GIP and GLP-1 as incretin hormones: lessons from single and double incretin receptor knockout mice

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut-derived incretins secreted in response to nutrient ingestion. Both incretins potentiate glucose-dependent insulin secretion and enhance beta-cell mass through regulation of beta-cell proliferation, neogenesis and apoptosis. In contrast, GLP-1, but not GIP, inhibits gastric emptying, glucagon secretion, and food intake. Furthermore, human subjects with Type 2 diabetes exhibit relative resistance to the actions of GIP, but not GLP-1R agonists. The physiological importance of both incretins has been investigated through generation and analysis of incretin receptor knockout mice. Elimination of incretin receptor action in GIPR-/- or GLP-1R-/- mice produces only modest impairment in glucose homeostasis. Similarly, double incretin receptor knockout (DIRKO) mice exhibit normal body weight and normal levels of plasma glucagon and hypoglycemic responses to exogenous insulin. However, glucose-stimulated insulin secretion is significantly decreased following oral but not intraperitoneal glucose challenge in DIRKO mice and the glucose lowering actions of dipeptidyl peptidase-IV (DPP-IV) inhibitors are extinguished in DIRKO mice. Hence, incretin receptor signaling exerts physiologically relevant actions critical for glucose homeostasis, and represents a pharmacologically attractive target for development of agents for the treatment of Type 2 diabetes.     

The separate and combined impact of the intestinal hormones, GIP, GLP-1, and GLP-2, on glucagon secretion in type 2 diabetes

Type 2 diabetes mellitus (T2DM) is associated with reduced suppression of glucagon during oral glucose tolerance test (OGTT), whereas isoglycemic intravenous glucose infusion (IIGI) results in normal glucagon suppression in these patients. We examined the role of the intestinal hormones glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 (GLP-2) in this discrepancy. Glucagon responses were measured during a 3-h 50-g OGTT (day A) and an IIGI (day B) in 10 patients with T2DM [age (mean ± SE), 51 ± 3 yr; body mass index, 33 ± 2 kg/m(2); HbA(1c), 6.5 ± 0.2%]. During four additional IIGIs, GIP (day C), GLP-1 (day D), GLP-2 (day E) and a combination of the three (day F) were infused intravenously. Isoglycemia during all six study days was obtained. As expected, no suppression of glucagon occurred during the initial phase of the OGTT, whereas significantly (P < 0.05) lower plasma levels of glucagon during the first 30 min of the IIGI (day B) were observed. The glucagon response during the IIGI + GIP + GLP-1 + GLP-2 infusion (day F) equaled the inappropriate glucagon response to OGTT (P = not significant). The separate GIP infusion (day C) elicited significant hypersecretion of glucagon, whereas GLP-1 infusion (day D) resulted in enhancement of glucagon suppression during IIGI. IIGI + GLP-2 infusion (day E) resulted in a glucagon response in the midrange between the glucagon responses to OGTT and IIGI. Our results indicate that the intestinal hormones, GIP, GLP-1, and GLP-2, may play a role in the inappropriate glucagon response to orally ingested glucose in T2DM with, especially, GIP, acting to increase glucagon secretion.     

DPP-4 inhibition increases GIP and decreases GLP-1 incretin effects during intravenous glucose tolerance test in Wistar rats

