Ewa Pruszyńska-Oszmałek

Glucagon increases circulating fibroblast growth factor 21 independently of endogenous insulin levels: a novel mechanism of glucagon-stimulated lipolysis?

Aims/hypothesisGlucagon reduces body weight by modifying food intake, glucose/lipid metabolism and energy expenditure. All these physiological processes are also controlled by fibroblast growth factor 21 (FGF-21), a circulating hepatokine that improves the metabolic profile in obesity and type 2 diabetes. Animal experiments have suggested a possible interaction between glucagon and FGF-21 however, the metabolic consequences of this crosstalk are not understood.MethodsThe effects of exogenous glucagon on plasma FGF-21 levels and lipolysis were evaluated in healthy volunteers and humans with type 1 diabetes, as well as in rodents with streptozotocin (STZ)-induced insulinopenic diabetes. In vitro, the role of glucagon on FGF-21 secretion and lipolysis was studied using isolated primary rat hepatocytes and adipocytes. Fgf-21 expression in differentiated rat pre-adipocytes was suppressed by small interfering RNA and released FGF-21 was immunoneutralised by polyclonal antibodies.ResultsGlucagon induced lipolysis in healthy human volunteers, patients with type 1 diabetes, mice and rats with STZ-induced insulinopenic diabetes, and in adipocytes isolated from diabetic and non-diabetic animals. In addition, glucagon increased circulating FGF-21 in healthy humans and rodents, as well as in patients with type 1 diabetes, and insulinopenic rodents. Glucagon stimulated FGF-21 secretion from isolated primary hepatocytes and adipocytes derived from animals with insulinopenic diabetes. Furthermore, FGF-21 stimulated lipolysis in primary adipocytes isolated from non-diabetic and diabetic rats. Reduction of Fgf-21 expression (by approximately 66%) or immunoneutralisation of released FGF-21 markedly attenuated glucagon-stimulated lipolysis in adipocytes.Conclusions/interpretationThese results indicate that glucagon increases circulating FGF-21 independently of endogenous insulin levels. FGF-21 participates in glucagon-induced stimulation of lipolysis.

Genistein restricts leptin secretion from rat adipocytes.

The isoflavones--genistein and daidzein -- compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein-- an analogue of genistein -- used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2h in Krebs--Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25--1mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.

A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes

Background: An increased glucagon/insulin ratio is known to contribute to hyperglycemia in diabetes. Results: NOX-G15, a mirror-image mixed DNA/RNA glucagon-neutralizing aptamer, was identified. It improved glucose tolerance in models of type 1 and 2 diabetes. Conclusion: NOX-G15 may be useful for treatment of type 1 and 2 diabetes. Significance: The new therapeutic candidate may help to reduce insulin need in diabetes. Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2′-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.

Effects of high-fat diet-induced obesity and diabetes on Kiss1 and GPR54 expression in the hypothalamic–pituitary–gonadal (HPG) axis and peripheral organs (fat, pancreas and liver) in male rats

Recent data indicates that kisspeptin, encoded by the KISS1 gene, could play a role in transducing metabolic information into the hypothalamic-pituitary-gonadal (HPG) axis, the mechanism that controls reproductive functions. Numerous studies have shown that in a state of negative energy balance, the hypothalamic kisspeptin system is impaired. However, data concerning positive energy balance (e.g. diabetes and obesity) and the role of kisspeptin in the peripheral tissues is scant. We hypothesized that: 1) in diet-induced obese (DIO) male rats and/or rats with diabetes type 1 (DM1) and type 2 (DM2), altered reproductive functions are related to an imbalance in Kiss1 and GPR54 mRNA in the HPG axis; and 2) in DIO and/or DM1 and/or DM2 rats, Kiss1 and GPR 54 expression are altered in the peripheral tissues involved in metabolic functions (fat, pancreas and liver). Animals were fed a high-fat or control diets and STZ (streptozotocin - toxin, which destroys the pancreas) was injected in high or low doses to induce diabetes type 1 (DM1) or diabetes type 2 (DM2), respectively. RT-PCR and Western blot techniques were used to assess the expression of Kiss1 and GRP54 in tissues. At the level of mRNA, we found that diabetic but not obese rats have alterations in Kiss1 and/or GPR54 mRNA levels in the HPG axis as well as in peripheral tissues involved in metabolic functions (fat, pancreas and liver). The most severe changes were seen in DM1 rats. However, in the case of protein levels in the peripheral tissues (fat, pancreas and liver), changes in Kiss1/GPR54 expression were noticed in DIO, DM1 and DM2 animals and were tissue-specific. Our data support the hypothesis that alterations in Kiss1/GPR54 balance may account for both reproductive and metabolic abnormalities reported in obese and diabetic rats.

