak fullness, r = 0.45, P = 0.007) but the dialysis patients did not. PD nutrient intake (energy and protein intake, r = -0.56, P < 0.0001) demonstrated significant negative correlation with leptin. CONCLUSION: Increased CCK levels appear to influence fullness and hunger perception in PD patients and thus may contribute to malnutrition. Leptin does not appear to affect perceived appetite in dialysis patients but it may influence nutrient intake in PD patients via central feeding centres.     

Mutations in the human leptin and leptin receptor genes as models of serum leptin receptor regulation

A part of serum Ob leptin, an adipocyte-secreted peptide, is bound to a soluble Ob receptor (sObR). Immunoreactive sObR was measured in 125 lean or obese control subjects (group 1), 18 individuals with a mutation in the leptin gene impairing leptin secretion (group 2), and 10 individuals with a mutation in the ObR gene, leading to production of a truncated ObR not anchored to cell membranes (group 3). In group 1, sObR levels were negatively correlated with age and BMI in children and with BMI in adults. sObR levels were also negatively correlated with leptin levels. Leptin binding activity and sObR levels coeluted in gel-filtration chromatography. In group 2, sObR levels did not differ from those in lean control subjects and were not correlated with BMI. A single peak was detected in chromatographic fractions. In group 3, sObR levels were high and positively correlated with BMI. Immunoreactive sObR coeluted with leptin binding activity. These data demonstrate that leptin is not needed for ObR gene expression, and they suggest that leptin plays a role in receptor downregulation because sObR levels are negatively correlated with leptin levels and BMI in control subjects, whereas sObR levels are not depressed in obese leptin-deficient or leptin receptor-deficient individuals.     

Leptin treatment induces loss of bone marrow adipocytes and increases bone formation in leptin-deficient ob/ob mice.

Normal mice and leptin-deficient ob/ob mice were treated with leptin to study effects on osteogenesis and adipogenesis in bone marrow. Leptin treatment significantly decreased bone marrow adipocyte size and number in ob/ob mice while increasing bone formation, BMC, and BMD. The results suggest that, in leptin-sensitive animals, the reduction in marrow adipocytes has positive effects on bone formation. INTRODUCTION: Adipocytes, osteoblasts, and osteoclasts have leptin receptors, and leptin can also affect bone metabolism indirectly through its receptors in the hypothalamus. We examined the effects of leptin treatment on bone formation, BMD, and marrow adipocyte population in normal mice and leptin-deficient ob/ob mice. MATERIALS AND METHODS: At the age of 15 weeks, mice were implanted with Alzet osmotic pumps for subcutaneous delivery of treatment solutions (saline, 2.5 microg leptin/day, or 10 microg leptin/day) for 14 days at a delivery rate of 0.25 microl/h. Bone formation was assessed using fluorochrome labels, cell populations were quantified using histomorphometry, and bone densitometry was measured using DXA. We also used a Luminex Beadlyte assay system to quantify cell survival markers in bone marrow samples. RESULTS AND CONCLUSIONS: Results indicate that both doses of leptin decreased the number of marrow adipocytes in ob/ob mice by >20% (p < 0.05) compared with PBS-treated ob/ob mice. The decrease in adipocyte number with leptin treatment is accompanied by an increase in concentration of the apoptosis marker caspase-3 in bone marrow adipocytes and hematopoietic cells. Both leptin doses also significantly (p < 0.05) increased the percentage of fluorochrome-labeled tibial endosteal surface by >30% compared with PBS-treated ob/ob mice. Leptin treatment increased whole body BMC by >30% in the ob/ob mice receiving the highest leptin dose. Leptin treatment provided no increase in bone formation, BMC, or BMD in normal, leptin-replete mice.     