GIP metabolite [GIP (3-42)] and GLP-1 metabolite [GLP-1 (9-36) amide] have been reported to differ with regard to biological actions. Systemic DPP-4 inhibition can therefore reveal different actions of GIP and GLP-1. In catheter wearing Wistar rats, insulinotropic effects of equipotent doses of GIP (2.0 nmol/kg) and GLP-1 (7-36) amide (4.0 nmol/kg) and vehicle were tested in the absence/presence of DPP-4 inhibition. Blood glucose and insulin were frequently sampled. DPP-4 inhibitor was given at -20 min, the incretin at -5 min and the intravenous glucose tolerance test (0.4 g glucose/kg) commenced at 0 min. G-AUC and I-AUC, insulinogenic index and glucose efflux, were calculated from glucose and insulin curves. Systemic DPP-4 inhibition potentiated the acute GIP incretin effects: I-AUC (115±34 vs. 153±39 ng·min/ml), increased the insulinogenic index (0.74±0.24 vs. 0.99±0.26 ng/mmol), and improved glucose efflux (19.8±3.1 vs. 20.5±5.0 min?1). The GLP-1 incretin effects were diminished: I-AUC (124±18 vs. 106±38 ng·min/ml), the insulinogenic index was decreased (0.70±0.18 vs. 0.50±0.19 ng/mmol), and glucose efflux declined (14.9±3.1 vs. 11.1±3.7 min?1). GLP-1 and GIP differ remarkably in their glucoregulatory actions in healthy rats when DPP-4 is inhibited. These previously unrecognized actions of DPP-4 inhibitors could have implications for future use in humans.     

Human duodenal enteroendocrine cells: source of both incretin peptides, GLP-1 and GIP

Among the products of enteroendocrine cells are the incretins glucagon-like peptide-1 (GLP-1, secreted by L cells) and glucose-dependent insulinotropic peptide (GIP, secreted by K cells). These are key modulators of insulin secretion, glucose homeostasis, and gastric emptying. Because of the rapid early rise of GLP-1 in plasma after oral glucose, we wished to definitively establish the absence or presence of L cells, as well as the relative distribution of the incretin cell types in human duodenum. We confirmed the presence of proglucagon and pro-GIP genes, their products, and glucosensory molecules by tissue immunohistochemistry and RT-PCR of laser-captured, single duodenal cells. We also assayed plasma glucose, incretin, and insulin levels in subjects with normal glucose tolerance and type 2 diabetes for 120 min after they ingested 75 g of glucose. Subjects with normal glucose tolerance (n=14) had as many L cells (15+/-1), expressed per 1,000 gut epithelial cells, as K cells (13+/-1), with some containing both hormones (L/K cells, 5+/-1). In type 2 diabetes, the number of L and L/K cells was increased (26+/-2; P<0.001 and 9+/-1; P < 0.001, respectively). Both L and K cells contained glucokinase and glucose transporter-1, -2, and -3. Newly diagnosed type 2 diabetic subjects had increased plasma GLP-1 levels between 20 and 80 min, concurrently with rising plasma insulin levels. Significant coexpression of the main incretin peptides occurs in human duodenum. L and K cells are present in equal numbers. New onset type 2 diabetes is associated with a shift to the L phenotype.     

Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are both incretin hormones regulating postprandial insulin secretion. Their relative importance in this respect under normal physiological conditions is unclear, however, and the aim of the present investigation was to evaluate this. Eight healthy male volunteers (mean age: 23 (range 20-25) years; mean body mass index: 22.2 (range 19.3-25.4) kg/m2) participated in studies involving stepwise glucose clamping at fasting plasma glucose levels and at 6 and 7 mmol/l. Physiological amounts of either GIP (1.5 pmol/kg/min), GLP-1(7-36)amide (0.33 pmol/kg/min) or saline were infused for three periods of 30 min at each glucose level, with 1 h "washout" between the infusions. On a separate day, a standard meal test (566 kcal) was performed. During the meal test, peak insulin concentrations were observed after 30 min and amounted to 223+/-27 pmol/l. Glucose+saline infusions induced only minor increases in insulin concentrations. GLP-1 and GIP infusions induced significant and similar increases at fasting glucose levels and at 6 mmol/l. At 7 mmol/l, further increases were seen, with GLP-1 effects exceeding those of GIP. Insulin concentrations at the end of the three infusion periods (60, 150 and 240 min) during the GIP clamp amounted to 53+/-5, 79+/-8 and 113+/-15 pmol/l, respectively. Corresponding results were 47+/-7, 95+/-10 and 171+/-21 pmol/l, respectively, during the GLP-1 clamp. C-peptide responses were similar. Total and intact incretin hormone concentrations during the clamp studies were higher compared to the meal test, but within physiological limits. Glucose infusion alone significantly inhibited glucagon secretion, which was further inhibited by GLP-1 but not by GIP infusion. We conclude that during normal physiological plasma glucose levels, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide contribute nearly equally to the incretin effect in humans, because their differences in concentration and potency outweigh each other.     

