Effect of metformin on adipose tissue resistin expression in db/db mice

Resistin, a novel adipose-derived protein, has been proposed to cause insulin-resistant states in obesity. To evaluate whether an insulin-sensitizing drug, metformin, regulates adipose tissue resistin expression, murine models of obesity and diabetes, db/db mice, were treated with metformin (metformin group), insulin (insulin group), and vehicle (control group) for 4 weeks, followed by analyzing resistin protein expression in their adipose tissues. Unexpectedly, resistin protein expression was increased by 66% in the metformin group relative to the control group, while it did not differ between the insulin and control groups. Hyperinsulinemia was improved in the metformin group, while the insulin group exhibited severe hyperinsulinemia, similar to the control group. Furthermore, in comparison between obese mice (db/db mice) and age-matched lean controls, resistin protein expression was reduced by 58% in the obese mice with severe hyperinsulinemia. These data collectively suggest that resistin expression may be suppressed by hyperinsulinemia and that metformin may upregulate resistin expression via the improvement of hyperinsulinemia in obesity.     

Loss of resistin ameliorates hyperlipidemia and hepatic steatosis in leptin-deficient mice

Resistin has been linked to components of the metabolic syndrome, including obesity, insulin resistance, and hyperlipidemia. We hypothesized that resistin deficiency would reverse hyperlipidemia in genetic obesity. C57Bl/6J mice lacking resistin [resistin knockout (RKO)] had similar body weight and fat as wild-type mice when fed standard rodent chow or a high-fat diet. Nonetheless, hepatic steatosis, serum cholesterol, and very low-density lipoprotein (VLDL) secretion were decreased in diet-induced obese RKO mice. Resistin deficiency exacerbated obesity in ob/ob mice, but hepatic steatosis was drastically attenuated. Moreover, the levels of triglycerides, cholesterol, insulin, and glucose were reduced in ob/ob-RKO mice. The antisteatotic effect of resistin deficiency was related to reductions in the expression of genes involved in hepatic lipogenesis and VLDL export. Together, these results demonstrate a crucial role of resistin in promoting hepatic steatosis and hyperlipidemia in obese mice.     

Preliminary experience with resistin assessment in common population

Resistin is a signal peptide produced by adipose tissue. Mice models have confirmed that resistin may play an important role in insulin resistance. Its function in the human organism has not been elucidated yet. Since in common population the resistin concentrations are not known (no validated commercial set is available), we performed resistin assessment using the ELISA method (with satisfying analytical characteristics) in a population of 123 non-obese probands without signs of insulin resistance and/or inflammation. Mean resistin values amounted to 14.3 ng/ml (reference limit of 7.3-21.3 ng/ml).     

The effects of designed angiopoietin-1 variant on lipid droplet diameter, vascular endothelial cell density and metabolic parameters in diabetic db/db mice

Metabolic syndrome consists of metabolic abnormality with central obesity, hypertriglyceridemia, insulin resistance and hypertension. Adipose tissue has been known as a primary site of insulin resistance and its adipocyte size may be correlated with the degree of insulin resistance. A designed angiopoietin-1, COMP-Angiopoietin-1 (COMP-Ang1), mitigated high-fat diet-induced insulin resistance in skeletal muscle. In this study, we examined effects of COMP-Ang1 on adipocyte droplet size, vascular endothelial cell density in adipose tissue and metabolic parameters in db/db mice by administering COMP-Ang1 or LacZ (as a control) adenovirus. Administration of COMP-Ang1 decreased fat droplet diameter in epididymal and abdominal visceral adipocyte and visceral fat content in db/db mice. The density of vascular endothelial cell in adipose tissue was increased in db/db mice after treatment with COMP-Ang1. Serum resistin and tumor necrosis factor-α level was lower after treatment with COMP-Ang1 in db/db mice. COMP-Ang1 caused a restoration of fasting glycemic control in db/db mice and decreased serum insulin level and insulin resistance measured by HOMA index. These findings indicate that COMP-Ang1 regulates adipocyte fat droplet diameter, vascular endothelial cell density and metabolic parameters in db/db mice.     

Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists

Elevated levels of the hormone resistin, which is secreted by fat cells, are proposed to cause insulin resistance and to serve as a link between obesity and type 2 diabetes. In this report we show that resistin expression is significantly decreased in the white adipose tissue of several different models of obesity including the ob/ob, db/db, tub/tub, and KKA(y) mice compared with their lean counterparts. Furthermore, in response to several different classes of antidiabetic peroxisome proliferator-activated receptor gamma agonists, adipose tissue resistin expression is increased in both ob/ob mice and Zucker diabetic fatty rats. These data demonstrate that experimental obesity in rodents is associated with severely defective resistin expression, and decreases in resistin expression are not required for the antidiabetic actions of peroxisome proliferator-activated receptor gamma agonists.     

Weight loss in tumour-bearing mice is not associated with changes in resistin gene expression in white adipose tissue.

Resistin, a product of white adipose tissue, is postulated to induce insulin resistance in obesity and regulate adipocyte differentiation. The aim of this study was to examine resistin gene expression in adipose tissue from mice bearing the MAC16 adenocarcinoma, which induces cancer cachexia with marked wasting of adipose tissue and skeletal muscle mass. MAC16-bearing mice lost weight progressively over the period following tumour transplantation, while the weight of control mice remained stable. Leptin mRNA in gonadal fat was 50 % lower in MAC16 mice than in controls (p < 0.05). Plasma insulin concentrations were also significantly lower in the MAC16 group (p < 0.05). However, resistin mRNA level in gonadal fat in MAC16 mice was similar to controls (94 % of controls). Thus, despite severe weight loss and significant falls in leptin expression and insulin concentration, resistin gene expression appears unchanged in white adipose tissue of mice with MAC16 tumour. Maintenance of resistin production may help inhibit the formation of new adipocytes in cancer cachexia.     

Regulation of adiponectin and its receptors in response to development of diet-induced obesity in mice

Adiponectin and its receptors play an important role in energy homeostasis and insulin resistance, but their regulation remains to be fully elucidated. We hypothesized that high-fat diet would decrease adiponectin but increase adiponectin receptor (AdipoR1 and AdipoR2) expression in diet-induced obesity (DIO)-prone C57BL/6J and DIO-resistant A/J mice. We found that circulating adiponectin and adiponectin expression in white adipose tissue are higher at baseline in C57BL/6J mice compared with A/J mice. Circulating adiponectin increases at 10 wk but decreases at 18 wk in response to advancing age and high-fat feeding. However, adiponectin levels corrected for visceral fat mass and adiponectin mRNA expression in WAT are affected by high-fat feeding only, with both being decreased after 10 wk in C57BL/6J mice. Muscle AdipoR1 expression in both C57BL/6J and A/J mice and liver adipoR1 expression in C57BL/6J mice increase at 18 wk of age. High-fat feeding increases both AdipoR1 and AdipoR2 expression in liver in both strains of mice and increases muscle AdipoR1 expression in C57BL/6J mice after 18 wk. Thus advanced age and high-fat feeding, both of which are factors that predispose humans to obesity and insulin resistance, are associated with decreasing adiponectin and increasing AdipoR1 and/or AdipoR2 levels.     

Resistin expression in different adipose tissue depots during rat development

Resistin is a hormonal factor synthesised by adipocytes that was first thought to be related with the resistance to insulin in obesity, but whose function is not yet completely established. Here we have studied the ontogenic pattern of resistin mRNA expression in different white adipose tissue depots (WAT) – epididymal, inguinal, mesenteric and retroperitoneal – and in brown adipose tissue (BAT), as well as the circulating resistin levels, in rats of different ages (from the suckling period to one year of age). Resistin mRNA was determined by Northern blotting, and serum levels by enzyme immunoassay. In WAT, resistin expression remains almost constant with age, except in early development, where there is a peak of expression in the epididymal and retroperitoneal depots, and a decrease in the inguinal one, while the expression remains constant for the mesenteric depot. Moreover, there is a site-specific difference regarding resistin expression: all the depots express characteristic levels of mRNA, especially at the age of 2 months, the moment when resistin mRNA levels are significantly higher in the epididymal and the retroperitoneal than in the inguinal and mesenteric WAT and than in the BAT. The transient increased resistin expression in the epididymal and the retroperitoneal WAT at a period of time in which there is a change in diet (from milk to chow) suggests a common nutritional regulation of the resistin gene. Circulating resistin levels increase with age probably reflecting the increase in the body fat content.     

