Amylin exerts osteogenic actions with different efficacy depending on the diabetic status.

Amylin displays osteogenic features, but its role in diabetic osteopenia is unclear. We examined the possible osteogenic action of amylin infusion for 3days into fructose-induced insulin-resistant (IR) and streptozotocin-induced type 2 diabetic (T2D) and normal (N) rats. Amylin failed to affect glycaemia or parathyroid hormone levels in any group, but reduced hyperinsulinemia in IR rats. In N rats, amylin increased bone formation rate and reduced osteoclast surface and erosive surface in the femoral metaphysis, and increased osteoprotegerin (OPG)/receptor activator of NFκB ligand (RANKL) mRNA ratio in the tibia. In T2D rats, amylin normalized trabecular structure parameters and increased osteoblast number and osteocalcin (OC) expression in long bones. In contrast, in IR rats, no apparent osteogenic effect of amylin in the femur was observed, although both OC and OPG/RANKL ratio were increased in the tibia. Our findings demonstrate a different osteogenic efficacy of amylin in two diabetic settings.

Amylin effect in extrapancreatic tissues participating in glucose homeostasis, in normal, insulin-resistant and type 2 diabetic state.

Amylin is co-secreted with insulin, responds to the same stimuli, is anorectic, lowers body weight by reducing fat mass, and is proposed for diabetes treatment. We examined the effect of a 3-day constant infusion of close to physiological doses of amylin in Wistar rats, on glucotransporter expression, glycogen content (G), glycogen synthase a activity (GSa) and glucose transport (GT), in liver, muscle and fat from insulin resistant (IR) and type 2 diabetic (T2D) models, compared to normal (N) animals; plasma glucose and insulin were measured. Plasma insulin in IR was higher than in N or T2D, and amylin normalized the value. In both, IR and T2D, liver G was lower than normal, accompanied by GLUT-2, mRNA and protein, higher and lower, respectively, than in N; amylin normalized G in both groups, without changes in GLUT-2, except for an mRNA increase in T2D. In IR and T2D, muscle GSa was reduced, together with respective over- and under-GLUT-4 expression; amylin induced only a trend toward GSa normalization in both groups. In isolated adipocytes, GT and GLUT-4 in IR and T2D were lower and higher, respectively, than in N; after amylin, not only GT was normalized in both groups but also the response to insulin was much more pronounced, including that in N, without major changes in GLUT-4. This suggests that the beneficial effect of amylin in states running with altered glucose homeostasis could occur by partially acting on the hexose metabolism of the liver and mainly on that of the adipose tissue.

Induction and reversibility of insulin resistance in rats exposed to exogenous D-fructose.

Long-term exposure of normal rats to a fructose-enriched diet or drinking water is currently used as an animal model for experimental insulin resistance. The present study deals with a comparison between rats given access to either a fructose-enriched diet or fructose-enriched drinking water. In both situations, a decrease in food intake and body weight gain, and the induction of insulin resistance with intolerance to D-glucose despite increased secretory response to the aldohexose of insulin-producing cells were documented. Moreover, the rats exposed to exogenous D-fructose displayed a lesser sensitivity to overnight fasting than control animals, in terms of the alteration of glucose homeostasis and reduction of the ratio between plasma insulin and D-glucose concentration. It is also shown that the fructose-induced insulin resistance, as assessed in a hyperinsulinemic-euglycemic clamp, represents a phenomenon reversed within 15-30 days after removal of the keto-hexose from the drinking water.

Glucose intolerance associated to insulin resistance and increased insulin secretion in rats depleted in long-chain omega3 fatty acids.

An intragastric D-glucose tolerance test was performed, after overnight starvation, in female rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3D rats) and control rats of same age and gender. The plasma D-glucose and insulin concentrations, insulinogenic index, and HOMA for insulin resistance were all higher, after overnight starvation, in omega3D rats than in control animals. Over the 120-minute period following the intragastric administration of D-glucose, the area under the curve for the same four variables was also higher in omega3D rats than in control animals. In addition to visceral obesity, liver steatosis, hypertension, and cardiac hypertrophy, the omega3D rats thus display further features of the metabolic syndrome, namely glucose intolerance and insulin resistance, despite hyperinsulinemia.

Effects of diet supplementation with olive oil and guar upon fructose-induced insulin resistance in normal rats.