Metabolic abnormalities in first-degree relatives of type 2 diabetics

Diabetic relatives and obese subjects are at increased risk for development of diabetes mellitus, and therefore are classed as potential abnormality of glucose tolerance (POT-AGT). Disturbances of lipid and purine metabolisms have been reported in diabetic and obese non-diabetic subjects. In obese subjects above alterations are probably due to hyperinsulinemia. This study aimed at verifying whether similar metabolic abnormalities could be found in relatives of non-insulin dependent diabetic patients and whether they could be related to possible glucose intolerance. We have studied 10706 outpatients and 95 hospitalized subjects, aged between 20 and 50 years. We have selected 4 groups according to diabetic relationship and body mass index: A (normal weight subjects), B (obese subjects), C (normal weight NIDDM-relatives), D (overweight NIDDM-relatives). The NIDDM-relatives showed higher prevalence of hyperglycemia, as expected; furthermore the relatives with normal glucose tolerance had higher glucose area during OGTT. Serum levels of uric acid and insulin response to oral glucose were increased in all obese subjects, but abnormalities of lipid metabolism and fasting hyperinsulinemia were found only in obese NIDDM-relatives. These results suggest that family history of diabetes mellitus can be a risk for metabolic disturbance even in absence of glucose intolerance. Furthermore some metabolic disorders observed in obese subjects could be due to an associated and not sufficiently investigated family history of diabetes.     

Effect of glycine on insulin secretion and action in healthy first-degree relatives of type 2 diabetes mellitus patients.

The aim of this study was to identify the effect of glycine on insulin secretion and action in healthy first-degree relatives of Type 2 diabetes mellitus patients. A randomized, double-blind, placebo-controlled clinical trial was performed in 12 healthy, non-obese volunteers who were first-degree relatives of Type 2 diabetes mellitus patients. Six volunteers received a morning dose of glycine 5 g orally and the other six received placebo. At baseline without drugs and after pharmacological intervention, a metabolic profile and, to assess insulin secretion and action, a hyperglycemic-hyperinsulinemic clamp study were performed. There were no significant differences in baseline metabolic profile, insulin secretion or action between groups. Changes from baseline of early (p < 0.05), late (p < 0.05), and total insulin (p < 0.02) responses were higher in the glycine group than in controls. There were no significant differences in the changes from baseline of insulin action between groups. In conclusion, a morning dose of glycine 5 g orally increased early, late and total insulin responses without changes in insulin action in healthy first-degree relatives of Type 2 diabetes mellitus patients.     

Exogenously imposed postprandial-like rises in systemic glucose and GLP-1 do not produce an incretin effect, suggesting an indirect mechanism of GLP-1 action

The insulinotropic intestinal hormone GLP-1 is thought to exert one of its effects by direct action on the pancreatic beta-cell receptors. GLP-1 is rapidly degraded in plasma, such that only a small amount of the active form reaches the pancreas, making it questionable whether this amount is sufficient to produce a direct incretin effect. The aim of our study was to assess, in a dog model, the putative incretin action of GLP-1 acting directly on the beta-cell in the context of postprandial rises in GLP-1 and glucose. Conscious dogs were fed a high-fat, high-carbohydrate meal, and insulin response was measured. We also infused systemic glucose plus GLP-1, or glucose alone, to simulate the meal test values of these variables and measured insulin response. The results were as follows: during the meal, we measured a robust insulin response (52 +/- 9 to 136 +/- 14 pmol/l, P < 0.05 vs. basal) with increases in portal glucose and GLP-1 but only limited increases in systemic glucose (5.3 +/- 0.1 to 5.7 +/- 0.1 mmol/l, P = 0.1 vs. basal) and GLP-1 (6 +/- 0 to 9 +/- 1 pmol/l, P = 0.5 vs. basal). Exogenous infusion of systemic glucose and GLP-1 produced a moderate increase in insulin (43 +/- 5 to 84 +/- 15 pmol/l, 43% of the meal insulin). However, infusion of glucose alone, without GLP-1, produced a similar insulin response (37 +/- 6 to 82 +/- 14 pmol, 53% of the meal insulin, P = 0.7 vs. glucose and GLP-1 infusion). In conclusion, in dogs with postprandial rises in systemic glucose and GLP-1, the hormone might not have a direct insulinotropic effect and could regulate glycemia via indirect, portohepatic-initiated neural mechanisms.     