Neuropeptide B and W regulate leptin and resistin secretion, and stimulate lipolysis in isolated rat adipocytes

Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.

Glucagon regulates orexin A secretion in humans and rodents

Aims/hypothesisOrexin A (OXA) modulates food intake, energy expenditure, and lipid and glucose metabolism. OXA regulates the secretion of insulin and glucagon, while glucose regulates OXA release. Here, we evaluate the role of glucagon in regulating OXA release both in vivo and in vitro.MethodsIn a double-blind crossover study, healthy volunteers and type 1 diabetic patients received either intramuscular glucagon or placebo. Patients newly diagnosed with type 2 diabetes underwent hyperinsulinaemic–euglycaemic clamp experiments, and insulin–hypoglycaemia tests were performed on healthy volunteers. The primary endpoint was a change in OXA levels after intramuscular glucagon or placebo administration in healthy participants and patients with type 1 diabetes. Secondary endpoints included changes in OXA in healthy participants during insulin tolerance tests and in patients with type 2 diabetes under hyperinsulinaemic–euglycaemic conditions. Participants and staff conducting examinations and taking measurements were blinded to group assignment. OXA secretion in response to glucagon treatment was assessed in healthy and obese mice, the streptozotocin-induced mouse model of type 1 diabetes, and isolated rat pancreatic islets.ResultsPlasma OXA levels declined in lean volunteers and in type 1 diabetic patients injected with glucagon. OXA levels increased during hyperinsulinaemic hypoglycaemia testing in healthy volunteers and during hyperinsulinaemic euglycaemic conditions in type 2 diabetic patients. Plasma OXA concentrations in healthy lean and obese mice and in a mouse model of type 1 diabetes were lower after glucagon treatment, compared with vehicle control. Glucagon decreased OXA secretion from isolated rat pancreatic islets at both low and high glucose levels. OXA secretion declined in pancreatic islets exposed to diazoxide at high and low glucose levels, and after exposure to an anti-insulin antibody. Glucagon further reduced OXA secretion in islets pretreated with diazoxide or an anti-insulin antibody.Conclusions/interpretationGlucagon inhibits OXA secretion in humans and animals, irrespective of changes in glucose or insulin levels. Through modifying OXA secretion, glucagon may influence energy expenditure, body weight, food intake and glucose metabolism.

Does somatostatin confer insulinostatic effects of neuromedin u in the rat pancreas

Objectives: Neuromedin U (NmU) is a neuropeptide with anorexigenic activity. Two receptor subtypes (NmUR1 and NmUR2) confer the effects of NmU on target cells. We have recently demonstrated that NmU reduces insulin secretion from isolated pancreatic islets. Aim of our current study is to investigate the role of somatostatin at mediating the effects of NmU on insulin secretion. Methods: Expression of NmU in the pancreas was detected by immunohistochemistry. Insulin and somatostatin secretion from in situ perfused rat pancreas and isolated pancreatic islets was measured by radioimmunoassay. The paracrine effects of somatostatin within pancreatic islets were blocked by cyclosomatostatin, a somatostatin receptor antagonist. Results: Receptor subtype NmUR1, but not NmUR2, was expressed in the endocrine pancreas, predominantly in the periphery. Neuromedin U reduced insulin secretion from in situ perfused rat pancreas and stimulated somatostatin secretion from isolated pancreatic islets. Neuromedin U stimulated somatostatin secretion at both physiological and supraphysiological glucose concentrations. Cyclosomatostatin increased insulin secretion and reduced NmU-induced inhibition of insulin secretion. Conclusions: Neuromedin U reduces insulin and increases somatostatin secretion. Blockade of somatostatin action abolishes the inhibition of insulin secretion by NmU. The results of the study suggest that somatostatin mediates the inhibitory action of NmU on insulin secretion.

