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.     

Gallbladder disease in the morbidly obese

During a 7 year period, 200 consecutive morbidly obese patients underwent a standardized gastric exclusion procedure. Group A was composed of the first 120 patients and Group B of the last 80 patients. In Group A, 22 patients had undergone a previous cholecystectomy and 12 patients had a cholecystectomy at the time of gastric exclusion because of positive diagnostic studies or palpation of stones. Of the remaining 87 patients in this initial group who were at risk for the development of gallbladder disease, 24 (27.6 percent) required a cholecystectomy in the first 3 postoperative years (mean 15.6 months). Twelve patients had acute cholecystitis, 3 patients had choledocholithiasis, and 1 patient had acute gallstone pancreatitis. In Group B, 18 patients had a previous cholecystectomy, 15 had positive diagnostic studies (ultrasonography and oral cholecystography) preoperatively, and 47 had negative studies. Cholecystectomy was routinely performed at the time of gastric exclusion surgery in the 62 patients with gallbladders in Group B. Of the 47 patients who had normal preoperative diagnostic studies, 40 (85.1 percent) had abnormal histologic findings in the gallbladder. Only seven patients in Group B had a normal gallbladder (14.7 percent). We conclude that gallbladder disease is considerably more frequent in the morbidly obese population (91.3 percent) than has previously been recognized, that diagnostic studies are frequently inaccurate, and that postoperative gallbladder disease is common (28.7 percent). On the basis of these results, routine cholecystectomy at the time of gastric exclusion surgery is recommended.     

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/cm2)	CCK 10−8 M (N/cm2)
Saline C57	17 ± 0	110 ± 12	.39 ± .04	1.14 ± .13
CNTF C57	13 ± 0∗	101 ± 7	.20 ± .04∗	0.59 ± .08∗
Saline Lepdb	41 ± 2†	440 ± 26†	.13 ± .03∗	0.46 ± .08∗
CNTF Lepdb	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.

Full-size table

     

Hepatic expression of microsomal triglyceride transfer protein and in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice

Secondary hyperlipidemia is a major cardiovascular risk factor in individuals with type 2 diabetes. Increased hepatic production of apolipoprotein B (apoB)-containing lipoproteins contributes to the elevated plasma levels, but the mechanism is poorly understood. Recent results have established that microsomal triglyceride transfer protein (MTP) is rate limiting for the assembly and secretion of apoB-containing lipoproteins. To better understand the mechanism of type 2 diabetes-associated hyperlipidemia, we quantified hepatic MTP mRNA levels, hepatic microsomal triglyceride transfer activity, and in vivo triglyceride secretion from the liver in two diabetic mouse models. Obese diabetic (ob/ob) mice had 45% higher (P = 0.006) hepatic MTP mRNA levels, 54% higher (P < 0.0001) microsomal triglyceride transfer activity, and 70% higher (P < 0.0001) in vivo triglyceride secretion rates compared with ob/+ control mice. In contrast, in lean streptozotocin-treated diabetic mice, hepatic MTP mRNA levels were unchanged, whereas microsomal triglyceride transfer activity and in vivo triglyceride secretion rates were marginally decreased. These studies suggest that obesity-induced type 2 diabetes in mice confers increases in hepatic MTP expression and secretion of triglyceride-rich lipoproteins. High blood glucose and altered hepatic expression of sterol regulatory element binding protein genes play a minor role in this diabetic response.     

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.     

Immunoreactive neurotensin in the pancreas of genetically obese and diabetic mice. A longitudinal study

Immunoreactive neurotensin (IR-NT) content in 2 N acetic acid extracts of pancreas was measured in genetically diabetic (C57BL/KsJ db/db and ob/ob) and obese (C57BL/6J ob/ob and db/db) mice and normal littermate controls from 5 to 24 wk of age to determine the relationship of any changes to the development of metabolic abnormalities. Pancreatic IR-NT in obese mice showed no consistent change compared with lean littermate controls. In contrast, diabetic mice demonstrated an increase in pancreatic IR-NT that occurred at 6-8 wk of age, and maximal about the time of islet B-cell failure (8-10 wk), and persisted over the study period. Pancreatic IR-NT eluted in two peaks on reverse phase high-pressure liquid chromatography, one of which exhibited a retention time similar to that of synthetic NT. These findings suggest that pancreatic IR-NT concentration is regulated by insulin, with elevated levels occurring in association with insulin deficiency and its metabolic consequences but not with insulin resistance. Taken together with the previous demonstration that NT influences pancreatic islet hormone secretion, the present findings support a possible role of endogenous NT in islet hormone regulation.     