Adiponectin expression in adipose tissue is reduced in first-degree relatives of type 2 diabetic patients

Adiponectin is suggested to be an important mediator of insulin resistance. Therefore, we investigated the association between adiponectin and insulin sensitivity in 22 healthy first-degree relatives (FDR) to type 2 diabetic patients and 13 matched control subjects. Subcutaneous adipose tissue biopsies were taken before and after a hyperinsulinemic euglycemic clamp. FDR subjects were insulin resistant, as indicated by a reduced M value (4.44 vs. 6.09 mg x kg(-1) x min(-1), P < 0.05). Adiponectin mRNA expression was 45% lower in adipose tissue from FDR compared with controls (P < 0.01), whereas serum adiponectin was similar in the two groups (6.4 vs. 6.6 microg/ml, not significant). Insulin infusion reduced circulating levels of adiponectin moderately (11-13%) but significantly in both groups (P < 0.05). In the control group, adiponectin mRNA levels were negatively correlated with fasting insulin (P < 0.05) and positively correlated with insulin sensitivity (P < 0.05). In contrast, these associations were not found in the FDR group. In conclusion, FDR have reduced adiponectin mRNA in subcutaneous adipose tissue but normal levels of circulating adiponectin. Adiponectin mRNA levels are positively correlated with insulin sensitivity in control subjects but not in FDR. These findings indicate dysregulation of adiponectin gene expression in FDR.     

Moderate effect of duodenal-jejunal bypass surgery on glucose homeostasis in patients with type 2 diabetes

Gastric bypass surgery causes resolution of type 2 diabetes (T2DM), which has led to the hypothesis that upper gastrointestinal (UGI) tract diversion, itself, improves glycemic control. The purpose of this study was to determine whether UGI tract bypass without gastric exclusion has therapeutic effects in patients with T2DM. We performed a prospective trial to assess glucose and β-cell response to an oral glucose load before and at 6, 9, and 12 months after duodenal-jejunal bypass (DJB) surgery. Thirty-five overweight or obese adults (BMI: 27.0 ± 4.0 kg/m(2)) with T2DM and 35 sex-, age-, race-, and BMI-matched subjects with normal glucose tolerance (NGT) were studied. Subjects lost weight after surgery, which was greatest at 3 months (6.9 ± 4.9%) with subsequent regain to 4.2 ± 5.3% weight loss at 12 months after surgery. Glycated hemoglobin (HbA(1c)) decreased from 9.3 ± 1.6% before to 7.7 ± 2.0% at 12 months after surgery (P < 0.001), in conjunction with a 20% decrease in the use of diabetes medications (P < 0.05); 7 (20%) subjects achieved remission of diabetes (no medications and HbA(1c) <6.5%). The area under the curve after glucose ingestion was ~20% lower for glucose but doubled for insulin and C-peptide at 12 months, compared with pre-surgery values (all P < 0.01). However, the β-cell response was still 70% lower than subjects with NGT (P < 0.001). DJB surgery improves glycemic control and increases, but does not normalize the β-cell response to glucose ingestion. These findings suggest that altering the intestinal site of delivery of ingested nutrients has moderate therapeutic effects by improving β-cell function and glycemic control.     

