Leptin-deficient Obese Mice Research Articles

Pioglitazone does not improve gallbladder motility in leptin-deficient obese diabetic mice

Pioglitazone does not improve gallbladder motility in leptin-deficient obese diabetic mice

Leptin increases small intestinal response to cholecystokinin in leptin-deficient obese mice 1

Leptin receptors are present in the jejunum, ileum, and vagal neurons. Leptin increases duodenal secretion of cholecystokinin (CCK) and acts with CCK on vagal mechanoreceptors in the regulation of small intestinal motility. We have demonstrated that leptin-deficient (Lepob) obese mice have increased jejunal and normal ileal responses to CCK. Therefore, we hypothesized that leptin administration alters small intestinal motility observed in leptin-deficient obese mice. Twelve-week-old female leptin-deficient (Lepob) obese mice received either saline (n=12) or 5 microg/g leptin ip (n=12) injections daily. After 4 weeks, jejunal and ileal segments were harvested, mounted in an organ bath, and reacted with acetylcholine (ACh, 10(-5)M) and CCK (10(-8,-7,-6)M). Data were expressed as N/cm2 and compared by ANOVA and Student's t test. The average body weights in the leptin-treated group were significantly decreased compared to those of the saline-treated group (34 versus 49 g, P <0.01). Jejunal responses to ACh within each group were significantly decreased (P <0.05) when compared to ileal responses. No significant differences in responses to ACh were observed between groups. Jejunal responses to 10(-7,-6)M CCK in the leptin-treated group were significantly greater than those in the saline-treated group. Ileal responses in the leptin group were similarly increased at all CCK concentrations. These data suggest that daily leptin administration for 4 weeks in leptin-deficient (Lepob) obese mice increases jejunal and ileal responses to CCK and does not alter responses to ACh. Therefore, we conclude that regulation of small intestinal motility may be influenced by synergistic action of cholecystokinin and leptin.

Altered intestinal motility in leptin-deficient obese mice 1

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. 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. 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). 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.

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.

Intestinal transit time is decreased in leptin-deficient obese mice

Background. Obesity is associated with multiple comorbidities, altered leptin metabolism, and increased intestinal absorption of nutrients. We have recently demonstrated that leptin-deficient (Lep ob) obese mice have increased ileal sucrase activity resulting in greater absorption of carbohydrate than lean controls. We have also demonstrated altered in vitro jejunal responses to cholecystokinin in leptin-deficient obese mice. Leptin-deficiency has also previously been shown to increase gastric emptying, but its influence on intestinal transit time is unknown. Therefore, we hypothesized that leptin deficiency would alter intestinal transit time. Methods. Gastric emptying and intestinal transit time experiments were performed in 12- to 14-week-old leptin-deficient ( Lep ob ) obese mice ( n = 10) and C57Bl/6J ( n = 10) lean control mice. Fluorescent-labeled liquid dextran (200 μL of 25 mg/4 mL) was gavaged into the stomach and intestine was harvested after 1 h. The contents of the stomach and entire small intestine were divided into 10 equal parts, including the cecum and colon. The fluorescence distributed along the gastrointestinal tract was obtained. Percentage gastric emptying was calculated as the amount of diet gavaged divided by the amount remaining. The geometric center was calculated as the sum of the % fluorescence per segment × segment number. Results. Data were analyzed by Student’s t-test and are presented in the table. Conclusions. These data suggest that leptin-deficiency results in (1) increased gastric emptying and (2) decreased intestinal transit time. Therefore, we conclude that decreased intestinal time transit time in leptin-deficient obese mice may contribute to enhanced nutrient absorptive efficiency commonly observed in obesity. TABLE—ABSTRACT P15 Strain % Gastric emptying Geometric center Lean 90.9 ± 1.7 8.35 ± 0.28 Obese 97.1 ± 0.6 ∗ 7.33 ± 0.30 ∗ ∗ P < 0.02 versus Lean.