Temporal relationship of tissue somatostatin-like immunoreactivity to metabolic changes in genetically obese and diabetic mice

Somatostatin-like immunoreactivity (SRIF-LI) content in 2 N acetic acid extracts of hypothalamus, gastric antrum, and pancreas was measured in genetically obese (C57BL/6J ob/ob and db/db) and diabetic (C57BL/KsJ db/db and ob/ob) mice and normal littermate controls from 5 to 24 wk to determine the relationship of previously reported changes to the development of metabolic abnormalities. Hypothalamic SRIF-L concentration was similar in control, diabetic, and obese mice at all ages and increased progressively with age in all groups. Gastric antrum SRIF-LI was similar in all groups of mice at all ages. Obese mice gained weight progressively and showed moderate hyperglycemia and marked hyperinsulinemia from 5 wk of age. Pancreatic SRIF-LI content in obese (C57BL/6J) animals was similar to that in lean littermate controls, but pancreatic SRIF-LI concentration (expressed by weight or protein content) was decreased until 8 (6J ob/ob) and 10 (6J db/db) wk. Diabetic (C57BL/KsJ) mice showed a similar metabolic pattern until 10 wk with no change in pancreatic SRIF-LI content or concentration. Thereafter a progressive fall in serum insulin and a marked rise in serum glucose was associated with increasing pancreatic SRIF-LI content and concentration. These studies suggest that the genetically hyperphagic syndromes are unassociated with any change in hypothalamic or gastric SRIF-LI; that pancreatic SRIF-LI increases occur in response to, rather than as the cause of, relative hypoinsulinemia; and that the genetic background of the mice (KsJ or 6J) rather than the mutant gene (db or ob) determines the defect in carbohydrate metabolism and the pancreatic SRIF-LI response.     

Long-term renal effects of a neutralizing RAGE antibody in obese type 2 diabetic mice

Advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic kidney disease. The actions of AGEs are mediated both through a non-receptor-mediated pathway and through specific receptors for AGE (RAGEs). To explore a specific role for RAGE in renal changes in type 2 diabetes, we examined the renal effects of a neutralizing murine RAGE antibody in db/db mice, a model of obese type 2 diabetes. One group of db/db mice was treated for 2 months with the RAGE antibody, and another db/db group was treated for the same period with an irrelevant IgG. Two groups of nondiabetic db/+ mice were treated with either RAGE antibody or isotype-matched IgG for 2 months. Placebo-treated db/db mice showed a pronounced increase in kidney weight, glomerular volume, basement membrane thickness (BMT), total mesangial volume, urinary albumin excretion (UAE), and creatinine clearance compared with nondiabetic controls. In RAGE antibody-treated db/db mice, the increase in kidney weight, glomerular volume, mesangial volume, and UAE was reduced, whereas the increase in creatinine clearance and BMT was fully normalized. Notably, these effects in db/db mice were seen without impact on body weight, blood glucose, insulin levels, or food consumption. In conclusion, RAGE is an important pathogenetic factor in the renal changes in an animal model of type 2 diabetes.     

A role for iNOS in fasting hyperglycemia and impaired insulin signaling in the liver of obese diabetic mice

Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat diet-induced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, L-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5- and 2-fold, respectively, and enhanced IRS-1- and IRS-2-mediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice.     

Combination drug treatment in obese diabetic patients

Drug combinations that include antiobesity drugs (such as orlistat and sibutramine) and target cardiovascular disease (CVD) risk factors may be a good approach to patients with type 2 diabetes and/or metabolic syndrome (MetS). Our group has investigated the orlistat-fenofibrate combination treatment in obese patients with MetS and the orlistat-ezetimibe and the sibutramine-antihypertensive combination treatment in obese patients with hyperlipidaemia with promising results in CVD risk factor reduction. In these studies, the combination treatment significantly improved the lipid and lipoprotein profile, the carbohydrate metabolism parameters and many other variables playing a role in the atherosclerotic process. Small studies give promising results but double-blind, randomized trials examining the effects of such multifactorial treatment in hard CVD endpoints in diabetic or MetS patients are missing.     

Roux-en-Y gastric bypass with a long versus a short biliopancreatic limb improves weight loss and glycemic control in obese mice

