Associations between serum leptin levels, hyperlipidemia, and cholelithiasis in dogs

Leptin and its receptor play several physiological roles in the canine gallbladder, and the dysregulation of leptin might play a role in the pathogenesis of gallbladder diseases such as gallbladder mucocele. Previous studies revealed a positive association between hyperlipidemia and gallstones in humans. However, the latter is still unclear in dogs with cholelithiasis. In this study, we examined the differences in leptin, leptin receptor, total cholesterol, and triglyceride levels between healthy dogs and dogs with cholelithiasis, and evaluated the correlation between leptin and hyperlipidemia. Twenty-eight healthy dogs and 34 client-owned dogs with cholelithiasis were enrolled in the study. Leptin concentrations and lipid profiles were determined from sera, and leptin and leptin receptor expression levels were quantified in gallbladder tissue. In dogs with cholelithiasis, serum concentrations of leptin (p < 0.001), total cholesterol (p < 0.001), and triglycerides (p < 0.001) were significantly higher compared with those in healthy dogs. Positive correlations were observed between serum leptin and total cholesterol (95% confidence interval (CI) = 0.61–0.89, r = 0.725, p < 0.001), and between leptin and triglycerides (95% CI = 0.63–0.89, r = 0.782, p < 0.001) in the cholelithiasis group. Hypercholesterolemia (Odds Ratio (OR) = 9.720; 95% CI = 1.148–82.318) and hypertriglyceridemia (OR = 12.913; 95% CI = 1.548–107.722) were shown to be risk factors for gallstone disease. In cholelithiasis patients who underwent cholecystectomy, serum leptin levels were significantly higher than in patients that had not undergone surgery (p < 0.001). Leptin and leptin receptor expression was upregulated in the gallbladder tissues of cholelithiasis patients (p < 0.01 and p < 0.001, respectively). These results indicate that increased serum leptin concentrations and hyperlipidemia (hypercholesterolemia or hypertriglyceridemia) are associated with canine cholelithiasis and that homeostatic imbalance of these parameters might affect the pathogenesis of gallstones.


Introduction
Cholelithiasis is a pathological condition in which choleliths are found within the gallbladder or intrahepatic or extrahepatic ductal system [1]. Most dogs with cholelithiasis are asymptomatic, and choleliths are discovered incidentally during diagnostic imaging procedures or a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 one investigator using a 9-point scaling system [25]. All dogs with a normal BCS (5-6/9) were selected and underwent physical examination, imaging examinations such as X-ray and abdominal ultrasonography, complete blood counts, and serum biochemical analysis. All gallbladders of the control group were confirmed to be normal based on abdominal ultrasonography and blood analyses. Dogs with cholelithiasis were diagnosed using abdominal X-ray and ultrasonography at the Department of Veterinary Radiology, College of Veterinary Medicine, SNU. Diabetes mellitus (DM) was diagnosed based on the presence of appropriate clinical signs, persistent fasting hyperglycemia (> 200 mg/dL), and concomitant glycosuria [26,27]. DM patients showed the classical clinical signs of polyuria, polydipsia, polyphagia, and weight loss [27]. Insulin resistance was determined by persistent observation of blood glucose levels over 200 mg/dL when measured every 2 h over 12 h period in diabetic patients treated with an insulin dose greater than 1.0 U/kg [28].
For biochemical analysis, jugular venipuncture was performed in 34 dogs diagnosed with cholelithiasis, 18 control client-owned healthy dogs and 10 control laboratory beagle healthy dogs. Food was withheld from the animals 12 h prior to venipuncture. Blood was collected in the serum separation tubes and centrifuged for 10 min at 1000 ×g. One portion of serum sample was immediately used for the determination of triglyceride, total cholesterol, and glucose levels, and the remainder was frozen at −80˚C until further ELISA analyses and insulin concentration measurements. Total serum cholesterol, triglyceride, and glucose levels were measured spectrophotometrically using a Hitachi 7180 automated biochemical analyzer (Hitachi, Tokyo, Japan) with commercial kits (JW Pharmaceutical, Seoul, Republic of Korea), and serum insulin levels were measured using radioimmunoassay (HI-14K, Millipore, Billerica, MA, USA) at IDEXX Laboratories, Inc. (Westbrook, ME, USA). For the quantification of leptin and leptin receptor expression, gallbladder samples were obtained from 10 dogs diagnosed with cholelithiasis that underwent therapeutic cholecystectomy and 10 healthy control beagle dogs that were euthanized because of other reasons (SNU-160720-13) not related to this study. Dogs with cholelithiasis underwent surgical interventions when clinical symptoms, abnormalities in serum biochemistry and results of imaging tests associated with biliary obstruction were observed, or when gallbladder rupture was suspected. Gallbladder samples were stored at −80˚C until their use in real-time PCR analysis. Informed consents were obtained from the owners, and all procedures were approved by the Seoul National University Institutional Animal Care and Use Committees (SNU-170220-1).