Leptin administration increases small intestinal response to cholecystokinin in leptin-deficient obese mice

Introduction. Leptin receptors are present in the jejunum, to a lesser degree in the ileum, and also in vagal neurons. Leptin has been shown to increase the duodenal secretion of cholecystokinin (CCK) in rats and to act with CCK on vagal mechanoreceptors in the regulation of small intestinal motility in cats. We have recently demonstrated that leptin-deficient (Lep ob) obese mice have increased jejunal and normal ileal responses to CCK. To confirm that these abnormalities were due to leptin deficiency, we hypothesized that leptin administration would alter the abnormality in small intestinal motility observed in leptin-deficient obese mice. Methods. Twelve-week-old female leptin-deficient (Lep ob) obese mice received daily injections of saline ( n = 12) or 5 μg/g recombinant murine leptin IP ( n = 12). All animals were weighed daily. After 1 month of injections, whole thickness jejunal and ileal longitudinal segments were harvested and mounted in an organ bath. Optimal length and tension were determined by response to acetylcholine (ACh 10 −5 M). Contractile response to CCK 10 −8,−7,−6 M also was measured. Data are expressed as N/cm 2 and were compared by Student’s t-test and two-way ANOVA. Results. Body weights, and jejunal and ileal responses to 10 −5 M ACh and 10 −7 M CCK for both groups are presented below. Conclusions. These data suggest that daily leptin administration for 1 month in leptin-deficient (Lep ob) obese mice (1) increases both jejunal and ileal responses to cholecystokinin and (2) does not alter responses to acetylcholine. Therefore, we conclude that regulation of small intestinal motility may be influenced by the synergistic action of cholecystokinin and leptin. TABLE—ABSTRACT P74 Body Wt (g) Jejunum Ileum ACh CCK ACh CCK Saline 48.9 ± 0.1 532 ± 54 † 174 ± 28 1292 ± 103 211 ± 49 Leptin 33.8 ± 0.1 ∗ 649 ± 51 † 290 ± 50 ∗ 1191 ± 87 352 ± 46 ∗ ∗ P < 0.05 versus Saline; † P < 0.05 versus Ileum.

Expression of Interleukin-6 Is Greater in Preadipocytes than in Adipocytes of 3T3-L1 Cells and C57BL/6J and ob/ob Mice

Inflammation plays a major role in the development of chronic diseases such as cardiovascular disease and Type 2 diabetes. Further, it was demonstrated that obese animals and humans have significantly higher levels of circulating proinflammatory cytokines, such as interleukin-6 (IL-6). The aim of this study was to determine whether adipose tissue could be a major source of circulating IL-6 in leptin-deficient obese (ob/ob) mice by comparing the expression of IL-6 in different tissues of ob/ob mice. Our secondary goal was to determine whether preadipocytes are the source of adipose tissue IL-6. The ob/ob mice had higher levels of plasma IL-6 (P < 0.05) and adipose tissue IL-6 mRNA (P < 0.05) compared with lean mice. Interestingly, IL-6 mRNA levels of liver and spleen were not different between ob/ob and lean mice, whereas adipose tissue IL-6 mRNA levels were higher in the ob/ob mice compared with lean mice (P < 0.05). In addition, we showed that IL-6 secretion from the adipose tissue stromal vascular fraction cells was higher than that from fully differentiated adipocytes (P < 0.001). We further demonstrated that 3T3-L1 preadipocytes had significantly higher levels of lipopolysaccharide (LPS)-stimulated IL-6 mRNA and IL-6 secretion than differentiated 3T3-L1 adipocytes. Taken together, these data suggest that adipose tissue and preadipocytes from the adipose tissue stromal vascular fraction may contribute significantly to the increased plasma IL-6 levels in ob/ob mice.