Identification of variables influencing resistin serum levels in patients with type 1 and type 2 diabetes mellitus.

BACKGROUND: Resistin, a peptide hormone, has been discussed controversially as a missing link between obesity and insulin resistance. In contrast to resistin mRNA expression in adipose tissue, data on human serum levels in obesity and diabetes mellitus is scarce. The physiological range of serum resistin levels, reference values or adjusted percentiles have not yet been determined, making the interpretation of serum resistin concentrations quite difficult. METHODS: Resistin serum concentrations were measured systematically by ELISA in 216 healthy controls, 555 patients with type 2 diabetes and 114 patients with type 1 diabetes. Mean values, median, and range were determined, and BMI-, gender-, and disease-adapted percentiles were calculated for all subgroups. RESULTS: Age and gender did not have any influence on resistin levels. BMI and resistin levels were positively correlated in healthy controls (p = 0.02), albeit with a weak correlation coefficient. This correlation was absent in patients with type 1 and type 2 diabetes. In both genders, healthy controls had significantly higher resistin levels than patients with type 1 and type 2 diabetes (7.9 +/- 0.2 ng/ml vs. 5.7 +/- 0.2 ng/ml and 5.5 +/- 0.1 ng/ml, respectively; p < 0.0001). There was no correlation between resistin levels and occurrence of diabetic retinopathy or nephropathy. CONCLUSIONS: Serum resistin levels can be measured by ELISA over a broad range from 0.6 ng/ml up to 27.7 ng/ml, suggesting that percentiles might be helpful in the interpretation of an individuals resistin value. While age and gender do not influence resistin levels, BMI and occurrence of diabetes have to be considered.     

Cyanidin 3-glucoside attenuates obesity-associated insulin resistance and hepatic steatosis in high-fat diet-fed and db/db mice via the transcription factor FoxO1

Obesity is a major risk factor for the development of type 2 diabetes, and both conditions are now recognized to possess significant inflammatory components underlying their pathophysiologies. Here, we hypothesized that cyanidin 3-glucoside (C3G), a typical anthocyanin reported to possess potent anti-inflammatory properties, would ameliorate obesity-associated inflammation and metabolic disorders, such as insulin resistance and hepatic steatosis in mouse models of diabesity. Male C57BL/6J obese mice fed a high-fat diet for 12 weeks and genetically diabetic db/db mice at an age of 6 weeks received dietary C3G supplementation (0.2%) for 5 weeks. We found that dietary C3G lowered fasting glucose levels and markedly improved the insulin sensitivity in both high-fat diet fed and db/db mice as compared with unsupplemented controls. White adipose tissue messenger RNA levels and serum concentrations of inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) were reduced by C3G, as did macrophage infiltration in adipose tissue. Concomitantly, hepatic triglyceride content and steatosis were alleviated by C3G. Moreover, C3G treatment decreased c-Jun N-terminal kinase activation and promoted phosphorylation and nuclear exclusion of forkhead box O1 after refeeding. These findings clearly indicate that C3G has significant potency in antidiabetic effects by modulating the c-Jun N-terminal kinase/forkhead box O1 signaling pathway and the related inflammatory adipocytokines.     