Inhibition by beta-CCM of cholecystokinin-induced release of acetylcholine from the longitudinal muscle-myenteric plexus preparation in the guinea-pig ileum

The antagonism between cholecystokinin (CCK) and methyl beta-carboline-3-carboxylate (beta-CCM) in the nervous system was studied by measuring the release of acetylcholine (ACh) from the longitudinal muscle-myenteric plexus preparation of guinea-pig. The ACh release was assessed by measuring [3H] output from the preparation preincubated with [3H] choline. Thirty mM of KCl caused a pronounced release of [3H] ACh from the preparation. Sulfated cholecystokinin octapeptide (CCK8) also increased the release of [3H] ACh in a dose-dependent manner at concentrations ranging from 10(-10)M to 10(-8)M. CCK8 at a concentration of 10(-8)M released [3H] ACh by about 300% of the 30 mM KCl-induced [3H] ACh release. In the presence of beta-CCM (10(-8)M to 10(-4)M), the release of [3H] ACh by KCl was not affected, but that by CCK8 was significantly inhibited depending on the concentrations of beta-CCM. These results show that the action of CCK8 to stimulate neurons in the myenteric plexus can be selectively antagonized by beta-CCM.     

Impairment of host resistance to Listeria monocytogenes infection in liver of db/db and ob/ob mice

Leptin is an adipocyte-derived hormone that regulates a number of physiological functions, including energy homeostasis and immune function. In immune responses, leptin plays a role in the induction of inflammation. We investigated a role of leptin in Listeria monocytogenes infection using leptin receptor-deficient db/db mice and leptin-deficient ob/ob mice. These mutant mice were highly susceptible to L. monocytogenes, and the elimination of bacteria from the liver was inhibited. After infection, the induction of monocyte chemoattractant protein-1 (MCP-1) and KC mRNA in the liver of db/db mice and the MCP-1 mRNA expression in the liver of ob/ob mice was decreased compared with their heterozygote littermates. Leptin replacement in ob/ob mice resulted in improvement of anti-listerial resistance and the MCP-1 mRNA expression. The elimination of L. monocytogenes was significantly enhanced, and the expression of MCP-1 and KC mRNA was completely reversed in db/db mice by insulin treatment. These results suggest that leptin is required for host resistance to L. monocytogenes infection and that hyperglycemia caused by leptin deficiency is involved in the inefficient elimination of bacteria from the liver. Moreover, defect of MCP-1 expression in the liver may be involved in the attenuated host resistance in these mutant mice.     

Hypothalamic control of bone formation: distinct actions of leptin and y2 receptor pathways.

Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2-/- anabolic pathways in modulating bone formation. INTRODUCTION: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient ob/ob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms. MATERIALS AND METHODS: The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2-/- ob/ob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2-/- mice. Cancellous bone volume and bone cell function were assessed. RESULTS: Osteoblast activity was comparably elevated in ob/ob, Y2-/-, and Y2-/- ob/ob mice. However, greater bone resorption in ob/ob and Y2-/- ob/ob mice reduced cancellous bone volume compared with Y2-/-. Both wildtype and Y2-/- AAV-NPY mice exhibited marked elevation of white adipose tissue accumulation and hence leptin expression, thereby reducing osteoblast activity. Despite this anti-osteogenic leptin effect in the obese AAV-NPY model, osteoblast activity in Y2-/- AAV-NPY mice remained significantly greater than in wildtype AAV-NPY mice. CONCLUSIONS: This study suggests that NPY is not a key regulator of the leptin-dependent osteoblast activity, because both the leptin-deficient stimulation of bone formation and the excess leptin inhibition of bone formation can occur in the presence of high hypothalamic NPY. The Y2-/- pathway acts consistently to stimulate bone formation; in contrast, leptin continues to suppress bone formation as circulating levels increase. As a result, they act increasingly in opposition as obesity becomes more marked. Thus, in the absence of leptin, the cancellous bone response to loss of Y2 receptor and leptin activity can not be distinguished. However, as leptin levels increase to physiological levels, distinct signaling pathways are revealed.