Exposure of normal rats to fructose-containing drinking water represents a current model of insulin resistance. The major aim of the present study was to assess the possible effect of diet supplementation with either olive oil or guar upon the metabolic consequences of exposure to exogenous fructose. For this purpose, the changes in body weight, plasma D-glucose and insulin concentrations, and D-glucose infusion rate during a hyperinsulinemic-euglycemic clamp were measured after 65 days exposure to exogenous fructose and either olive oil- or guar-enriched diet. The results were compared to those previously collected in control animals exposed for the same period to either tap water or the fructose-containing drinking water and a standard diet. Diet supplementation with olive oil or guar failed to affect the increase in the insulinogenic index and the decrease in insulin sensitivity and fasted/fed ratio for plasma insulin concentration caused by exogenous fructose. In the rats exposed to exogenous fructose, the olive oil-fed rats differed from other animals by the absence of a decrease in food intake and body weight gain, whilst the guar-fed rats differed from other animals in a lower plasma D-glucose concentration in fed state and an absence, at day 65, of a higher plasma D-glucose concentration than that at day 0 measured in after overnight fasting state. These findings argue in favour of guar, rather than olive oil, to oppose the effect of exogenous fructose on glucose homeostasis.

Alteration of adipocyte metabolism in omega3 fatty acid-depleted rats.

Presently an insufficient supply of long-chain polyunsaturated omega3 fatty acid is prevalent in Western populations leading to potential metabolic consequences. Based on this fact, this study deals mainly with various aspects of lipid metabolism in second generation female omega3-depleted rats. The parametrial fat and body weights were higher in omega3-depleted than control animals. This coincided with liver steatosis but did not alter heart triglyceride/phospholipid ratio. The net uptake of [U-14C] palmitate by adipocytes was also higher in omega3-depleted rats than in control animals. The uptake of D-[U- 4C] glucose or [1,2 (-14)C] acetate by adipocytes was lower, however in omega3-depleted than control animals and was unaffected by insulin in the former as distinct from latter animals. Despite comparable basal lipolysis, the increase in glycerol output from adipocytes provoked by theophylline was higher in omega3-depleted than control rats. The fatty acid pattern of lipids in adipose tissue was characterized in the omega3-depleted rats by a much lower omega3 content, higher apparent Delta 9-saturase and elongase activities, lower efficiency for the conversion of C18:2omega6 to C20:4omega6 and higher efficiency for the conversion of C18:3omega3 to C20:5omega3. These features were compared to those prevailing in liver and plasma lipids. The present study thus extends knowledge on the alteration of lipid metabolism resulting from a deficiency in long-chain polyunsaturated omega3 fatty acids.

Short-term and long-term effects of guar on postprandial plasma glucose, insulin and glucagon-like peptide 1 concentration in healthy rats.

Ingestion of guar gum decreases postprandial glycemia and insulinemia and improves sensitivity to insulin in diabetic patients and several animal models of diabetes. The aim of the present study was to compare the short-term and long-term effects of guar on plasma insulin and glucagon-like peptide 1 concentration in healthy rats. In the short-term experiments, the concomitant intragastric administration of glucose and guar reduced the early increment in plasma glucose, insulin and glucagon-like peptide 1 concentration otherwise induced by glucose alone. Comparable findings were made after twelve days of meal training exposing the rats to either a control or guar-enriched diet for fifteen minutes. Mean plasma glucose concentrations were lower while mean insulin concentrations were higher in the guar group than in the controls according to intragastric glucose tolerance tests conducted in overnight fasted rats maintained for 19 to 36 days on either the control or guar-enriched diet. The intestinal content of glucagon-like peptide 1 at the end of the experiments was also lower in the guar group. Changes in body weight over 62 days of observation were comparable in the control and guar rats. Thus, long-term intake of guar improves glucose tolerance and insulin response to glucose absorption, without improving insulin sensitivity, in healthy rats.

Effects of an olive oil-enriched diet on glucagon-like peptide 1 release and intestinal content, plasma insulin concentration, glucose tolerance and pancreatic insulin content in an animal model of type 2 diabetes.