Combination therapy with PPARgamma and PPARalpha agonists increases glucose-stimulated insulin secretion in db/db mice

Although peroxisome proliferator-activated receptor (PPAR)gamma agonists ameliorate insulin resistance, they sometimes cause body weight gain, and the effect of PPAR agonists on insulin secretion is unclear. We evaluated the effects of combination therapy with a PPARgamma agonist, pioglitazone, and a PPARalpha agonist, bezafibrate, and a dual agonist, KRP-297, for 4 wk in male C57BL/6J mice and db/db mice, and we investigated glucose-stimulated insulin secretion (GSIS) by in situ pancreatic perfusion. Body weight gain in db/db mice was less with KRP-297 treatment than with pioglitazone or pioglitazone + bezafibrate treatment. Plasma glucose, insulin, triglyceride, and nonesterified fatty acid levels were elevated in untreated db/db mice compared with untreated C57BL/6J mice, and these parameters were significantly ameliorated in the PPARgamma agonist-treated groups. Also, PPARgamma agonists ameliorated the diminished GSIS and insulin content, and they preserved insulin and GLUT2 staining in db/db mice. GSIS was further increased by PPARgamma and -alpha agonists. We conclude that combination therapy with PPARgamma and PPARalpha agonists may be more useful with respect to body weight and pancreatic GSIS in type 2 diabetes with obesity.     

Resistin Production from Adipose Tissue Is Decreased in db/db Obese Mice,and Is Reversed by Rosiglitazone

Objective

This study was designed to (1) investigate the expression profiles of resistin in db/db mice and its dynamic association with metabolic parameters; and (2) evaluate the effects of Rosiglitazone on production of resistin.

Methods

Db/db mice and their lean litter mates were used for this study. Epididymal fat tissue was excised from mice of different age (from 5 to 12 weeks) for ex vivo incubation. Resistin,along with adiponectin,in serum and conditioned culture medium of epididymal fat pads were measured with immunoassays. The gene expression of resistin was determined by real-time PCR. Rosiglitazone or the vehicle (PBS) was administered into db/db mice by daily intra-gastric gavage. Differentiated 3T3-L1 adipocytes were used for in vitro evaluation.

Results

The secretion of resistin from the fat pads in db/db mice was significantly lower than that in lean mice (P<0.01). The mRNA expression of the resistin gene in fat tissue of db/db mice at the age of 5 weeks was decreased by 60.5% compared to lean controls (p<0.05). Serum levels of resistin were comparable between the obese and lean groups, perhaps due to the increased total fat mass in db/db mice. Correlation analysis showed that serum resistin levels were positively correlated to resistin secretion from fat pads(r = 0.844,P = 0.000), while negatively associated with the body weight (r = −0.515, P = 0.000) and fasting glucose level (r = −0.357, P = 0.002). Notably, treatment with rosiglitazone increased the serum resistin levels by 66.4%(P<0.05)in db/db mice. In 3T3-L1 adipocytes, Rosiglitazone (10 uM) markedly enhanced the secretion of resistin by 120% (P<0.01) and its gene expression by 78.1% (P<0.05).

Conclusion

Both resistin gene expression and its secretion from the epididymal adipose tissue were decreased in db/db obese mice, while the insulin-sensitizing drug rosiglitazone increased resistin production. Our results do not support the role of resistin as an etiological link between obesity and diabetes.     

Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice

Conjugated linoleic acid (CLA) causes insulin resistance and hepatic steatosis in conjunction with depletion of adipokines in some rodent models. Our objective was to determine whether the maintenance of adipokines, mainly leptin and adiponectin, by either removing CLA from diets or using an adiponectin enhancer, rosiglitazone (ROSI), could attenuate CLA-induced insulin resistance. Male C57BL/6 mice were consecutively fed two experimental diets containing 1.5% CLA mixed isomer for 4 weeks followed by a diet without CLA for 4 weeks. CLA significantly depleted adiponectin but not leptin and was accompanied by hepatic steatosis and insulin resistance. These effects were attenuated after switching mice to the diet without CLA along with restoration of adiponectin. To further elucidate the role of adiponectin in CLA-mediated insulin resistance, ROSI was used in a subsequent study in male ob/ob mice fed either control (CON) or CLA diet. ROSI maintained significantly higher adiponectin levels in CON- and CLA-fed mice and prevented the depletion of epididymal adipose tissue and the development of insulin resistance. In conclusion, we show that insulin resistance induced by CLA may be related more to adiponectin depletion than to leptin and that maintaining adiponectin levels alone either by removing CLA or using ROSI can attenuate these effects.     

