Measurement of salivary resistin, visfatin and adiponectin levels

Hormonal determination in saliva offers several advantages. Peptides enter the salivary glands either by active transport mechanisms or are expressed and secreted by the salivary glands themselves. The collection of saliva is a noninvasive, easily repeatable and less stressful technique than blood withdrawal. The purpose of the present study was to introduce a method for measuring salivary resistin, visfatin and adiponectin levels and to evaluate their associations with serum levels. Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for serum with minor modifications. The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary levels with serum levels (r=0.441, p<0.01 and r=0.347, p<0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. To our knowledge, this is the first study to report the determination of resistin and visfatin in saliva and the significant correlation of salivary resistin with serum levels, while it confirmed the significant association between salivary and serum adiponectin. The introduction of salivary determinations of adipokines could contribute to the elucidation of the physiology and the role of the specific adipokines in various clinical conditions (obesity, insulin resistance, inflammation, reproduction, energy imbalance and stress response).     

Sub-chronic administration of stable GIP analog in mice decreases serum LPL activity and body weight

GIP receptor knockout mice were shown to be protected from the development of obesity on a high fat diet, suggesting a role of GIP in the development of obesity. In our study we aimed to test the hypothesis if excess of GIP could accelerate development of obesity and to identify GIP gene targets in adipose tissue. Therefore, mice were kept on a chow or a high fat diet and during the last 2 weeks D-Ala²-GIP or PBS injections were performed. Afterwards, serum LPL activity and several biochemical parameters (TG, FFA, cholesterol, glucose, insulin, resistin, IL-6, IL-1β, TNFα, GIP) were measured. Fat tissue was isolated and QPCR was performed for a set of genes involved in energy metabolism and inflammation. A DNA-microarray was used to identify GIP gene targets in adipose tissue of the chow diet group. We found that the D-Ala²-GIP injections caused a significant decrease in both body weight and LPL activity compared to controls. Serum biochemical parameters were not affected by D-Ala²-GIP, with an exception for resistin and insulin. The set of inflammatory genes were significantly decreased in adipose tissue in the D-Ala²-GIP injected animals on a chow diet. A DNA-microarray revealed that APO-genes and CYP-genes were affected by D-Ala²-GIP treatment in adipose tissue. These results suggest that the body weight-reducing effect of D-Ala²-GIP may be explained by lower LPL activity and insulin serum level. Moreover, the identified GIP candidate gene targets in adipose tissue link GIP action to lipid metabolism exerted by APO and CYP genes.     

Conditioned medium obtained from in vitro differentiated adipocytes and resistin induce insulin resistance in human hepatocytes

Adipocyte-derived factors might play a role in the development of hepatic insulin resistance. Resistin was identified as an adipokine linking obesity and insulin resistance. Resistin is secreted from adipocytes in rodents but in humans it was proposed to originate from macrophages and its impact for insulin resistance has remained elusive. To analyze the role of adipokines in general and resistin as a special adipokine, we cultured the human liver cell line HepG2 with adipocyte-conditioned medium (CM) containing various adipokines such as IL-6 and MCP-1, and resistin. CM and resistin both induce insulin resistance with a robust decrease in insulin-stimulated phosphorylation of Akt and GSK3. Insulin resistance could be prevented by co-treatment with troglitazone but not by co-stimulation with adiponectin. As human adipocytes do not secrete resistin, HepG2 cells were also treated with resistin added into CM. CM with resistin addition induced stronger insulin resistance than CM alone pointing to a specific role of resistin in the initiation of hepatic insulin resistance in humans.     

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.     

Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet

Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE^−/−) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE^−/− mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE^−/− mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE^−/− mice. In fat-induced CatE^−/− mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE^−/− mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia.     

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.     

Proteomic and functional characterization of endogenous adiponectin purified from fetal bovine serum

Adiponectin is a plasma protein exclusively secreted from fat tissue. Many recent pharmacological studies suggest that recombinant adiponectin has multiple therapeutic potentials for obesity-related metabolic disorders, including type 2 diabetes, dyslipidemia, insulin resistance and atherosclerosis. However, the physiological relevance of these findings remains to be further established. In the present study, we have purified endogenous adiponectin from fetal bovine serum and characterized its post-translational modifications and physiological functions in animal models. Endogenous bovine serum adiponectin consists predominantly of full-length proteins that form multiple oligomeric complexes, including trimers, hexamers and higher molecular species. Two-dimensional gel electrophoresis revealed that bovine serum adiponectin exists as multiple post-translationally modified isoforms with distinct molecular weight and isoelectric point. Further analysis using mass spectrometry and Edman degradation sequencing demonstrated that five conserved lysine residues (Lys 28, 60, 63, 72 and 96) within the collagenous domain of bovine adiponectin are hydroxylated and glycosylated by a glucosyl alpha(1-2)galactosyl group. Injection of endogenous bovine adiponectin into C57 mice potently decreased circulating glucose levels and enhanced lipid clearance after a high fat meal. Chronic administration of this protein for a period of two weeks significantly increased insulin sensitivity and glucose tolerance, and depleted hepatic lipid accumulation in high-fat fed mice. These results provide direct evidence that endogenous bovine adiponectin is a physiological hormone that can regulate lipid and glucose metabolism.     

