A Human Monoclonal Antibody That Improves Glycemic Control
A Human Monoclonal Antibody That Improves Glycemic Control
Allosteric antibodies to the INSR were identified by screening phage display libraries for human scFv fragments that bound the insulin-INSR complex. The highest affinity scFv fragment was then reformatted into a fully human IgG2 monoclonal antibody, XMetA. By flow cytometry, we determined that XMetA, but not an isotype control monoclonal antibody, specifically bound to CHO cells expressing the human INSR (CHO-hINSR) with an half-maximal effective concentration (EC50) of 0.10 nmol/L (Fig. 1A). With the use of KinExA methodology to determine the binding affinity of XMetA to the hINSR, XMetA bound to the hINSR with a KD of 0.040 nmol/L (data not shown). Neither XMetA nor the control antibody altered the affinity of insulin binding to the hINSR in the absence of antibodies (KD of 0.17 nmol/L) (Fig. 1B). XMetA also had no effect on the binding of labeled insulin to the INSR on these same cells by flow cytometry (data not shown). These data demonstrate, therefore, that XMetA binds to hINSR with high affinity, and this binding is to an allosteric site, which is distinct from the orthosteric insulin binding site.
(Enlarge Image)
Figure 1.
XMetA binds to the INSR at an allosteric site. A: CHO-hINSR cells were incubated with increasing concentrations of either XMetA (▪) or isotype control antibody (○) and antibody binding measured by flow cytometry (n = 4). B: Increasing concentrations of CHO-hINSR cells were incubated with 80 pmol/L insulin and either 70 nmol/L XMetA (▪) or isotype control antibody (○). Insulin binding to the INSR was determined by KinExA (n = 3). mAb, monoclonal antibody.
The ability of XMetA to activate the hINSR was evaluated in CHO-hINSR cells. We first studied hINSR autophosphorylation. Insulin activated this function with an EC50 of 0.18 nmol/L (Fig. 2A). XMetA activated INSR autophosphorylation with an EC50 of 1.3 nmol/L and maximal activation of ~20% that of insulin, indicating that XMetA is a partial agonist of the hINSR. An isotype control antibody was without effect. When XMetA was used at a maximally stimulating concentration, insulin was still able to fully activate the hINSR with similar sensitivity (Fig. 2B). This observation strongly supports the notion that XMetA binds to the hINSR at an allosteric site and does not interfere with insulin binding its site. Similarly, XMetA, but not the control antibody, stimulated the phosphorylation of Akt, a major intracellular mediator of INSR-dependent glucoregulatory signaling, with an EC50 of 1.1 nmol/L (Fig. 2C). Maximal activation was ~40% that of insulin, further demonstrating that XMetA is a partial agonist of the INSR. As with INSR autophosphorylation, when XMetA was used at a maximally stimulating concentration, insulin was still able to fully phosphorylate Akt with similar sensitivity (Fig. 2D). In contrast with insulin, XMetA did not stimulate the phosphorylation of Erk, which mediates INSR-dependent mitogenic properties (Fig. 2E), nor did it affect the capacity of insulin to phosphorylate Erk (data not shown).
(Enlarge Image)
Figure 2.
XMetA is a partial agonist of the INSR that selectively activates the PI3K/Akt pathway. A: CHO-hINSR cells were incubated with increasing concentrations of either XMetA (▪), isotype control antibody (○), or insulin (▴), and INSR autophosphorylation was measured by ELISA (n = 3). B: CHO-hINSR cells were incubated with either 33 nmol/L XMetA (▪) or isotype control antibody (○) with increasing concentrations of insulin. INSR autophosphorylation was then measured (n = 3). C: CHO-hINSR cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and Akt phosphorylation was measured by ELISA (n = 3). D: CHO-hINSR cells were incubated with either 33 nmol/L XMetA (▪) or isotype control antibody (○) with increasing concentrations of insulin. Akt phosphorylation was then measured (n = 3). E: CHO-hINSR cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and extracellular signal–related kinase (Erk)1/2 phosphorylation was measured by ELISA (n = 3). mAb, monoclonal antibody.
The IGF-IR has structural and functional similarity to INSR. We therefore investigated whether XMetA would activate the IGF-IR. For this purpose, we used CHO cells that expressed the human IGF-IR (CHO-hIGF-IR). Under conditions in which XMetA maximally activated INSR autophosphorylation in CHO-hINSR cells (Fig. 2A), XMetA neither directly activated autophosphorylation of the IGF1-R nor influenced the ability of the ligand, IGF-I, to activate this function (Fig. 3).