Compensation for partial lipectomy in mice with genetic alterations of leptin and its receptor subtypes

One hypothesis for the regulation of total body fat suggests that leptin is a lipostatic feedback signal that acts at brain sites involved in regulation of energy balance. The importance of leptin in recovery from partial surgical lipectomy was tested by performing bilateral epididymal lipectomy or sham surgery on wild-type and leptin-deficient ob/ob mice. Eight weeks later, nonexcised pads of lipectomized mice were increased but total carcass fat was lower than in sham-operated ob/ob mice. In experiment 2, ob/ob mice, wild-type mice, and two db/db mutants, C57BL/6J db(Lepr)/db(Lepr) (BL/6J) mice possessing short-form and circulating leptin receptors and C57BL/6J db(3J)/db(3J) (BL/3J) mice expressing only circulating receptors, were lipectomized or sham operated. Sixteen weeks later, body mass and carcass lipid were not different between sham and lipectomized ob/ob mice, wild-type mice, or BL/6J db/db mice, whereas there was incomplete (decreased carcass fat) but suggestive recovery (increased retroperitoneal fat mass and cell number) in lipectomized BL/3J db/db mice. These data indicate that leptin is not required for the regulation of total body fat.     

Differential regulation of leptin expression and function in A/J vs. C57BL/6J mice during diet-induced obesity

Obesity-resistant (A/J) and obesity-prone (C57BL/6J) mice were weaned onto low-fat (LF) or high-fat (HF) diets and studied after 2, 10, and 16 wk. Despite consuming the same amount of food, A/J mice on the HF diet deposited less carcass lipid and gained less weight than C57BL/6J mice over the course of the study. Leptin mRNA was increased in white adipose tissue (WAT) in both strains on the HF diet but to significantly higher levels in A/J compared with C57BL/6J mice. Uncoupling protein 1 (UCP1) and UCP2 mRNA were induced by the HF diet in brown adipose tissue (BAT) and WAT of A/J mice, respectively, but not in C57BL/6J mice. UCP1 mRNA was also significantly higher in retroperitoneal WAT of A/J compared with C57BL/6J mice. The ability of A/J mice to resist diet-induced obesity is associated with a strain-specific increase in leptin, UCP1, and UCP2 expression in adipose tissue. The findings indicate that the HF diet does not compromise leptin-dependent regulation of adipocyte gene expression in A/J mice and suggest that maintenance of leptin responsiveness confers resistance to diet-induced obesity.     

Increased resistin expression in the adipose tissue of male prolactin transgenic mice and in male mice with elevated androgen levels.

The aim of this study was to investigate the regulation of resistin, a recently identified adipocyte-secreted peptide, in the adipose tissue of prolactin (PRL)-transgenic (tg) mice using ribonuclease protection assay. The level of resistin mRNA increased 3.5-fold in the adipose tissue of untreated male PRL-tg mice compared to controls. However, there was no difference in resistin expression in the adipose tissue of female PRL-tg mice compared to control mice. PRL-tg male mice have elevated serum testosterone levels and we therefore analyzed the effects of testosterone alone on resistin mRNA expression. Furthermore, the effects of elevated androgen levels on PRL receptor (PRLR) mRNA expression in the adipose tissue were investigated. Resistin mRNA increased 2.6-fold in the adipose tissue of control male mice with elevated serum androgen levels. In addition, PRLR mRNA expression was increased in the adipose tissue of male mice with elevated testosterone. These results suggest testosterone to be a regulator of resistin and PRLR mRNA expression in the adipose tissue of male mice.     