IL-12 serum levels in patients with type 2 diabetes treated with sulphonylureas

Interleukin-12 (IL-12) has been identified as a pro-inflammatory cytokine which is thought to contribute to the development of atherosclerosis. However, to date, the various associations between factors related to the course of type 2 diabetes, like metabolic compensation, beta cell secretory dysfunction, insulin resistance and IL-12 serum levels, remain unclear. Our study involved 41 patients with type 2 diabetes, 19 patients with coronary artery disease (CAD), and 19 healthy controls. We measured serum levels of fasting glucose, HbA₁c, 1,5-anhydro-d-glucitol, and lipids. In addition, serum levels of C-peptide, insulin, proinsulin and IL-12 were assayed. HOMA_IR score was calculated. The serum concentrations of IL-12 were higher in diabetics than in either patients with CAD or healthy controls, and were correlated with BMI, C-peptide, insulin, HOMA_IR, proinsulin and HDL serum levels. Multiple regression analysis revealed that the IL-12 serum level in type 2 diabetics primarily is dependent upon fasting proinsulin concentration. Our results demonstrate that elevated IL-12 serum levels in type 2 diabetics treated with sulphonylureas are induced especially by peripheral insulin resistance and beta cells dysfunction, as expressed by fasting serum proinsulin levels. This finding gives us hope that treatment to decrease peripheral insulin resistance and to avoid excessive proinsulin secretion might be successful in the prevention of IL-12-induced atherosclerosis.     

Magainin-AM2 improves glucose homeostasis and beta cell function in high-fat fed mice

Background

Magainin-AM2, a previously described amphibian host-defense peptide, stimulates insulin- and glucagon-like peptide-1-release in vitro. This study investigated anti-diabetic effects of the peptide in mice with diet-induced obesity and glucose intolerance.

Methods

Male National Institute of Health Swiss mice were maintained on a high-fat diet for 12-weeks prior to the daily treatment with magainin-AM2. Various indices of glucose tolerance were monitored together with insulin secretory responsiveness of islets at conclusion of study.

Results

Following twice daily treatment with magainin-AM2 for 15 days, no significant difference in body weight and food intake was observed compared with saline-treated high fat control animals. However, non-fasting blood glucose was significantly (P < 0.05) decreased while plasma insulin concentrations were significantly (P < 0.05) increased. Oral and intraperitoneal glucose tolerance and insulin secretion following glucose administration via both routes were significantly (P < 0.05) enhanced. The peptide significantly (P < 0.001) improved insulin sensitivity as well as the beta cell responses of islets isolated from treated mice to a range of insulin secretagogues. Oxygen consumption, CO₂ production, respiratory exchange ratio and energy expenditure were not significantly altered by sub-chronic administration of magainin-AM2 but a significant (P < 0.05) reduction in fat deposition was observed.

Conclusion

These results indicate that magainin-AM2 improves glucose tolerance, insulin sensitivity and islet beta cells secretory responsiveness in mice with obesity-diabetes.

General significance

The activity of magainin-AM2 suggests the possibility of exploiting this peptide for treatment of type 2 diabetes.     

Decreased AMP-activated protein kinase activity is associated with increased inflammation in visceral adipose tissue and with whole-body insulin resistance in morbidly obese humans

Inflammation and infiltration of immune cells in white adipose tissue have been implicated in the development of obesity-associated insulin resistance. Likewise, dysregulation of the fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been proposed as a pathogenetic factor for these abnormalities based on both its links to insulin action and its anti-inflammatory effects. In this study, we examined the relationships between AMPK activity, the expression of multiple inflammatory markers in visceral (mesenteric and omental) and abdominal subcutaneous adipose tissue, and whole-body insulin sensitivity in morbidly obese patients (BMI 48 ± 1.9 kg/m²) undergoing gastric bypass surgery. AMPK activity was assessed by Western-blots (P-AMPK/T-AMPK) and mRNA levels of various markers of inflammation by qRT-PCR. Patients were stratified as insulin sensitive obese or insulin-resistant obese according to their HOMA-IR values. The results indicate that AMPK activity is lower in visceral than in subcutaneous abdominal adipose tissue of these patients and that this is associated with an increased expression of multiple inflammatory genes. They also revealed that AMPK activity is lower in adipose tissue of obese patients who are insulin resistant (HOMA-IR > 2.3) than in BMI-matched insulin sensitive subjects. Furthermore, this difference was evident in all three fat depots. In conclusion, the data suggest that there are close links between reduced AMPK activity and inflammation in white adipose tissue, and whole-body insulin resistance in obese humans. Whether adipose tissue AMPK dysregulation is a causal factor for the development of the inflammation and insulin resistance remains to be determined.     