(Enlarge Image)
Figure 3.
XMetA does not activate the IGF-IR. CHO-hIGF-IR cells were incubated with either 33 nmol/L XMetA or isotype control antibody in the presence or absence of 100 nmol/L IGF-I. IGF-IR autophosphorylation was measured by ELISA (n = 3). p, phosphorylation.
A major metabolic function of insulin is to enhance glucose transport. Accordingly, the uptake of 2-deoxy-D-glucose was analyzed in 3T3-HIR cells, a cell line that is known to be responsive to insulin. Insulin stimulated 2-deoxy-D-glucose uptake with an EC50 of 0.15 nmol/L. XMetA stimulated this function in a manner similar to that of insulin, whereas the control antibody was without effect (Fig. 4A). These data suggest that the lesser effects of XMetA on receptor phosphorylation may not fully indicate the effect of XMetA on insulin action.
(Enlarge Image)
Figure 4.
XMetA promotes glucose uptake, but not cell growth. A: 3T3 cells expressing hINSR were incubated with increasing concentrations of either XMetA (▪), isotype control antibody (○), or insulin (▴), and 2-deoxy-D-glucose uptake was measured (n = 5). B: MCF-7 cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and cell proliferation was determined by CellTiter Glo assay (n = 6). C: MCF-7 cells were incubated with either 33 nmol/L XMetA (▪) or isotype control antibody (○) with increasing concentrations of insulin. Cell proliferation was then measured (n = 3). mAb, monoclonal antibody.
In addition to its metabolic effects, insulin, via its own receptor, stimulates cell growth, in particular the growth of cancer cells. MCF-7 human breast cancer cells, which express the hINSR, have been extensively used to study this effect of insulin. In these cells, insulin stimulated growth with an EC50 of 1.9 nmol/L (Fig. 4B). In contrast with insulin, neither XMetA nor the control antibody stimulated the growth of MCF-7 cells. Moreover XMetA did not potentiate the effect of insulin on cell proliferation (Fig. 4C). Similar results were obtained using methods that evaluate DNA content as a surrogate for proliferation (data not shown).
XMetA was next evaluated for its ability to bind to and activate the mINSR. For these studies we used CHO cells that expressed the mINSR (CHO-mINSR). By flow cytometry, we determined that XMetA, but not the isotype control antibody, specifically bound to CHO-mINSR cells with an EC50 of 0.085 nmol/L (data not shown), a value similar to that of its binding to CHO-hINSR cells. In CHO-mINSR cells, insulin stimulated the phosphorylation of Akt with an EC50 of 1.7 nmol/L. XMetA, but not the control antibody, stimulated the phosphorylation of Akt with a maximal effect of ~40% that of insulin, with an EC50 of 1.4 nmol/L (Fig. 5A). These data indicated, therefore, that diabetic mice could be used to study the effects of XMetA on metabolic regulation.
(Enlarge Image)
Figure 5.
XMetA improves hyperglycemia and other metabolic markers of disease in diabetic mice. A: CHO-mINSR cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and Akt phosphorylation was measured by ELISA (n = 3). B: Fasting blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). C: After 3 weeks of treatment, fasting blood glucose was measured in control mice treated with 10 mg/kg isotype control antibody (white bar), diabetic mice treated with 10 mg/kg isotype control antibody (gray bar), and diabetic mice treated with the indicated doses of XMetA (black bars). D: Nonfasted blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). After 6 weeks of treatment, blood hemoglobin A1c (E) and nonfasted plasma β-hydroxybutyrate (F) were measured in control mice treated with 10 mg/kg isotype control antibody (white bar) and diabetic mice treated with either 10 mg/kg isotype control antibody (gray bar) or XMetA (black bar). Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control; n = 8 mice/group. mAb, monoclonal antibody.