In the light of a recent study conducted in normal rats, the present investigations were aimed at exploring the immediate and long-term effects of an olive oil-enriched diet (OO diet) on GLP-1 release and intestinal content, plasma insulin concentration, glucose tolerance and pancreatic insulin content in adult rats that had been injected with streptozotocin during the neonatal period (STZ rats). The OO diet, when compared to a standard diet, increased the immediate GLP-1 response in meal-trained rats, but decreased GLP-1 content in the intestinal tract after 50 days. Over 50 days, the body weight gain was lower in the rats fed the OO diet compared to standard diet. In the former, however, no improvement of glucose tolerance or insulin response during an oral glucose tolerance test was observed. Thus, a paradoxical lowering of the insulinogenic index, i. e. the paired ratio between plasma insulin and glucose concentration, was recorded during the oral glucose tolerance test in rats fed either standard or OO diet. Moreover, the insulin content of the pancreas was equally low in the STZ rats fed either standard or OO diet. These findings will be discussed in the framework of possible differences in the pathophysiology of B-cell dysfunction in most patients with type-2 diabetes and the present animal model of non-insulin-dependent diabetes.

Participation of protein kinases in the stimulant action of GLP-1 on 2-deoxy-D-glucose uptake by normal rat skeletal muscle.

Several protein kinases were recently proposed for involvement in GLP-1-stimulated D-glucose transport in skeletal muscle from both normal subjects and type 2 diabetic patients. This study was mainly aimed at investigating the effect of potential inhibitors of distinct protein kinases and protein phosphatase-1 upon insulin- and GLP-1-stimulated 2-deoxy-D-glucose net uptake by normal rat skeletal muscle. The basal uptake of the D-glucose analog was decreased by wortmannin--a phosphatidylinositol-3-kinase inhibitor--, PD98059--a mitogen-activated protein kinases inhibitor--, and TNFalpha--a protein phosphatase-1 inhibitor--, but not by either rapamycin--a p70s6 kinase inhibitor--, or H-7--, a protein kinase C inhibitor--. The enhancing action of both insulin and GLP-1 upon 2-deoxy-D-glucose transport was abolished by PD98059 and H-7, but largely unaffected by TNFalpha. Wortmannin and rapamycin preferentially affected the response to GLP-1 and insulin, respectively. These findings thus document both analogies and dissimilarities in the participation of the concerned enzymes in the stimulant action of insulin versus GLP-1 upon D-glucose transport in normal rat skeletal muscle.

Cell signalling of glucagon-like peptide-1 action in rat skeletal muscle.

Glucagon-like peptide-1 (GLP-1), an incretin with glucose-dependent insulinotropic and insulin-independent antidiabetic properties, has insulin-like effects on glucose metabolism in extrapancreatic tissues participating in overall glucose homeostasis. These effects are exerted through specific receptors not associated with cAMP, an inositol phosphoglycan being a possible second messenger. In rat hepatocytes, activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB), protein kinase C (PKC) and protein phosphatase 1 (PP-1) has been shown to be involved in the GLP-1-induced stimulation of glycogen synthase. We have investigated the role of enzymes known or suggested to mediate the actions of insulin in the GLP-1-induced increase in glycogen synthase a activity in rat skeletal muscle strips. We first explored the effect of GLP-1, compared with that of insulin, on the activation of PI3K, PKB, p70s6 kinase (p70s6k) and p44/42 mitogen-activated protein kinases (MAPKs) and the action of specific inhibitors of these kinases on the insulin- and GLP-1-induced increment in glycogen synthase a activity. The study showed that GLP-1, like insulin, activated PI3K/PKB, p70s6k and p44/42. Wortmannin (a PI3K inhibitor) reduced the stimulatory action of insulin on glycogen synthase a activity and blocked that of GLP-1, rapamycin (a 70s6k inhibitor) did not affect the action of GLP-1 but abolished that of insulin, PD98059 (MAPK inhibitor) was ineffective on insulin but blocked the action of GLP-1, okadaic acid (a PP-2A inhibitor) and tumour necrosis factor-alpha (a PP-1 inhibitor) were both ineffective on GLP-1 but abolished the action of insulin, and Ro 31-8220 (an inhibitor of some PKC isoforms) reduced the effect of GLP-1 while completely preventing that of insulin. It was concluded that activation of PI3K/PKB and MAPKs is required for the GLP-1-induced increment in glycogen synthase a activity, while PKC, although apparently participating, does not seem to play an essential role; unlike in insulin signaling, p70s6k, PP-1 and PP-2A do not seem to be needed in the action of GLP-1 upon glycogen synthase a activity in rat muscle.