Reduced adipose tissue mass and hypoleptinemia in iNOS deficient mice: effect of LPS on plasma leptin and adiponectin concentrations

The AAA+ chaperone ClpB solubilizes in cooperation with the DnaK chaperone system aggregated proteins. The mechanistic features of the protein disaggregation process are poorly understood. Here, we investigated the mechanism of ClpB/DnaK-dependent solubilization of heat-aggregated malate dehydrogenase (MDH) by following characteristics of MDH aggregates during the disaggregation reaction. We demonstrate that disaggregation is achieved by the continuous extraction of unfolded MDH molecules and not by fragmentation of large MDH aggregates. These findings support a ClpB-dependent threading mechanism as an integral part of the disaggregation reaction.     

Proteomic analysis of adipose tissue: informing diabetes research

Diabetes, one of the most common endocrine diseases worldwide, results from complex pathophysiological mechanisms that are not fully understood. Adipose tissue is considered a major endocrine organ and plays a central role in the development of diabetes. The identification of the adipose tissue-derived factors that contribute to the onset and progression of diabetes will hopefully lead to the development of preventive and therapeutic interventions. Proteomic techniques may be useful tools for this purpose. In the present review, we have summarized the studies conducting adipose tissue proteomics in subjects with diabetes and insulin resistance, and discussed the proteins identified in these studies as candidates to exert important roles in these disorders.     

Maximum activities of enzymes involved in adenosine metabolism in adipose tissue of rats and mice under conditions of variations in insulin sensitivity

Changes in the maximum activities of 5′-nucleotidase, adenosine kinase and adenosine deaminase in adipose tissue from obese mice, starved mice and rats, and adrenalectomised rats lead to the suggestion that changes in the adenosine concentration may be involved in changes in insulin sensitivity in these conditions.     

Effects of diet and exercise on metabolic disturbances in high-fat diet-fed mice

Consumption of a high-fat diet (HFD) is associated with white adipose tissue (WAT) inflammation, which contributes to key components of the metabolic syndrome, including insulin resistance (IR) and hepatic steatosis (HS). To determine the differential effects of exercise training (EX), low-fat diet (LFD), and their combination on WAT inflammation, Balb/cByJ male mice (n = 34) were fed an HFD for 12 wks before they were randomized into one of four intervention groups: HFD-EX, LFD-EX, HFD-sedentary (SED), or LFD-SED. EX mice performed 12 wks of exercise training on a motorized treadmill (1 h/d, 5 d/wk, 12 m/min, 5% grade, 65% VO₂ max), while SED mice remained sedentary in their home cages. WAT gene expression of adipokines was assessed using rt-PCR. IR was measured using HOMA-IR, and HS via hepatic triglyceride content. EX significantly reduced (53%) WAT gene expression of MCP-1, and LFD significantly reduced (50%) WAT gene expression of the macrophage specific marker, F4/80 as well as the adipocytokine IL-1ra (25%). EX independently improved IR, while both EX and LFD improved HS. These findings suggest that both diet and exercise have unique beneficial effects on WAT inflammatory markers and the mechanism by which each treatment improves metabolic complications associated with chronic consumption of an HFD may be different.     

Adiponectin: a biomarker of obesity-induced insulin resistance in adipose tissue and beyond

Adiponectin is one of the most thoroughly studied adipocytokines. Low plasma levels of adiponectin are found to associate with obesity, metabolic syndrome, diabetes and many other human diseases. From animal experiments and human studies, adiponectin has been shown to be a key regulator of insulin sensitivity. In this article, we review the evidence and propose that hypo-adiponectinemia is not a major cause of obesity. Instead, it is the result of obesity-induced insulin resistance in the adipose tissue. Hypo-adiponectinemia then mediates the metabolic effects of obesity on the other peripheral tissues, such as liver and skeletal muscle and may also exert some direct effects on end-organ damage. We propose that deciphering the molecular details governing the adiponectin gene expression and protein secretion will lead us to more comprehensive understanding of the mechanisms of insulin resistance in the adipose tissue and provide us new avenues for the therapeutic intervention of obesity and insulin resistance-related human disorders     

Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans

Macrophage infiltration of white adipose tissue (WAT) is implicated in the metabolic complications of obesity. The precipitating event(s) and function(s) of macrophage infiltration into WAT are unknown. We demonstrate that >90% of all macrophages in WAT of obese mice and humans are localized to dead adipocytes, where they fuse to form syncytia that sequester and scavenge the residual "free" adipocyte lipid droplet and ultimately form multinucleate giant cells, a hallmark of chronic inflammation. Adipocyte death increases in obese (db/db) mice (30-fold) and humans and exhibits ultrastructural features of necrosis (but not apoptosis). These observations identify necrotic-like adipocyte death as a pathologic hallmark of obesity and suggest that scavenging of adipocyte debris is an important function of WAT macrophages in obese individuals. The frequency of adipocyte death is positively correlated with increased adipocyte size in obese mice and humans and in hormone-sensitive lipase-deficient (HSL-/-) mice, a model of adipocyte hypertrophy without increased adipose mass. WAT of HSL-/- mice exhibited a 15-fold increase in necrotic-like adipocyte death and formation of macrophage syncytia, coincident with increased tumor necrosis factor-alpha gene expression. These results provide a novel framework for understanding macrophage recruitment, function, and persistence in WAT of obese individuals.