To evaluate the in vivo activity of XMetA, we used an animal model of insulinopenic, insulin-resistant diabetes, the multi-low dose STZ, high-fat diet (MLDS/HFD) mouse. Ten days after the last dose of STZ, blood glucose levels after a 14-h fast were elevated to ~200 mg/dL, and these levels increased over the course of the 6-week study (Fig. 5B). In contrast, nondiabetic control animals maintained fasting glucose levels of ~100 mg/dL. XMetA was administered by intraperitoneal injection twice weekly to the diabetic mice. Seven days after treatment, fasting blood glucose levels in the XMetA-treated diabetic mice were near normal and remained near normal for up to 28 days, but were slightly elevated at 42 days. At this time, anti-human IgG antibodies were detected in the treated mice (data not shown). These antibodies likely enhanced XMetA clearance, causing this elevation in blood glucose. An effect of XMetA was detected at doses as low as 0.1 mg/kg (Fig. 5C). XMetA maximally improved fasting blood glucose levels at a dose of 1.0 mg/kg. At this and higher doses of XMetA, there was no evidence of hypoglycemia.
Glucose levels in diabetic mice allowed free access to food and water were also measured. Nondiabetic control animals maintained glucose levels in the range of 150 mg/dL (Fig. 5D). Diabetic animals had glucose values in the range of 600 mg/dL. Treatment with XMetA lowered nonfasted glucose values in diabetic mice, but in contrast with the results observed in fasted diabetic mice, XMetA did not normalize nonfasted glucose values.
Various parameters in the mice were studied at the end the 6 weeks of treatment. In addition to improving fasting blood glucose to near normal levels and reducing nonfasted glucose levels, treatment with XMetA improved other metabolic indices in the diabetic animals. Consistent with the decrease in glucose levels, XMetA had a major effect on hemoglobin A1c (Fig. 5E). The diabetic animals were markedly ketotic as measured by β-hydroxybutyrate; XMetA normalized this value (Fig. 5F). XMetA treatment increased insulin levels (295 ± 37 vs. 158 ± 38 pg/mL, P < 0.05) without increasing the level of C-peptide (334 ± 40 vs. 353 ± 29 pmol/L) ( Table 1 ). Diabetes reduced weight gain in diabetic animals, and XMetA treatment of diabetic animals did not change this parameter. However, other manifestations of diabetes were improved by XMetA. Food intake was decreased (4.8 ± 0.6 vs. 7.7 ± 0.3 g/day, P < 0.05), and water intake was decreased (11.1 ± 2.2 vs. 21.3 ± 0.9 g/day, P < 0.05). In addition, XMetA also improved non-HDL cholesterol (78 ± 4 vs. 107 ± 6 mg/dL, P < 0.05) and free fatty acids (21.2 ± 2.4 vs. 32.9 ± 1.3 mg/dL, P < 0.05).
After 3 weeks of XMetA treatment, fasted diabetic animals underwent glucose tolerance tests, with either intraperitoneal (Fig. 6A) or oral (Fig. 6B) glucose. Diabetic animals developed markedly elevated glucose levels, reaching 600 mg/dL or greater. In contrast, during both types of glucose tolerance tests, animals treated with XMetA maintained near normal blood glucose concentrations.
(Enlarge Image)
Figure 6.
XMetA improves glucose tolerance in diabetic mice. A: After 3 weeks of treatment, glucose was administered intraperitoneally (IP) at 1 g/kg to fasted mice. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). B: After 3 weeks of treatment, glucose was administered orally at 1 g/kg to fasted mice. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control antibody; n = 8 mice/group.
We next studied the effect of XMetA administration on the glucose response to exogenous insulin administration. After 5 weeks of treatment, animals were given intraperitoneal exogenous insulin, and the fall in blood glucose was measured for up to 120 min (Fig. 7A). Control, diabetic, and diabetic XMetA-treated animals all responded to insulin with a fall in blood glucose. When expressed as the percent change from initial glucose levels, the response to insulin in the diabetic animals was blunted compared with normal mice. This blunted response was corrected by XMetA treatment, suggesting that XMetA effects in this context are additive (Fig. 7B).
(Enlarge Image)
Figure 7.
XMetA improves insulin tolerance in diabetic mice. A: After 5 weeks of treatment, insulin was administered intraperitoneally at 0.75 units/kg. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). B: Data normalized to preinsulin glucose values. Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control antibody; n = 8 mice/group.