Glucagon-like peptide-1 (GLP-1) and glucose metabolism in human myocytes.

Glucagon-like peptide-1 (GLP-1) has been shown to have insulin-like effects upon the metabolism of glucose in rat liver, muscle and fat, and on that of lipids in rat and human adipocytes. These actions seem to be exerted through specific receptors which, unlike that of the pancreas, are not - at least in liver and muscle - cAMP-associated. Here we have investigated the effect, its characteristics, and possible second messengers of GLP-1 on the glucose metabolism of human skeletal muscle, in tissue strips and primary cultured myocytes. In muscle strips, GLP-1, like insulin, stimulated glycogen synthesis, glycogen synthase a activity, and glucose oxidation and utilization, and inhibited glycogen phosphorylase a activity, all of this at physiological concentrations of the peptide. In cultured myotubes, GLP-1 exerted, from 10(-13) mol/l, a dose-related increase of the D-[U-(14)C]glucose incorporation into glycogen, with the same potency as insulin, together with an activation of glycogen synthase a; the effect of 10(-11) mol/l GLP-1 on both parameters was additive to that induced by the equimolar amount of insulin. Synthase a was still activated in cells after 2 days of exposure to GLP-1, as compared with myotubes maintained in the absence of peptide. In human muscle cells, exendin-4 and its truncated form 9-39 amide (Ex-9) are both agonists of the GLP-1 effect on glycogen synthesis and synthase a activity; but while neither GLP-1 nor exendin-4 affected the cellular cAMP content after 5-min incubation in the absence of 3-isobutyl-1-methylxantine (IBMX), an increase was detected with Ex-9. GLP-1, exendin-4, Ex-9 and insulin all induced the prompt hydrolysis of glycosylphosphatidylinositols (GPIs). This work shows a potent stimulatory effect of GLP-1 on the glucose metabolism of human skeletal muscle, and supports the long-term therapeutic value of the peptide. Further evidence for a GLP-1 receptor in this tissue, different from that of the pancreas, is also illustrated, suggesting a role for an inositolphosphoglycan (IPG) as at least one of the possible second messengers of the GLP-1 action in human muscle.

Resistance of succinic acid dimethyl ester insulinotropic action to exendin (9-39) amide.

Exendin (9-39) amide (Ex [9-39]) was recently proposed for use in the treatment of alimentary or reactive hypoglycaemia. It was indeed found to antagonise the insulinotropic action of GLP-1 in rats infused with the dimethyl ester of succinic acid (SAD). We have now investigated whether, under comparable experimental conditions, Ex (9-39) also opposes the insulin-releasing action of SAD itself. Since this was not the case, Ex (9-39) could be safely used to abolish the incretin effect of GLP-1 without interfering with the control of insulin secretion by circulating nutrients.

Pancreatic and extrapancreatic effects of GLP-1.

Glucagon-like peptide-1 (GLP-1), an incretin hormone which helps to regulate plasma glucose levels, is considered a potential agent for the treatment of type-2 diabetes mellitus, because of its insulinotropic capacity and insulinomimetic actions. In normal conditions, the beta-cell secretory response to GLP-1 is modulated by the extracellular concentration of D-glucose; however, the recognition of D-glucose by the beta-cell is often impaired in type-2 diabetes, and this could impede the full GLP-1 insulinotropic action. Non-glucidic substrates, such as the dimethyl ester of succinic acid, restore the effect of GLP-1 in the isolated perfused rat pancreas of normal or diabetic rats, in the absence of any other exogenous nutrient; likewise, the dimethyl ester of succinic or L-glutamic acid, and the monomethyl ester of pyruvic acid, potentiate the in vivo beta-cell secretory response to GLP-1 in normal and diabetic rats. Therefore, it was proposed that nutrients susceptible to bypass the site-specific defects of the diabetic beta-cell, could be used to potentiate and/or prolong the insulinotropic action of antidiabetic agents such as GLP-1. In vitro, GLP-1 insulin-like effects on glucose metabolism have been documented in normal and diabetic rat liver, and in rat and human skeletal muscle. In rat and human adipocytes, GLP-1 is lipolytic and/or lipogenic, and also stimulates parameters involved in the glucose metabolism. In liver, muscle and fat, GLP-1 seems to act through specific receptors, apparently different--at least in liver and muscle--in structure or signaling pathway from the pancreatic one. It is proposed that an inositolphosphoglycan might be a second messenger of GLP-1 action in extrapancreatic tissues.