Sandwich ELISA
Serum leptin concentrations in all samples were analyzed in duplicate using a commercial canine-specific leptin sandwich ELISA kit (Canine Leptin ELISA, Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The intra-and inter-assay coefficients of variation were 5% and 7%, respectively. The absorbances were determined using an automated microplate spectrophotometer (Epoch, BioTek Instruments Inc., Winooski, VT, USA) at 450 nm.

Real-time PCR
Total RNA extraction was performed using a Hybrid-R RNA extraction kit (GeneAll Biotechnology, Seoul, Republic of Korea) according to manufacturer's protocol, and RNA levels were determined using an automated microplate spectrophotometer (Epoch, BioTek Instruments Inc., Winooski, VT, USA). cDNA was synthesized using the RNA to cDNA EcoDry™ Premix (Clontech Laboratories Inc., Mountain View, CA, USA) with 1000 ng of extracted total RNA as a template, and the newly synthesized cDNA was amplified using real-time PCR in triplicate with a SYBR 1 Premix Ex Taq™ II (Takara Bio Inc., Otsu, Japan) and StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The level of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used for the normalization of leptin and leptin receptor expression levels. Primers for leptin and the leptin receptor in the canine gallbladder were reported previously [18], and primer sequences are shown in Table 1.

Statistical analysis
Statistical analyses were performed using SPSS, version 23.0 (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk method was used to evaluate the normal distribution of the obtained data. The Mann-Whitney U-test was performed in order to analyze differences in age, body weight, BCS, and biochemical characteristics such as serum leptin, total cholesterol, triglyceride, glucose, and insulin concentrations between the control and cholelithiasis groups. The Kruskal-Wallis test was used for comparisons of serum leptin concentrations and serum insulin levels between three groups (healthy dogs, cholelithiasis patients that did not undergo a surgery, and cholelithiasis patients that underwent surgery; or healthy dogs, cholelithiasis patients without DM, and cholelithiasis patients with DM). When a significant difference was obtained, posthoc comparisons were performed using the Mann-Whitney test with Bonferroni correction. The data are presented as the median value of each group followed by the interquartile range. The independent t-test was used to compare the total cholesterol and triglyceride levels between dogs with cholelithiasis that did not undergo surgery and dogs with cholelithiasis that underwent surgery, and to analyze the relative mRNA expression levels of leptin and its receptor in gallbladder samples in the healthy and patient groups. These data are presented as mean and standard deviation. The relationship between the serum leptin concentration and total cholesterol, triglycerides, glucose, insulin, age, sex, breed, body weight, and BCS was estimated using the Spearman rank correlation test. The univariate odds ratio (OR) and 95% confidence interval (CI) for cholelithiasis associated with hypercholesterolemia, hypertriglyceridemia, hyperglycemia, or hyperinsulinemia were evaluated using Fisher's exact test. A p-value < 0.05 was considered statistically significant.