CNTF restores gallbladder contractility in leptin-resistant obese diabetic mice

Background. Obesity and diabetes are major risk factors for the development of cholesterol gallstones, and the majority of obese people have leptin-resistant obesity. Previous studies from our laboratory have demonstrated that both leptin-deficient (Lep ob) and leptin-resistant (Lep db) obese diabetic mice have decreased in vitro gallbladder motility. We have also shown that leptin administration to leptin-deficient (Lep ob) animals restores gallbladder motility and reverses obesity and hyperinsulinemia. However, the administration of additional leptin to leptin-resistant (Lep db) mice would not be expected to ameliorate their physiologic parameters. In contrast, recent studies have demonstrated that ciliary neurotrophic factor (CNTF) can reduce weight and hyperinsulinemia in leptin-resistant obesity. Hypothesis. CNTF would cause weight loss, lower blood sugars, and restore gallbladder contractility in leptin-resistant (Lep db) mice. Methods. To test this hypothesis, 20 C57b/6J and 20 Lep db 8-week-old mice were injected intraperitoneally daily with either saline or 0.3 μg/g CNTF Ax15 for 17 days. At 11 weeks, mice were weighed and underwent cholecystectomy. Intact gallbladders were mounted in 3 mL muscle baths and stimulated with acetylcholine (ACh) and cholecystokinin (CCK). Serum glucose levels were measured. Data were analyzed by two-way ANOVA. Results are shown in the table. Conclusions. These data suggest that daily administration of ciliary neurotrophic factor (CNTF) causes (1) significant weight loss, (2) improvement of diabetes, and (3) significant alterations in gallbladder motility which is decreased in lean nondiabetic mice and improved in obese diabetic mice. We conclude that ciliary neurotrophic factor may improve gallbladder contractility in leptin-resistant obese diabetes. TABLE—ABSTRACT 48 Strain Wt (g) Glucose (mg/dL) ACh 10 −5 M (N/cm 2) CCK 10 −8 M (N/cm 2) Saline C57 17 ± 0 110 ± 12 .39 ± .04 1.14 ± .13 CNTF C57 13 ± 0 ∗ 101 ± 7 .20 ± .04 ∗ 0.59 ± .08 ∗ Saline Lep db 41 ± 2 † 440 ± 26 † .13 ± .03 ∗ 0.46 ± .08 ∗ CNTF Lep db 22 ± 1 ‡ 200 ± 33 † .54 ± .10 ‡ 1.02 ± .09 ‡ ∗ P < 0.05 versus Saline C57; † P < 0.01 versus other groups; Ddagger; P < 0.001 versus Saline lep db.

Leptin receptor-deficient MMTV-TGF-alpha/Lepr(db)Lepr(db) female mice do not develop oncogene-induced mammary tumors.

Being overweight is a risk factor for postmenopausal breast cancer and is associated with an increased incidence and shortened latency of spontaneous and chemically induced mammary tumors in rodents. However, leptin-deficient obese Lep(ob)Lep(ob) female mice have reduced incidences of spontaneous and oncogene-induced mammary tumors. Of interest, leptin enhances the proliferation of human breast cancer cell lines in which leptin receptors are expressed, which suggests that leptin signaling plays a role in tumor development. We evaluated oncogene-induced mammary tumor development in obese MMTV-TGF-alpha/Lepr(db)Lepr(db) mice that exhibit a defect in OB-Rb, which is considered to be the major signaling isoform of the leptin receptor. Lepr and MMTV-TGF-alpha mice were crossed, and the offspring were genotyped for oncogene expression and the determination of Lepr status. Lean MMTV-TGF-alpha/Lepr(+)Lepr(+) (homozygous) and MMTV-TGF-alpha/Lepr(+)Lepr(db) (heterozygous) mice and obese MMTV-TGF-alpha/Lepr(db)Lepr(db) mice were monitored until age 104 weeks. Body weights of MMTV-TGF-alpha/ Lepr(db)Lepr(db) mice were significantly heavier than those of the lean groups. No mammary tumors were detected in MMTV-TGF-alpha/Lepr(db)Lepr(db) mice, whereas the incidence of mammary tumors in MMTV-TGF-alpha/Lepr(+)Lepr(+) and MMTV-TGF-alpha/ Lepr(+)Lepr(db) mice was 69% and 82%, respectively. Examination of mammary tissue whole mounts indicated an absence of duct formation and branching for MMTV-TGF-alpha/Lepr(db)Lepr(db) mice. Both age at mammary tumor detection and tumor burden (tumors/mouse and tumor weights) were similar for the lean genotypes. Serum leptin levels of MMTV-TGF-alpha/Lepr(db)Lepr(db) mice were 12-20-fold higher than levels of lean mice. Thus, despite elevated serum leptin levels, leptin receptor-deficient MMTV-TGF-alpha/Lepr(db)Lepr(db) mice do not develop mammary tumors. This study provides additional evidence that leptin and its cognate receptor may be involved in mammary tumorigenesis.