BackgroundRoux-en-Y gastric bypass (RYGB) results in long-term weight loss and reduced obesity related co-morbidities. However, little is known about how the lengths of the biliopancreatic limb (BPL), the alimentary limb (AL), and the common limb (CL) affect weight loss and glucose metabolism.ObjectivesOur aim was to establish a RYGB obese mouse model with defined proportions of the AL and BPL and a constant CL to assess the effects on weight loss,glucose metabolism, and obesity-related co-morbidities.SettingIn vivo mouse study.MethodsSix-week-old male C57BL/6J mice fed with a high-fat diet (HFD) underwent bariatric surgery with defined BPL lengths: a very long, long, and short BPL (35%, 25%, and 15% of total bowel length), or sham surgery. The length of the AL was adjusted to achieve the same CL length. Mice were analyzed for weight loss, glycemic control, and obesity-related co-morbidities.ResultsMice undergoing RYGB surgery with a very long BPL had excessive weight loss and mortality and were therefore not further analyzed. Mice with a long BPL showed a significantly increased total weight loss when compared with mice with a short BPL. In addition, a long BPL improved glucose tolerance, particularly early after surgery. A long BPL was also associated with lower triglyceride levels. Resolution of hepatic steatosis and adipose tissue inflammation was, however, not statistically significant. Of note, bariatric surgery dramatically changed gut microbiota, regardless of limb length.ConclusionIn obese mice, a long BPL results in enhanced weight loss and improved glucose tolerance. These findings could potentially be translated to humans by tailoring the BPL length according to body weight, obesity-related co-morbidities, and total bowel length of an individual patient.     

Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility

Obesity is a strong risk factor for the development of type 2 diabetes. We have previously reported that in adipose tissue of obese (ob/ob) mice, the expression of adipogenic genes is decreased. When made genetically obese, the BTBR mouse strain is diabetes susceptible and the C57BL/6J (B6) strain is diabetes resistant. We used DNA microarrays and RT-PCR to compare the gene expression in BTBR-ob/ob versus B6-ob/ob mice in adipose tissue, liver, skeletal muscle, and pancreatic islets. Our results show: 1) there is an increased expression of genes involved in inflammation in adipose tissue of diabetic mice; 2) lipogenic gene expression was lower in adipose tissue of diabetes-susceptible mice, and it continued to decrease with the development of diabetes, compared with diabetes-resistant obese mice; 3) hepatic expression of lipogenic enzymes was increased and the hepatic triglyceride content was greatly elevated in diabetes-resistant obese mice; 4) hepatic expression of gluconeogenic genes was suppressed at the prediabetic stage but not at the onset of diabetes; and 5) genes normally not expressed in skeletal muscle and pancreatic islets were expressed in these tissues in the diabetic mice. We propose that increased hepatic lipogenic capacity protects the B6-ob/ob mice from the development of type 2 diabetes.     

Wound collagen accumulation in obese hyperglycemic mice

We used C57-BL ob/ob mice as a model to study wound healing in type II (adult-onset) diabetes. Planimetry was used to assess rate of closure in standard open skin wounds. In agreement with previous subcutaneous wound collagen-accumulation studies, closure was slower in the ob/ob mice. Subcutaneous implants were used to evaluate wound collagen accumulation. Weanling mice have collagen accumulation similar to lean littermates (mean 3.43 micrograms/cm vs. 3.46 micrograms/cm), but the same ob/ob animals had decreased wound collagen (mean 2.39 micrograms/cm vs. 3.02 micrograms/cm, P less than 0.04) when mature. Other ob/ob animals fed a restricted diet (and thus not obese) had normal collagen accumulation at the same age. Neither insulin nor diet restriction restored wound collagen accumulation in phenotypically obese mice. Because collagen accumulation is not improved by measures that control hyperglycemia (insulin and diet restriction) and the defect was seen only in phenotypically obese ob/ob mice, the decreased wound collagen accumulation may be due in part to structural changes in adipose tissue.     

Macrophage scavenger receptor-a-deficient mice are resistant against diabetic nephropathy through amelioration of microinflammation

Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications, including diabetic nephropathy. Macrophage scavenger receptor-A (SR-A) is a multifunctional receptor expressed on macrophages. This study aimed to determine the role of SR-A in diabetic nephropathy using SR-A-deficient (SR-A(-/-)) mice. Diabetes was induced in SR-A(-/-) and wild-type (SR-A(+/+)) mice by streptozotocin injection. Diabetic SR-A(+/+) mice presented characteristic features of diabetic nephropathy: albuminuria, glomerular hypertrophy, mesangial matrix expansion, and overexpression of transforming growth factor-beta at 6 months after induction of diabetes. These changes were markedly diminished in diabetic SR-A(-/-) mice, without differences in blood glucose and blood pressure levels. Interestingly, macrophage infiltration in the kidneys was dramatically decreased in diabetic SR-A(-/-) mice compared with diabetic SR-A(+/+) mice. DNA microarray revealed that proinflammatory genes were overexpressed in renal cortex of diabetic SR-A(+/+) mice and suppressed in diabetic SR-A(-/-) mice. Moreover, anti-SR-A antibody blocked the attachment of monocytes to type IV collagen substratum but not to endothelial cells. Our results suggest that SR-A promotes macrophage migration into diabetic kidneys by accelerating the attachment to renal extracellular matrices. SR-A may be a key molecule for the inflammatory process in pathogenesis of diabetic nephropathy and a novel therapeutic target for diabetic vascular complications.