Short-term pioglitazone treatment prevents free fatty acid-induced hepatic insulin resistance in normal rats: possible role of the resistin and adiponectin

We have evaluated the effects of a 2 week treatment with pioglitazone (Pio, 4mg/kg x d) on hepatic and peripheral insulin sensitivity, plasma adiponectin, and resistin concentrations in lipid-infused rats. Lipid infusion caused a large (60% in 4h) decrease in whole-body insulin sensitivity. Hepatic and peripheral insulin resistance contributed about equally to the whole-body insulin resistance. Pio treatment significantly improved whole-body insulin sensitivity due to normalization of hepatic insulin action, whereas peripheral insulin action remained unchanged and inhibited. Basal plasma resistin levels were approximately 4-fold lower in Pio-treated than in untreated rats. During lipid infusion, resistin levels rose in both Pio-treated and untreated rats, but remained significantly lower in Pio-treated than in untreated rats (P<0.01). Dot-blot analyses revealed a marked decrease in resistin protein levels in the liver of Pio-treated rats. Resistin levels were higher in muscle tissue in lipid group compared with control and Pio-treated rats (P<0.05). Fasting plasma adiponectin levels were 1.5-fold higher in Pio-treated than in untreated rats. We conclude that short-term treatment of rats with Pio prevented lipid-induced hepatic insulin resistance and that Pio mediated lowering of blood resistin and raising of adiponectin levels may have contributed to that effect.     

Momordica charantia (Bitter Melon) Reduces Obesity-Associated Macrophage and Mast Cell Infiltration as well as Inflammatory Cytokine Expression in Adipose Tissues

Obesity is a world-wide epidemic disease that correlates closely with type 2 diabetes and cardiovascular diseases. Obesity-induced chronic adipose tissue inflammation is now considered as a critical contributor to the above complications. Momordica charantia (bitter melon, BM) is a traditional Chinese food and well known for its function of reducing body weight gain and insulin resistance. However, it is unclear whether BM could alleviate adipose tissue inflammation caused by obesity. In this study, C57BL/6 mice were fed high fat diet (HFD) with or without BM for 12 weeks. BM-contained diets ameliorated HFD-induced obesity and insulin resistance. Histological and real-time PCR analysis demonstrated BM not only reduced macrophage infiltration into epididymal adipose tissues (EAT) and brown adipose tissues (BAT). Flow cytometry show that BM could modify the M1/M2 phenotype ratio of macrophages in EAT. Further study showed that BM lowered mast cell recruitments in EAT, and depressed pro-inflammatory cytokine monocyte chemotactic protein-1 (MCP-1) expression in EAT and BAT as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in EAT. Finally, ELISA analysis showed BM-contained diets also normalized serum levels of the cytokines. In summary, in concert with ameliorated insulin resistance and fat deposition, BM reduced adipose tissue inflammation in diet-induced obese (DIO) mice.     

Long-time alcohol intake modifies resistin secretion and expression of resistin gene in adipose tissue

Elevated serum resistin is implicated in insulin resistance associated with obesity and type 2 diabetes mellitus. Alcohol consumption interferes with the nutritional status, metabolic and hormonal activity of the drinker. Impact of ethanol intake on resistin level and resistin metabolic effects is unknown. Effect of long-time (28 days) ad libitum moderate alcohol (6% ethanol solution) intake on serum resistin and resistin mRNA level in adipose tissue of rats (A) was compared to control (C) and pair-fed (PF) animals. PF rats were fed the same caloric amount as A rats on previous day. Alcohol consumption resulted in reduction of food and energy intake, decreased body mass gain, epididymal fat pads mass and smaller adipocytes (vs. C rats). Alcohol intake significantly increased serum resistin and glucose, insulinemia remained unchanged. Systemic insulin resistance was not proved by HOMA, QUICKI and McAuley indexes, but impaired insulin effect on glucose transport in isolated adipocytes was present. Elevated serum resistin was positively correlated with glycemia (r = 0.88, p < 0.01) and negatively with fat cell size (r = -0.73, p < 0.05). High resistin level as the consequence of long-time alcohol intake could contribute to smaller adipocytes, higher glycemia, attenuation of insulin-stimulated glucose transport in adipocytes. Diminished resistin gene expression in adipose tissue of A and PF rats was present.