Leptin corrects increased gene expression of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase and -24-hydroxylase in leptin-deficient, ob/ob mice.

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We investigated leptin effects on bone metabolism using male leptin-deficient obese (ob/ob) mice, which had lower bone mineral density (BMD) and shorter femurs than lean (?/+) controls. Serum concentrations of calcium, phosphate, tartrate-resistant acid phosphatase (a bone resorption marker) and alkaline phosphatase, and urinary calcium and phosphate excretion were significantly elevated in ob/ob mice, whereas urinary concentrations of deoxypyridinoline did not differed between ob/ob and control mice. Because ob/ob mice develop severe hypogonadism, testosterone was administered to these mice for 10 wk (5 mg/kg, sc, twice weekly); this did not affect femoral BMD. Control and ob/ob mice showed similar vitamin D-receptor densities in bone and kidney; the obese mice had marked increases in serum 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] and in mRNA expression and activities of renal 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (CYP27B1) and -24-hydroxylase (CYP24) compared with control mice. Leptin administration to ob/ob mice (4 mg/kg body weight, ip, every 12 h for 2 d) greatly reduced mRNAs and activities of 1 alpha-hydroxylase and 24-hydroxylase. Elevated concentrations of serum calcium, phosphate, and 1,25-(OH)(2)D(3) were normalized by leptin treatment. Thus, leptin suppresses renal gene overexpression for 1 alpha-hydroxylase and 24-hydroxylase and corrects increased serum concentrations of calcium and phosphate in ob/ob mice. Therefore, low BMD in leptin-deficient mice appears to be related to stimulation of bone resorption by 1,25-(OH)(2)D(3).

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.

Leptin-deficient obese mice have altered intestinal metabolic enzyme activities

Introduction: Obesity is associated with multiple comorbidities, altered leptin metabolism, and increased intestinal absorption of nutrients. Leptin, the protein product of the mouse obese (ob) gene, inhibits carbohydrate absorption and improves the transport of oligopeptides across the intestinal epithelium via the PepT-1 transporter. Therefore, we hypothesized that leptin-deficient (Lepob) obese mice would have altered sucrase and brush border peptidase activities compared to lean controls. Methods: Sucrase and peptidase (aminopeptidase N (ApN), dipeptidyl peptidase IV (DPPIV)) activities were determined in jejunal and ileal segments from 16–18 week old male, lean control (C57BL/6J, n = 8) and leptin-deficient (Lepob, n = 8) obese mice. Data were analyzed using two-way ANOVA and Student-Newman-Keuls test for pair-wise multiple comparisons. Results: Lepob mice had significantly increased ileal (IL) sucrase activity and decreased jejunal (JE) ApN and DPPIV activity compared to lean controls. The activity for all three enzymes was greater in the jejunum than ileum for the lean control mice, whereas no difference was observed between the tissues for Lepob mice. Data (± SEM) are presented in the table below. Conclusions: These findings suggest 1) leptin deficient obese mice have greater absorption of carbohydrate in the ileum than lean controls and 2) diminished absorption for protein in the jejunum. Therefore, we conclude that the increased efficiency of carbohydrate absorption and not protein, may contribute to the obesity and diabetes evident in leptin-deficient obese mice. TABLE—ABSTRACT P58Strain/tissueSucrase (mcmol/cm/m in ×10−2)ApN (U/g)DPPIV (U/g)Lean/JE1.2 ± 0.1†17.3 ± 2.8†7.6 ± 0.5†Lean/IL0.5 ± 0.19.5 ± 2.83.9 ± 0.5Lepob/JE1.1 ± 0.19.6 ± 1.3∗4.0 ± 0.6∗Lepob/IL1.1 ± 0.1∗9.0 ± 0.72.5 ± 0.6∗P < 0.05 vs lean control.†P < 0.05 vs ileum same strain.