Cases
Detailed basic information and the results of blood analyses of 28 healthy control dogs and 34 dogs diagnosed with cholelithiasis are summarized in Table 2. The median age of the dogs with cholelithiasis was 12.5 years, which was significantly higher than that of the healthy control dogs (8 years old) (p < 0.001). Body weight and BCS did not significantly differ between the groups (p = 0.218 and p = 0.403, respectively). Seven of 34 dogs with cholelithiasis (20.6%) were found to have DM, and two of them showed insulin resistance. Of seven diabetic dogs, one had hypercholesterolemia, while two had hypertriglyceridemia. The gallbladder tissue samples used in this study were acquired from 10 healthy beagle dogs registered as experimental animals and 10 patients that underwent cholecystectomy. Information regarding these dogs is summarized in Table 3. The median age was significantly higher in the cholelithiasis group (14 years old) than in the healthy control group (8 years old) (p < 0.001). Mean body weight was significantly higher in the healthy control group (8.31 kg) than in the cholelithiasis group (5.21 kg) (p < 0.001). The median BCS of the healthy control group was 5/9, which was similar to that of the cholelithiasis group (p = 0.661). One of the 10 dogs with cholelithiasis (10%) that underwent surgery had DM, but insulin resistance was not observed.

Analysis of serum leptin, total cholesterol, triglyceride, glucose, and insulin levels in healthy control dogs and dogs with cholelithiasis
In the cholelithiasis group, serum leptin concentrations were significantly higher than those in the healthy group (p < 0.001; Fig 1). To determine the changes in serum leptin concentration according to the severity of cholelithiasis, the cholelithiasis group was further categorized into dogs that underwent cholecystectomy and dogs that did not; serum leptin levels were Table 2

Relative expression levels of leptin and leptin receptor in healthy dogs and dogs with cholelithiasis
The mRNA expression levels of leptin and its receptor were significantly upregulated in dogs with cholelithiasis, compared with those in the healthy control dogs (p < 0.01 and p < 0.001, respectively; Fig 4).

Correlations
A significant positive correlation was observed between serum leptin and total cholesterol in both control and patient groups (95% CI = 0.55-0.89, r = 0.794, p < 0.001 and 95%    Table 4.