Effect of GLP-1 treatment on GLUT2 and GLUT4 expression in type 1 and type 2 rat diabetic models.

Glucagon-like peptide-1 (G LP-1) is an incretin with glucose-dependent insulinotropic and insulin-independent antidiabetic properties that exerts insulin-like effects on glucose metabolism in rat liver, skeletal muscle, and fat. This study aimed to search for the effect of a prolonged treatment, 3 ds, with GLP-1 on glucotransporter GLUT2 expression in liver, and on that of GLUT4 in skeletal muscle and fat, in rats. Normal rats and streptozotocin-induced type 1 and type 2 diabetic models were used; diabetic rats were also treated with insulin for comparison. In normal rats, GLP-1 treatment reduced in the three tissues the corresponding glucotransporter protein level, without modifying their mRNA. In the type 2 diabetic model, GLP-1, like insulin, stimulated in liver and fat only the glucotransporter translational process, while in the muscle an effect at the GLUT4 transcriptional level was also observed. In the type 1 diabetic model, GLP-1 apparently exerted in the liver only a posttranslational effect on GLUT2 expression; in muscle and fat, while insulin was shown to have an action on GLUT4 at both transcriptional and translational levels, the effect of GLP-1 was restricted to glucotransporter translation. In normal and diabetic rats, exogenous GLP-1 controlled the glucotransporter expression in extrapancreatic tissues participating in the overall glucose homeostasis-liver, muscle, and fat-where the effect of the peptide seems to be exerted only at the translational and/or posttranslational level; in muscle and fat, the presence of insulin seems to be required for GLP-1 to activate the transcriptional process. The stimulating action of GLP-1 on GLUT2 and GLUT4 expression, mRNA or protein, could be a mechanism by which, at least in part, the peptide exerts its lowering effect on blood glucose.

Potentiation by glutamic acid dimethyl ester of GLP-1 insulinotropic action in fed anaesthetized rats.

The dimethyl ester of L-glutamic acid (DMG) stimulates insulin release and was proposed as a possible insulinotropic tool in the treatment of non-insulin-dependent diabetes. In such a perspective, it was investigated whether DMG enhances the B-cell secretory response to GLP-1 in fed anaesthetized rats. The primed constant infusion of DMG (1.0 micromol and then 0.5 micromol/min, both per g body wt.) provoked a rapid and sustained increase in plasma insulin concentration and augmented the release of insulin caused by GLP-1. Thus, DMG indeed appears as a suitable tool to potentiate the insulinotropic action of GLP-1.

Synergistic insulinotropic effects of succinic acid dimethyl ester and exendin-4 in anaesthetized rats.

It was recently proposed that suitable succinic acid esters could be used to potentiate the insulinotropic action of glucagon-like peptide 1 (GLP-1) in the treatment of type-2 diabetes mellitus. In such a perspective, the present study aimed mainly at investigating whether exendin-4 (Ex-4), a peptide structurally related to GLP-1(7-36)amide, and succinic acid dimethyl ester (SAD) also act synergistically upon insulin secretion in anaesthetized rats. Despite a higher plasma insulin concentration in SAD-infused rats (5.5+/-1.1 ng/ml) than in saline-infused animals (1.9+/-0.7 ng/ml), the intravenous injection of Ex-4 augmented to a greater extent the plasma concentration of insulin in the former rats (+7.4+/-2.5 ng/ml) than in the latter animals (+2.8+/-0.6 ng/ml). These findings document that the insulinotropic actions of Ex-4 and GLP-1 display comparable nutrient dependency, being both potentiated by a non-glucidic nutrient secretagogue such as SAD.

Potentiation by methyl pyruvate of GLP-1 insulinotropic action in normal rats.