Changes in obestatin gene and GPR39 receptor expression in peripheral tissues of rat models of obesity, type 1 and type 2 diabetes.

Obestatin has a role in regulating food intake and energy expenditure, but the roles of obestatin and the GPR39 receptor in obesity and type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively) are not well understood. The aim of the present study was to investigate changes in obestatin and GPR39 in pathophysiological conditions like obesity, T1DM, and T2DM.

Changes of agouti-related protein in hypothalamus, placenta, and serum during pregnancy in the rat

Agouti-related protein (AGRP) is a homolog of the agouti protein and acts as an antagonist of peptides derived from propiomelanocortin through melanocortin receptors. This peptide is produced mainly in the hypothalamus, particularly during negative energy balance and influences increased food intake. In the hypothalamus, this peptide is co-expressed in arcuate nuclei with neuropeptide Y, another important peptide that regulates energy metabolisms. In our study, we analyzed changes in the Agrp mRNA level in the hypothalamus as well as mRNA and protein levels in placenta during different stages of rat pregnancy. We also investigated the AGRP level in the blood serum. In this study, we found the AGRP level in serum increased, while its gene expression in the hypothalamus increased only up to the 13th day of pregnancy, and decreased on the 18th day. This study demonstrates that AGRP is expressed during late pregnancy in placenta. Moreover, we found that AGRP expression is higher on the 18th than on the 13th day of pregnancy. Our results indicate that AGRP may play an important role during pregnancy in the mothers and, possibly, also in the fetuss energy balance.

Long-term obestatin treatment of mice type 2 diabetes increases insulin sensitivity and improves liver function

PurposeObestatin and ghrelin are peptides encoded by the preproghrelin gene. Obestatin inhibits food intake, in addition to regulation of glucose and lipid metabolism. Here, we test the ability of obestatin at improving metabolic control and liver function in type 2 diabetic animals (type 2 diabetes mellitus).MethodsThe effects of chronic obestatin treatment of mice with experimentally induced type 2 diabetes mellitus on serum levels of glucose and lipids, and insulin sensitivity are characterized. In addition, alterations of hepatic lipid and glycogen contents are evaluated.ResultsObestatin reduced body weight and decreased serum glucose, fructosamine, and β-hydroxybutyrate levels, as well as total and low-density lipoprotein fractions of cholesterol. In addition, obestatin increased high-density lipoproteins cholesterol levels and enhanced insulin sensitivity in mice with type 2 diabetes mellitus. Moreover, obestatin diminished liver mass, hepatic triglycerides and cholesterol contents, while glycogen content was higher in livers of healthy and mice with type 2 diabetes mellitus treated with obestatin. These changes were accompanied by reduction of increased alanine aminotransferase, aspartate aminotransferase, and gamma glutamyl transpeptidase in T2DM mice with type 2 diabetes mellitus. Obestatin increased adiponectin levels and reduced leptin concentration. Obestatin influenced the expression of genes involved in lipid and carbohydrate metabolism by increasing Fabp5 and decreasing G6pc, Pepck, Fgf21 mRNA in the liver. Obestatin increased both, AKT and AMPK phosphorylation, and sirtuin 1 (SIRT1) protein levels as well as mRNA expression in the liver.ConclusionObestatin improves metabolic abnormalities in type 2 diabetes mellitus, restores hepatic lipid contents and decreases hepatic enzymes. Therefore, obestatin could potentially have a therapeutic relevance in treating of insulin resistance and metabolic dysfunctions in type 2 diabetes mellitus.