Discussion
The results of this study showed that serum leptin concentrations were not significantly correlated with age, sex, breed, body weight, or BCS in either dogs with cholelithiasis or healthy dogs. In previous investigations, plasma leptin levels were significantly higher in dogs with increased BCS values; however, no significant effects of age, sex, or breed were found [29,30]. These results confirm that plasma leptin levels may be a useful marker for evaluating adiposity regardless of variations in breed, age, or sex [29]. One study suggested that serum cholesterol, triglyceride, and glucose levels in dogs can be affected by the BCS increasing [31]. Based on previous studies suggesting that BCS is significantly associated with the circulating levels of leptin, cholesterol, triglycerides, and glucose [29][30][31], in order to rule out the influence of BCS on the measurement of these analytes, all examined dogs were selected based on similar BCS values (5-6/9). Cholelithiasis was diagnosed based on the combination of biochemical analysis results, medical imaging, and clinical signs such as abdominal pain, vomiting, anorexia, and icterus. Biochemical analyses vary widely with cholelithiasis, and abdominal radiography is occasionally limited in its ability to confirm the presence of choleliths depending on the relative composition of calcium salt [1,27]. Radiopaque gallstones account for 48% in dogs with cholelithiasis, which is higher than that in humans (15%). These results may be explained by a higher percentage of calcium bilirubinate-containing choleliths in dogs [2]. Abdominal  ultrasonography is one of the most useful tools for the diagnosis of cholelithiasis. Ultrasonographically, choleliths are observed as echogenic focal structures, usually with acoustic shadowing within the gallbladder lumen, and might be accompanied by gallbladder wall thickening, dilation of the biliary tract, or pericholecystic fluid [2,32]. However, even if these findings are observed via abdominal ultrasonography, they do not always correspond to the clinical symptoms and changes in laboratory findings. For this reason, in some cases, the use of ultrasonography is not sufficient to evaluate the prognosis of the disease. This study showed that the serum leptin concentrations in dogs with cholelithiasis were significantly higher than those in healthy dogs. To further estimate the association between serum leptin levels and the severity of cholelithiasis, dogs in the cholelithiasis group were divided into those that underwent cholecystectomy and those that did not, and leptin levels were found to be significantly increased in the operated group. These results are supported by previous studies suggesting that human patients with cholelithiasis exhibit higher serum leptin concentrations compared with those in the controls, and they indicate a relationship between leptin and the occurrence of gallstone disease [21,33]. Therefore, our results further suggest that serum leptin levels may represent an additional useful biomarker for the assessment of the severity of cholelithiasis in dogs.
Three potential pathophysiological factors may affect the development of gallstones, including hypomotility of gallbladder, changes in bile properties due to dysregulated absorption/ secretion, and hypersecretion of mucin. A growing number of studies showed that leptin levels may be associated with these abnormalities, either directly or indirectly. The contractile responses of gallbladder are triggered by neurotransmitters such as neuropeptide Y (NPY) and cholecystokinin (CCK), and both NPY and CCK have direct excitatory effects in the biliary tract through the stimulation of cholecystic contractility and the sphincter of Oddi [20,34]. Previous research reported that leptin-deficient or leptin-resistant obese mice exhibited decreased gallbladder responses to NPY and CCK compared with control mice with normal leptin metabolism, suggesting that leptin dysregulation is associated with gallbladder motility and gallstone formation [20,35]. The emptying of the postprandial gallbladder (up to 80% of the fasting gallbladder volume) is mostly controlled by CCK [36]. Leptin resistance decreases the sensitivity of vagal afferent neurons to CCK, thus inhibiting motility and emptying of the gallbladder [37]. One study [8] revealed leptin-regulated gallbladder-associated genes, such as aquaporin 1 water channel, aquaporin 4, epithelial sodium channel-α, and sodium hydrogen exchangers, which were shown to be related to absorption/secretion. Therefore, it was suggested that bile properties may be altered due to leptin dysregulation. Another study [19] reported an association between serum leptin and GBM suggesting that leptin imbalance is also related to the hypersecretion of mucus. We also demonstrated a relative increase in leptin and leptin receptor mRNA expression in the gallbladder tissues of dogs with cholelithiasis compared with that in healthy dogs. Based on these results, we can hypothesize that the upregulation of the expression of leptin and its receptor in the gallbladder of cholelithiasis patients is a response aimed at maintaining the homeostasis of gallbladder motility and bile composition under pathological conditions.
Hyperlipidemia represents an increased concentration of lipid fractions, such as total cholesterol and triglycerides in the blood [38]. The results of our study are consistent with those of previous studies in human patients, showing that the serum concentrations of total cholesterol and triglycerides in cholelithiasis patients were higher compared with those in the controls [21,39]. In addition, hypercholesterolemia and hypertriglyceridemia were found to represent high risk factors for the development of cholelithiasis [40,41]. The hypersecretion of cholesterol in the bile and the subsequent supersaturation of bile contribute to the formation of biliary sludge, which consequently reduces gallbladder motility [40]. The gallbladder motility of patients with hypertriglyceridemia is reduced compared to that of body mass index (BMI)matched controls [41]. In a study investigating the association between gallbladder dysmotility and hypertriglyceridemia, decreased sensitivity to CCK, rather than reduced CCK release, was revealed to be the cause of impaired gallbladder motility in patients with high triglyceride blood levels [41].