Methyl pyruvate (MP) stimulates insulin release both in vivo and in vitro. The present study aims at investigating whether MP is also able to enhance the B-cell secretory response to glucagon-like peptide 1 (GLP-1). In anaesthetized rats receiving a primed constant infusion of MP, the ester augmented plasma insulin concentration before GLP-1 injection and potentiated the insulinotropic action of the intestinal hormone. MP infusion also augmented plasma D-glucose concentration, whether in the absence or presence of GLP-1. A further rise in plasma D-glucose concentration was observed when the infusion of MP was halted, this coinciding with a fall in plasma insulin concentration. Whilst documenting that MP indeed enhances the B-cell secretory response to GLP-1, these findings do not suggest that MP is an appropriate tool for optimizing the hypoglycaemic action of the enteric hormone in the treatment of type-2 diabetes mellitus.

Pancreatic and hepatic glycogen content in normoglycemic and hyperglycemic rats.

As judged from morphological criteria, glycogen accumulates to a larger extent in insulin-producing B-cells than in acinar cells of the pancreas in situations of sustained hyperglycemia. In the present study, the glycogen content of the pancreatic gland and liver was measured in either euglycemic or glucose-infused hyperglycemic control rats, as well as in streptozotocin-induced diabetic rats. Whilst the glycogen content of the pancreas was significantly higher in STZ rats than in control euglycemic rats, it was further enhanced in glucose-infused control rats, despite the fact that the latter animals were not more severely hyperglycemic and for a shorter time than STZ rats. From these measurements, it was estimated that, relative to wet weight, the glycogen content was, under the present experimental conditions, about 75 times higher in insulin-producing than other pancreatic cells. Moreover, it is proposed that the intravenous administration of glucagon may help in distinguishing between the glycogen present in the endocrine and exocrine moieties of the pancreatic gland, this hormone being apparently unable to provoke glycogenolysis in the exocrine pancreas, at variance with the situation prevailing in isolated pancreatic islets.

Effect of GLP-1 on lipid metabolism in human adipocytes.

We have studied the effect of several doses of GLP-1, compared to that of insulin and glucagons, on lipogenesis, lipolysis and cAMP cellular content, in human adipocytes isolated from normal subjects. In human adipocytes, GLP-1 exerts a dual action, depending upon the dose, on lipid metabolism, being lipogenic at low concentrations of the peptide (ED50, 10(-12) M), and lipolytic only at doses 10-100 times higher (ED50, 10(-10) M); both effects are time- and GLP-1 concentration-dependent. The GLP-1 lipogenic effect is equal in magnitude to that of equimolar amounts of insulin; both hormones apparently act synergically, and their respective action is abolished by glucagon. The lipolytic effect of GLP-1 is comparable to that of glucagon, apparently additive to it, and the stimulated value induced by either one is neutralized by the presence of insulin. In the absence of IBMX, GLP-1, at 10(-13) and 10(-12) M, only lipogenic doses, does not modify the cellular content of cAMP, while from 10(-11) M to 10(-9) M, also lipolytic concentrations, it has an increasing effect; in the presence of IBMX, GLP-1 at already 10(-12) M increased the cellular cAMP content. In human adipocytes, GLP-1 shows glucagon- and also insulin-like effects on lipid metabolism, suggesting the possibility of GLP-1 activating two distinct receptors, one of them similar or equal to the pancreatic one, accounting cAMP as a second messenger only for the lipolytic action of the peptide.

Potentiation and prolongation of the insulinotropic action of glucagon-like peptide 1 by methyl pyruvate or dimethyl ester of L-glutamic acid in a type 2 diabetes animal model.

Methyl pyruvate and the dimethyl ester of L-glutamic acid were administered intravenously, as a primed constant infusion (1.0-2.0 micromol followed by 0.5-1.0 micromol/min, both expressed per gram of body wt), in adult rats that had been injected with streptozotocin during the neonatal period. Each ester augmented plasma insulin concentration and potentiated and/or prolonged the insulinotropic action of glucagon-like peptide 1 (GLP-1) injected intravenously (5 pmol/g of body wt) at min 5 of the test. It is proposed, therefore, that suitable nonglucidic nutrients, susceptible to bypassing the site-specific defects of D-glucose transport and metabolism responsible for the preferential impairment of the B-cell secretory response to D-glucose in non-insulin-dependent diabetes, could be used to optimize the insulinotropic action of GLP-1.