Similar incretin secretion in obese and non-obese Japanese subjects with type 2 diabetes

Incretin secretion and effect on insulin secretion are not fully understood in patients with type 2 diabetes. We investigated incretin and insulin secretion after meal intake in obese and non-obese Japanese patients with type 2 diabetes compared to non-diabetic subjects. Nine patients with type 2 diabetes and 5 non-diabetic subjects were recruited for this study. Five diabetic patients were obese (BMI ? 25) and 4 patients were non-obese (BMI < 25). In response to a mixed meal test, the levels of immunoreactive insulin during 15-90 min and C-peptide during 0-180 min in non-obese patients were significantly lower than those in obese patients. Total GLP-1 and active GIP levels showed no significant difference between obese and non-obese patients throughout the meal tolerance test. In addition, there were no significant differences between diabetic patients and non-diabetic subjects. In conclusion, incretin secretion does not differ between Japanese obese and non-obese patients with type 2 diabetes and non-diabetic subjects.     

Suppression of glucose production by GLP-1 independent of islet hormones: a novel extrapancreatic effect

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that stimulates insulin secretion and decreases glucagon release. It has been hypothesized that GLP-1 also reduces glycemia independent of its effect on islet hormones. Based on preliminary evidence that GLP-1 has independent actions on endogenous glucose production, we undertook a series of experiments that were optimized to address this question. The effect of GLP-1 on glucose appearance (Ra) and glucose disposal (Rd) was measured in eight men during a pancreatic clamp that was performed by infusing octreotide to suppress secretion of islet hormones, while insulin and glucagon were infused at rates adjusted to maintain blood glucose near fasting levels. After stabilization of plasma glucose and equilibration of [3H]glucose tracer, GLP-1 was given intravenously for 60 min. Concentrations of insulin, C-peptide, and glucagon were similar before and during the GLP-1 infusion (115 +/- 14 vs. 113 +/- 11 pM; 0.153 +/- 0.029 vs. 0.156 +/- 0.026 nM; and 64.7 +/- 11.5 vs. 65.8 +/- 13.8 ng/l, respectively). With the initiation of GLP-1, plasma glucose decreased in all eight subjects from steady-state levels of 4.8 +/- 0.2 to a nadir of 4.1 +/- 0.2 mM. This decrease in plasma glucose was accounted for by a significant 17% decrease in Ra, from 22.6 +/- 2.8 to 19.1 +/- 2.8 micromol. kg-1. min-1 (P < 0.04), with no significant change in Rd. These findings indicate that, under fasting conditions, GLP-1 decreases endogenous glucose production independent of its actions on islet hormone secretion.     

Urinary excretion of glucagon-like peptide 1 (GLP-1) 7-36 amide in human type 2 (non-insulin-dependent) diabetes mellitus.

The urinary excretion of insulinotropic glucagon-like peptide 1 (GLP-1) was investigated as an indicator of renal tubular integrity in 10 healthy subjects and in 3 groups of type 2 diabetic patients with different degrees of urinary albumin excretion rate. No significant difference emerged between the groups with respect to age of the patients, known duration of diabetes, metabolic control, BMI, or residual beta-cell pancreatic function. Endogenous creatinine clearance was significantly reduced under conditions of overt diabetic nephropathy, compared with normo and microalbuminuric patients (p < 0.01). Urinary excretion of GLP-1 was significantly higher in normoalbuminuric patients compared to controls (490.4 +/- 211.5 vs. 275.5 +/- 132.1 pg/min; p < 0.05), with further increase under incipient diabetic nephropathy conditions (648.6 +/- 305 pg/min; p < 0.01). No significant difference resulted, in contrast, between macroproteinuric patients and non-diabetic subjects. Taking all patients examined into account, a significant positive relationship emerged between urinary GLP-1 and creatinine clearance (p = 0.004). In conclusion, an early tubular impairment in type 2 diabetes would occur before the onset of glomerular permeability alterations. The tubular dysfunction seems to evolve with the development of persistent microalbuminuria. Finally, the advanced tubular involvement, in terms of urinary GLP1 excretion, under overt diabetic nephropathy conditions would be masked by severe concomitant glomerular damage with the coexistence of both alterations resulting in a peptide excretion similar to control subjects.     