In this study, we determined that both serum cholesterol and triglyceride levels positively correlate with leptin levels in both healthy and cholelithiasis patients. The association between the serum concentration of leptin and that of total cholesterol or triglycerides is controversial. In a study investigating leptin and cholesterol levels in lean (BCS 4-5/9) and obese (BCS 7-9/9) dogs, a positive correlation between leptin and cholesterol levels was reported [23]. The results of another study indicated the positive association between leptin and triglyceride levels in obese dogs as well [42]. In contrast, our previous GBM research revealed that, although many GBM patients had hyperlipidemia, no correlation between serum total cholesterol or triglyceride and leptin levels was detected [19]. One study showed that the associations between leptin and lipid [low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol, and triglyceride] levels were not statistically significant in patients with combined hyperlipidemia [39]. Relationships between leptin levels and lipid profiles may differ according to the pathological conditions of the patient, such as GBM [19], obesity [23,42], and hyperlipidemia [39].
In humans, previous studies investigating the associations between DM, insulin resistance, and leptin have been reported. Previously, it was suggested that leptin and insulin levels, and insulin resistance have different associations with obesity [43,44]. DM is a well-known risk factor for gallstone formation [45]. In diabetic patients, cholesterol is hypersecreted during liver metabolism, leading to an increase in its absolute concentration in bile and causing a change in its relative proportion, which in turn affects the composition of the bile [46]. Insulin affects cholesterol saturation and gallbladder motility, an important factor in the formation of gallstones. In particular, hyperinsulinemia reduces gallbladder motility induced by CCK and increases the incidence of gallstones [47]. Hyperinsulinemia due to the insulin resistance affects the Na + -K + pump activity, it regulates the release of neurotransmitters in presynaptic nerve terminals, and changes gallbladder smooth muscle tone. Therefore, it can lead to an increased gallbladder motility and decreased gallstone formation [48]. In one study in humans, serum leptin levels were shown to be directly related to insulin levels regardless of age and gender [49]. Other studies demonstrated that insulin resistance is most closely correlated with the leptin levels [50]. Taken together, these results suggest that leptin, insulin, and insulin resistance are associated with cholelithiasis. In our study, serum insulin levels were significantly higher in patients with cholelithiasis than those in the controls, but no statistically significant relationship between hyperinsulinemia and cholelithiasis was detected. Moreover, we showed that there were no significant differences in serum leptin and insulin concentrations between cholelithiasis patients with and without DM. These findings are consistent with previous investigations, showing that the leptin levels are associated with the disease status (chronic kidney disease, hyperadrenocorticism, and acute pancreatitis) [51] or obesity [52] rather than the presence of DM. Additionally, unlike the results showing a direct association between leptin and insulin levels [50], we observed no statistically significant associations between leptin and insulin concentrations in both the control and cholelithiasis group. Furthermore, only two of 34 dogs with cholelithiasis exhibited insulin resistance, and the number of dogs with hypercholesterolemia or hypertriglyceridemia of the total number of diabetic dogs was 1/7 and 2/7, respectively. Therefore, further studies on the associations between these factors are required.
This study has several limitations. First, we included a relatively small number of control and cholelithiasis patients. Analyses with more samples would provide more precise and reliable results. Due to the small number of samples, the associations between cholelithiasis, DM, and insulin resistance were not analyzed. Second, control over precise histories and analysis groups may be limited by the lack of complete medical records, including diagnostic and treatment histories. If this is the case, incorrect groupings may have influenced the results of the present study. If dogs had already begun receiving treatment for DM at the time of sample collection, this may have affected the observed differences in serum concentrations of leptin, insulin, and glucose between dogs with and without DM. Finally, our quantitative assessment of the relative expression differences of leptin and leptin receptor in the gallbladder tissues was performed using real-time PCR and not at the protein level.
In conclusion, in this study, we demonstrated that an increase in serum leptin concentrations and hyperlipidemia (hypercholesterolemia or hypertriglyceridemia) is associated with the occurrence of cholelithiasis in dogs, and the results of this study suggest that the homeostatic imbalance of these parameters may affect the pathogenesis of gallstones. The changes in the mRNA expression of leptin and leptin receptor within the gallbladder of dogs with cholelithiasis further underline that leptin may be a factor involved in the development of cholelithiasis, considering its important physiological role in gallbladder. However, further studies are required to definitively elucidate the relationship between hyperlipidemia and leptin in the pathophysiology of gallstones disease.