Results
XMetA Binds to INSR at an Allosteric Site
Allosteric antibodies to the INSR were identified by screening phage display libraries for human scFv fragments that bound the insulin-INSR complex. The highest affinity scFv fragment was then reformatted into a fully human IgG2 monoclonal antibody, XMetA. By flow cytometry, we determined that XMetA, but not an isotype control monoclonal antibody, specifically bound to CHO cells expressing the human INSR (CHO-hINSR) with an half-maximal effective concentration (EC50) of 0.10 nmol/L (Fig. 1A). With the use of KinExA methodology to determine the binding affinity of XMetA to the hINSR, XMetA bound to the hINSR with a KD of 0.040 nmol/L (data not shown). Neither XMetA nor the control antibody altered the affinity of insulin binding to the hINSR in the absence of antibodies (KD of 0.17 nmol/L) (Fig. 1B). XMetA also had no effect on the binding of labeled insulin to the INSR on these same cells by flow cytometry (data not shown). These data demonstrate, therefore, that XMetA binds to hINSR with high affinity, and this binding is to an allosteric site, which is distinct from the orthosteric insulin binding site.
(Enlarge Image)
Figure 1.
XMetA binds to the INSR at an allosteric site. A: CHO-hINSR cells were incubated with increasing concentrations of either XMetA (▪) or isotype control antibody (○) and antibody binding measured by flow cytometry (n = 4). B: Increasing concentrations of CHO-hINSR cells were incubated with 80 pmol/L insulin and either 70 nmol/L XMetA (▪) or isotype control antibody (○). Insulin binding to the INSR was determined by KinExA (n = 3). mAb, monoclonal antibody.
XMetA is a Partial Agonist of INSR Signaling That Selectively Activates the PI3K/Akt Pathway
The ability of XMetA to activate the hINSR was evaluated in CHO-hINSR cells. We first studied hINSR autophosphorylation. Insulin activated this function with an EC50 of 0.18 nmol/L (Fig. 2A). XMetA activated INSR autophosphorylation with an EC50 of 1.3 nmol/L and maximal activation of ~20% that of insulin, indicating that XMetA is a partial agonist of the hINSR. An isotype control antibody was without effect. When XMetA was used at a maximally stimulating concentration, insulin was still able to fully activate the hINSR with similar sensitivity (Fig. 2B). This observation strongly supports the notion that XMetA binds to the hINSR at an allosteric site and does not interfere with insulin binding its site. Similarly, XMetA, but not the control antibody, stimulated the phosphorylation of Akt, a major intracellular mediator of INSR-dependent glucoregulatory signaling, with an EC50 of 1.1 nmol/L (Fig. 2C). Maximal activation was ~40% that of insulin, further demonstrating that XMetA is a partial agonist of the INSR. As with INSR autophosphorylation, when XMetA was used at a maximally stimulating concentration, insulin was still able to fully phosphorylate Akt with similar sensitivity (Fig. 2D). In contrast with insulin, XMetA did not stimulate the phosphorylation of Erk, which mediates INSR-dependent mitogenic properties (Fig. 2E), nor did it affect the capacity of insulin to phosphorylate Erk (data not shown).
(Enlarge Image)
Figure 2.
XMetA is a partial agonist of the INSR that selectively activates the PI3K/Akt pathway. A: CHO-hINSR cells were incubated with increasing concentrations of either XMetA (▪), isotype control antibody (○), or insulin (▴), and INSR autophosphorylation was measured by ELISA (n = 3). B: CHO-hINSR cells were incubated with either 33 nmol/L XMetA (▪) or isotype control antibody (○) with increasing concentrations of insulin. INSR autophosphorylation was then measured (n = 3). C: CHO-hINSR cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and Akt phosphorylation was measured by ELISA (n = 3). D: CHO-hINSR cells were incubated with either 33 nmol/L XMetA (▪) or isotype control antibody (○) with increasing concentrations of insulin. Akt phosphorylation was then measured (n = 3). E: CHO-hINSR cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and extracellular signal–related kinase (Erk)1/2 phosphorylation was measured by ELISA (n = 3). mAb, monoclonal antibody.
XMetA Does not Activate the IGF-IR
The IGF-IR has structural and functional similarity to INSR. We therefore investigated whether XMetA would activate the IGF-IR. For this purpose, we used CHO cells that expressed the human IGF-IR (CHO-hIGF-IR). Under conditions in which XMetA maximally activated INSR autophosphorylation in CHO-hINSR cells (Fig. 2A), XMetA neither directly activated autophosphorylation of the IGF1-R nor influenced the ability of the ligand, IGF-I, to activate this function (Fig. 3).
(Enlarge Image)
Figure 3.