The reduction in hepatic insulin clearance after oral glucose is not mediated by gastric inhibitory polypeptide (GIP)

Since the C-peptide/insulin ratio is reduced after oral glucose ingestion, the incretin hormone gastric inhibitory polypeptide (GIP) has been assumed to decrease hepatic insulin extraction. It was the aim of the present study to evaluate the effects of GIP on insulin extraction. Seventy-eight healthy subjects (27 male, 51 female, 43+/-11 years) were subjected to (a). an oral glucose tolerance test and (b). an intravenous injection of 20 pmol GIP/kg body weight, with capillary and venous blood samples collected over 30 min for insulin, C-peptide and GIP (specific immunoassays). Following GIP administration, plasma concentrations of total and intact GIP reached to peak levels of 80+/-7 and 54+/-5 pmol/l, respectively (p<0.0001). The rise in insulin after oral glucose and after intravenous GIP administration significantly exceeded the rise in C-peptide (p<0.0001). Estimating insulin extraction from the total integrated insulin and C-peptide concentrations (AUCs), only the oral glucose load (p<0.0001), but not the intravenous GIP administration (p=0.18) significantly reduced insulin clearance. Therefore, insulin clearance is reduced after an oral glucose load. This effect does not appear to be mediated by GIP.     

Incretin hormones in the treatment of type 2 diabetes. Part I: influence of insulinotropic gut-derived hormones (incretins) on glucose metabolism

Insulinotropic gut-derived hormones (incretins) play a significant role in the regulation of glucose homeostasis in healthy subjects and are responsible for 50-70% of insulin response to a meal. The main mediators of the incretin effect are glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1). However, in patients with type 2 diabetes the effect of incretins action is to a large extent impaired, which seems to explain disturbed secretional activity of beta cells in pancreatic islets. Detailed analysis of incretin defect proved that GIP secretion remains within physiological limits, whereas GLP-1 secretion is significantly decreased. Nevertheless, GLP-1 insulinotropic effect is preserved and GIP effect is significantly impaired. In consequence, substitutional GLP-1 administration aiming at the reduction of its deficiency, seems to be logical therapeutic management, because despite a physiologically retained quantity response from GIP, resistance to this peptide is frequently found. Therefore, particularly promising are the results of clinical studies with the use of GLP-1 analogues , GLP-1 receptors activation, as well as the inhibitors of dipeptidyl peptidase-IV (DPP IV), the enzyme responsible for incretin proteolysis, which restores the proper function of the intestinal-pancreatic axis in subjects with type 2 diabetes and creates new possibilities of a glycaemia reducing therapy and improvement in quality of life in this group of patients.     

Chemokine (C-C motif) ligand 2 (CCL2) in sera of patients with type 1 diabetes and diabetic complications

Background

Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), has been implicated in the pathogenesis of many diseases characterized by monocytic infiltration. However, limited data have been reported on MCP-1 in type 1 diabetes (T1D) and the findings are inconclusive and inconsistent.

Methods

In this study, MCP-1 was measured in the sera from 2,472 T1D patients and 2,654 healthy controls using a Luminex assay. The rs1024611 SNP in the promoter region of MCP-1 was genotyped for a subset of subjects (1764 T1D patients and 1323 controls) using the TaqMan-assay.

Results

Subject age, sex or genotypes of MCP-1 rs1024611SNP did not have a major impact on serum MCP-1 levels in either healthy controls or patients. While hemoglobin A1c levels did not have a major influence on serum MCP-1 levels, the mean serum MCP-1 levels are significantly higher in patients with multiple complications (mean = 242 ng/ml) compared to patients without any complications (mean = 201 ng/ml) (p = 3.5×10⁻⁶). Furthermore, mean serum MCP-1 is higher in controls (mean = 261 ng/ml) than T1D patients (mean = 208 ng/ml) (p<10⁻²³). More importantly, the frequency of subjects with extremely high levels (>99^th percentile of patients or 955 ng/ml) of serum MCP-1 is significantly lower in the T1D group compared to the control group (odds ratio = 0.11, p<10⁻³³).

Conclusion

MCP-1 may have a dual role in T1D and its complications. While very high levels of serum MCP-1 may be protective against the development of T1D, complications are associated with higher serum MCP-1 levels within the T1D group.