Intermittent hypoxia increases insulin resistance in genetically obese mice.

Obstructive sleep apnoea, a syndrome that leads to recurrent intermittent hypoxia, is associated with insulin resistance in obese individuals, but the mechanisms underlying this association remain unknown. We utilized a mouse model to examine the effects of intermittent hypoxia on insulin resistance in lean C57BL/6J mice and leptin-deficient obese (C57BL/6J-Lepob) mice. In lean mice, exposure to intermittent hypoxia for 5 days (short term) resulted in a decrease in fasting blood glucose levels (from 173 +/- 11 mg dl-1 on day 0 to 138 +/- 10 mg dl-1 on day 5, P < 0.01), improvement in glucose tolerance without a change in serum insulin levels and an increase in serum leptin levels in comparison with control (2.6 +/- 0.3 vs. 1.7 +/- 0.2 ng ml-1, P < 0.05). Microarray mRNA analysis of adipose tissue revealed that leptin was the only upregulated gene affecting glucose uptake. In obese mice, short-term intermittent hypoxia led to a decrease in blood glucose levels accompanied by a 607 +/- 136 % (P < 0.01) increase in serum insulin levels. This increase in insulin secretion after 5 days of intermittent hypoxia was completely abolished by prior leptin infusion. Obese mice exposed to intermittent hypoxia for 12 weeks (long term) developed a time-dependent increase in fasting serum insulin levels (from 3.6 +/- 1.1 ng ml-1 at baseline to 9.8 +/- 1.8 ng ml-1 at week 12, P < 0.001) and worsening glucose tolerance, consistent with an increase in insulin resistance. We conclude that the increase in insulin resistance in response to intermittent hypoxia is dependent on the disruption of leptin pathways.

Alterations in nitric oxide-cGMP pathway in ventricular myocytes from obese leptin-deficient mice.

Leptin is a regulator of body weight and affects nitric oxide (NO) production. This study was designed to determine whether the myocardial NO-cGMP signal transduction system was altered in leptin-deficient obese mice. Contractile function, guanylyl cyclase activity, and cGMP-dependent protein phosphorylation were assessed in ventricular myocytes isolated from genetically obese (B6.V-Lepob) and age-matched lean (C57BL/6J) mice. There were no differences in baseline contraction between the lean and obese groups. After stimulation with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10-6 and 10-5 M) or a membrane-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10(-6) and 10(-5) M), cell contractility was depressed. However, 8-Br-cGMP had significantly greater effects in obese mice than in lean controls with percent shortening reduced by 47 vs. 39% and maximal rate of shortening decreased by 46 vs. 36%. The negative effects of SNAP were similar between the two groups. Soluble guanylyl cyclase activity was not attenuated. This suggests that the activity of the cGMP-independent NO pathway may be enhanced in obesity. The phosphorylated protein profile of cGMP-dependent protein kinase showed that four proteins were more intensively phosphorylated in obese mice, which suggests an explanation for the enhanced effect of cGMP. These results indicate that the NO-cGMP signaling pathway was significantly altered in ventricular myocytes from the leptin-deficient obese mouse model.