XMetA does not activate the IGF-IR. CHO-hIGF-IR cells were incubated with either 33 nmol/L XMetA or isotype control antibody in the presence or absence of 100 nmol/L IGF-I. IGF-IR autophosphorylation was measured by ELISA (n = 3). p, phosphorylation.
XMetA Promotes Glucose Uptake but not Cell Growth
A major metabolic function of insulin is to enhance glucose transport. Accordingly, the uptake of 2-deoxy-D-glucose was analyzed in 3T3-HIR cells, a cell line that is known to be responsive to insulin. Insulin stimulated 2-deoxy-D-glucose uptake with an EC50 of 0.15 nmol/L. XMetA stimulated this function in a manner similar to that of insulin, whereas the control antibody was without effect (Fig. 4A). These data suggest that the lesser effects of XMetA on receptor phosphorylation may not fully indicate the effect of XMetA on insulin action.
(Enlarge Image)
Figure 4.
XMetA promotes glucose uptake, but not cell growth. A: 3T3 cells expressing hINSR were incubated with increasing concentrations of either XMetA (▪), isotype control antibody (○), or insulin (▴), and 2-deoxy-D-glucose uptake was measured (n = 5). B: MCF-7 cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and cell proliferation was determined by CellTiter Glo assay (n = 6). C: MCF-7 cells were incubated with either 33 nmol/L XMetA (▪) or isotype control antibody (○) with increasing concentrations of insulin. Cell proliferation was then measured (n = 3). mAb, monoclonal antibody.
In addition to its metabolic effects, insulin, via its own receptor, stimulates cell growth, in particular the growth of cancer cells. MCF-7 human breast cancer cells, which express the hINSR, have been extensively used to study this effect of insulin. In these cells, insulin stimulated growth with an EC50 of 1.9 nmol/L (Fig. 4B). In contrast with insulin, neither XMetA nor the control antibody stimulated the growth of MCF-7 cells. Moreover XMetA did not potentiate the effect of insulin on cell proliferation (Fig. 4C). Similar results were obtained using methods that evaluate DNA content as a surrogate for proliferation (data not shown).
XMetA Binds to and Activates the Mouse INSR
XMetA was next evaluated for its ability to bind to and activate the mINSR. For these studies we used CHO cells that expressed the mINSR (CHO-mINSR). By flow cytometry, we determined that XMetA, but not the isotype control antibody, specifically bound to CHO-mINSR cells with an EC50 of 0.085 nmol/L (data not shown), a value similar to that of its binding to CHO-hINSR cells. In CHO-mINSR cells, insulin stimulated the phosphorylation of Akt with an EC50 of 1.7 nmol/L. XMetA, but not the control antibody, stimulated the phosphorylation of Akt with a maximal effect of ~40% that of insulin, with an EC50 of 1.4 nmol/L (Fig. 5A). These data indicated, therefore, that diabetic mice could be used to study the effects of XMetA on metabolic regulation.
(Enlarge Image)
Figure 5.
XMetA improves hyperglycemia and other metabolic markers of disease in diabetic mice. A: CHO-mINSR cells were incubated with increasing concentrations of XMetA (▪), isotype control antibody (○), or insulin (▴), and Akt phosphorylation was measured by ELISA (n = 3). B: Fasting blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). C: After 3 weeks of treatment, fasting blood glucose was measured in control mice treated with 10 mg/kg isotype control antibody (white bar), diabetic mice treated with 10 mg/kg isotype control antibody (gray bar), and diabetic mice treated with the indicated doses of XMetA (black bars). D: Nonfasted blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). After 6 weeks of treatment, blood hemoglobin A1c (E) and nonfasted plasma β-hydroxybutyrate (F) were measured in control mice treated with 10 mg/kg isotype control antibody (white bar) and diabetic mice treated with either 10 mg/kg isotype control antibody (gray bar) or XMetA (black bar). Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control; n = 8 mice/group. mAb, monoclonal antibody.