Meal patterns of lean and leptin-deficient obese mice in a simulated foraging environment

C57BL/6J lean and obese ( lep −/−) mice were studied in a closed economy operant protocol that simulates foraging. A predetermined number of presses on a procurement lever (PFR) activated a consumatory lever on which presses would produce 20-mg food pellets. Mice could eat as much as they wished but, once no responding occurred for an elapsed 10-min period, the consumatory lever was inactivated and the procurement or foraging cycle began again. Under these conditions, as has been shown for rats and other species, mice initiated relatively discrete meals (about nine per day) at the lowest PFR, and the number of meals initiated per day decreased with increasing PFR. Meal size increased reciprocally, so that total intake was conserved across the range of PFR examined. Obese mice ate larger meals than lean mice at low PFR, and showed further increases but only at the highest PFRs. The small and inconsistent literature on meal patterns in mice is reviewed, and we discuss the utility of the present protocol to study the interactions between genetic and environmental economic factors, and their implications for the etiology of human obesity.

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. ( J Gastrointest Surg 2002;6:438–444.)

Evaluation of Liver Fatty Acid Oxidation in the Leptin-Deficient Obese Mouse

We hypothesized that liver fatty acid oxidation (FAO) is compromised in the leptin-deficient obese (Lepob/Lepob) mouse model, and that this would be further challenged when these mice were fed a high-fat diet. Obese mice had a 3.8-fold increased body fat content and a 9-fold increased liver fat content as compared to control mice when both groups were fed a low-fat diet. The expression of liver FAO enzymes, carnitine palmitoyltransferase-1a, long-chain acyl-CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and short-chain acyl-CoA dehydrogenase, was not affected in obese mice as compared to controls on either a low-fat or a high-fat diet. The expression of very-long-chain acyl-CoA dehydrogenase was elevated in obese mice on the control diet, as compared to control mice. For all measures evaluated, increasing the level of fat in the diet had a smaller effect than leptin deficiency. In summary, despite obese mice having an excess of fat available for mitochondrial β-oxidation in liver, overall energy balance appeared to dictate that the net liver FAO remained at control levels.

Distribution and Regulation of Galanin-Like Peptide (GALP) in the Hypothalamus of the Mouse

Galanin-like peptide (GALP) is a newly discovered molecule whose expression in the brain is confined to the arcuate nucleus and median eminence. In the rat, cellular levels of GALP mRNA are reduced by fasting and reversed by peripheral administration of leptin. The purpose of this investigation was 1) to clone and map the distribution of GALP mRNA in the brain of the mouse; 2) to compare the pattern and magnitude of GALP mRNA expression in the leptin-deficient obese (ob/ob) mouse with that of wild-type controls; and 3) to examine the effects of leptin delivered into the brain on the expression of GALP mRNA in the ob/ob mouse. We report the sequence of a mouse GALP cDNA and show that GALP mRNA is expressed in the arcuate nucleus, median eminence, infundibular stalk, and the neurohypophysis of this species. The expression of GALP mRNA in the brain was markedly reduced in the ob/ob mice, compared with wild-type animals. Intracerebroventricular infusion of leptin to ob/ob mice increased both the number of GALP mRNA-expressing neurons and their content of GALP mRNA, compared with vehicle-treated controls. These observations demonstrate that GALP mRNA is induced by leptin through a direct action on the brain.

Effects of leptin deficiency and short-term repletion on hepatic gene expression in genetically obese mice.

By supplying most organs of the body with metabolic substrates, the liver plays a central role in maintaining energy balance. Hepatic metabolism of glucose, fatty acids, and lipoproteins is disrupted in the leptin-deficient obese (Lep(ob)/Lep(ob)) mouse, leading to hyperglycemia, steatosis, and hypercholesterolemia. Microarray expression profiles were used to identify transcriptional perturbations that underlie the altered hepatic physiology of Lep(ob)/Lep(ob) mice. A wide variety of genes involved in fatty acid metabolism are altered in expression, which suggests that both fatty acid synthesis and oxidation programs are activated in obese mice. The expression of a small subset of genes is upregulated by leptin deficiency, not modulated by caloric restriction, and markedly suppressed by short-term leptin treatment. Among these leptin-regulated genes, apolipoprotein A-IV is a strong candidate for mediating the atherogenic-resistant phenotype of Lep(ob)/Lep(ob) mice.

Decreased gallbladder response in leptin-deficient obese mice

Decreased gallbladder response in leptin-deficient obese mice