XMetA Improves Fasting Blood Glucose Levels in Diabetic Mice
To evaluate the in vivo activity of XMetA, we used an animal model of insulinopenic, insulin-resistant diabetes, the multi-low dose STZ, high-fat diet (MLDS/HFD) mouse. Ten days after the last dose of STZ, blood glucose levels after a 14-h fast were elevated to ~200 mg/dL, and these levels increased over the course of the 6-week study (Fig. 5B). In contrast, nondiabetic control animals maintained fasting glucose levels of ~100 mg/dL. XMetA was administered by intraperitoneal injection twice weekly to the diabetic mice. Seven days after treatment, fasting blood glucose levels in the XMetA-treated diabetic mice were near normal and remained near normal for up to 28 days, but were slightly elevated at 42 days. At this time, anti-human IgG antibodies were detected in the treated mice (data not shown). These antibodies likely enhanced XMetA clearance, causing this elevation in blood glucose. An effect of XMetA was detected at doses as low as 0.1 mg/kg (Fig. 5C). XMetA maximally improved fasting blood glucose levels at a dose of 1.0 mg/kg. At this and higher doses of XMetA, there was no evidence of hypoglycemia.
XMetA Improves Nonfasting Blood Glucose Levels in Diabetic Mice
Glucose levels in diabetic mice allowed free access to food and water were also measured. Nondiabetic control animals maintained glucose levels in the range of 150 mg/dL (Fig. 5D). Diabetic animals had glucose values in the range of 600 mg/dL. Treatment with XMetA lowered nonfasted glucose values in diabetic mice, but in contrast with the results observed in fasted diabetic mice, XMetA did not normalize nonfasted glucose values.
XMetA Improves Metabolic Markers of Diabetes
Various parameters in the mice were studied at the end the 6 weeks of treatment. In addition to improving fasting blood glucose to near normal levels and reducing nonfasted glucose levels, treatment with XMetA improved other metabolic indices in the diabetic animals. Consistent with the decrease in glucose levels, XMetA had a major effect on hemoglobin A1c (Fig. 5E). The diabetic animals were markedly ketotic as measured by β-hydroxybutyrate; XMetA normalized this value (Fig. 5F). XMetA treatment increased insulin levels (295 ± 37 vs. 158 ± 38 pg/mL, P < 0.05) without increasing the level of C-peptide (334 ± 40 vs. 353 ± 29 pmol/L) ( Table 1 ). Diabetes reduced weight gain in diabetic animals, and XMetA treatment of diabetic animals did not change this parameter. However, other manifestations of diabetes were improved by XMetA. Food intake was decreased (4.8 ± 0.6 vs. 7.7 ± 0.3 g/day, P < 0.05), and water intake was decreased (11.1 ± 2.2 vs. 21.3 ± 0.9 g/day, P < 0.05). In addition, XMetA also improved non-HDL cholesterol (78 ± 4 vs. 107 ± 6 mg/dL, P < 0.05) and free fatty acids (21.2 ± 2.4 vs. 32.9 ± 1.3 mg/dL, P < 0.05).
XMetA Improves Glucose Tolerance in Diabetic Mice
After 3 weeks of XMetA treatment, fasted diabetic animals underwent glucose tolerance tests, with either intraperitoneal (Fig. 6A) or oral (Fig. 6B) glucose. Diabetic animals developed markedly elevated glucose levels, reaching 600 mg/dL or greater. In contrast, during both types of glucose tolerance tests, animals treated with XMetA maintained near normal blood glucose concentrations.
(Enlarge Image)
Figure 6.
XMetA improves glucose tolerance in diabetic mice. A: After 3 weeks of treatment, glucose was administered intraperitoneally (IP) at 1 g/kg to fasted mice. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). B: After 3 weeks of treatment, glucose was administered orally at 1 g/kg to fasted mice. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control antibody; n = 8 mice/group.
XMetA Improves Insulin Tolerance in Diabetic Mice
We next studied the effect of XMetA administration on the glucose response to exogenous insulin administration. After 5 weeks of treatment, animals were given intraperitoneal exogenous insulin, and the fall in blood glucose was measured for up to 120 min (Fig. 7A). Control, diabetic, and diabetic XMetA-treated animals all responded to insulin with a fall in blood glucose. When expressed as the percent change from initial glucose levels, the response to insulin in the diabetic animals was blunted compared with normal mice. This blunted response was corrected by XMetA treatment, suggesting that XMetA effects in this context are additive (Fig. 7B).
(Enlarge Image)
Figure 7.
XMetA improves insulin tolerance in diabetic mice. A: After 5 weeks of treatment, insulin was administered intraperitoneally at 0.75 units/kg. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (▪) or isotype control antibody (•). B: Data normalized to preinsulin glucose values. Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control antibody; n = 8 mice/group.
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