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Endocrinology 147(6):2619 –2630 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2005-1556

Recombinant Human Insulin-Like Growth Factor-I Treatment Inhibits Gluconeogenesis in a Transgenic Mouse Model of Type 2 Diabetes Mellitus Patricia Pennisi, Oksana Gavrilova, Jennifer Setser-Portas, William Jou, Stefania Santopietro, David Clemmons, Shoshana Yakar, and Derek LeRoith Diabetes Branch (P.P., J.S.-P., S.S., S.Y., D.L.) and Mouse Metabolism Core Laboratory (O.G., W.J.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892; and Division of Endocrinology (D.C.), University of North Carolina, Chapel Hill, North Carolina 27599 IGF-I and insulin are structurally related polypeptides that mediate a similar pattern of biological effects via receptors that display considerably homology. Administration of recombinant human IGF-I (rhIGF-I) has been proven to improve glucose control and liver and muscle insulin sensitivity in patients with type 2 diabetes mellitus (DM). The effect of rhIGF-I treatment was evaluated in a mouse model of type 2 DM (MKR mouse), which expresses a dominant-negative form of the human IGF-I receptor under the control of the muscle creatine kinase promoter specifically in skeletal muscle. MKR mice have impaired IGF-I and insulin signaling in skeletal muscle, leading to severe insulin resistance in muscle, liver, and fat, developing type 2 DM at 5 wk of age. Six-week-old MKR mice were treated with either saline or rhIGF-I for 3 wk. Blood glucose levels were decreased in response to rhIGF-I treatment in MKR mice. rhIGF-I treatment also increased body weight in MKR with concomitant changes in body com-

I

GF-I IS A POLYPEPTIDE structurally related to insulin that plays an important role in the regulation of both growth and metabolism (1). Its biological actions are modulated by a family of binding proteins (2). The main regulators of liver IGF-I expression and secretion are GH, insulin, and nutritional status (1). GH regulates the expression and secretion of IGF-I in many tissues, including adipose tissue, skeletal muscle, and the liver. However, direct effects of IGF-I in the liver and adipose tissue are unlikely because these tissues lack or have only few functional IGF-I receptors (IGFIRs) (3, 4) . Based on its growth-promoting and anabolic effects, recombinant human IGF-I (rhIGF-I) has been proposed as a therapeutic agent for the treatment of several disorders such as Laron syndrome (5), catabolic states like trauma, postsur-

First Published Online March 2, 2006 Abbreviations: BW, Body weight; DM, diabetes mellitus; EGP, endogenous glucose production; FA, fatty acid; GIR, glucose infusion rate; GlnTT, glutamine tolerance test; G-6-Pase, glucose-6-phosphatase; PGTT, glucose tolerance test; H&E, hematoxylin and eosin staining; IGF-IR, IGF-I receptor; IR, insulin receptor; ITT, insulin tolerance test; NMR, nuclear magnetic resonance; PEPCK, phosphoenolpyruvate carboxykinase; TT, pyruvate tolerance test; rhIGF-I, recombinant human IGF-I; WT, wild type. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

position such as a decrease in fat mass and an increase in lean body mass. Insulin, fatty acid, and triglyceride levels were not affected by rhIGF-I, nor were insulin or glucose tolerance in MKR mice. Hyperinsulinemic-euglycemic clamp analysis demonstrated no improvement in overall insulin sensitivity. Pyruvate and glutamine tolerance tests proved that there was a decrease in the rate of glucose appearance in MKR mice treated with rhIGF-I, suggesting a reduction in the gluconeogenic capacity of liver, kidney, and small intestine. Taken together these results demonstrate that the improvement of the hyperglycemia was achieved by inhibition of gluconeogenesis rather than an improvement in insulin sensitivity. Also, these results suggest that a functional IGF-I receptor in skeletal muscle is required for IGF-I to improve insulin sensitivity in this mouse model of type 2 DM. (Endocrinology 147: 2619 –2630, 2006)

gical states, and burns (6) among others. Based on its structural and functional similarities with insulin, rhIGF-I has been proposed as a potential therapy for both type 1 and type 2 diabetes mellitus (DM) (7–12). Acutely a high dose of IGF-I results in hypoglycemia, decreased serum levels of fatty acids (FAs) and increased lipogenesis, effects that are similar to those of insulin (13, 14). In skeletal muscle tissue, IGF-I has direct insulin-like effects via the type I IGF-IR. These effects include increased translocation of glucose transporters, increased glucose uptake, and increased glycogen formation (15–17). Several studies showed that rhIGF-I increases insulin sensitivity and improves glycemic control in type 2 DM (11, 18) and syndromes of severe insulin resistance (19, 20). Given in combination with IGF binding protein-3 (9), it reduces the insulin requirements in patients with type 1 DM. Using clamp techniques in type 2 diabetic patients, Cusi and DeFronzo (10) suggested that the initial improvement in glycemic control results from direct effects of rhIGF-I on both liver and muscle, with secondary improvements due to the removal of glucose toxicity. However, the primary site of action of IGF-I in regard to its ability to improve glucose homeostasis and insulin insensitivity in type 2 DM remains unclear. We recently created an insulin-resistant diabetic mouse by expressing a dominant-negative mutant IGF-IR specifically

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in skeletal muscle (MKR mouse) (21). The formation of hybrid receptors between the mutated IGF-IR and endogenous IGF-IR and insulin receptors (IRs) causes impairment of both insulin and IGF-I signaling pathways in skeletal muscle. This leads to insulin resistance in skeletal muscle with hyperinsulinemia as early as 3 wk of age. At 5 wk of age, there is also insulin resistance in fat and liver with ␤-cell dysfunction and type 2 DM. Therefore, the MKR mouse is a useful model for the study of the mechanisms underlying the pathogenesis of type 2 DM and potential therapies. Because of its lack in functional IGF-IRs and its few functional IRs specifically in skeletal muscle, we used the MKR mouse as a suitable model to test the ability of rhIGF-I to improve glucose homeostasis in the absence of a functional IGF-IR in skeletal muscle and evaluate the relative contributions of liver vs. muscle to the improvement in glycemic control and insulin resistance in IGF-I-treated mice. We found that after 3 wk of treatment, rhIGF-I was able to reduce hyperglycemia by decreasing gluconeogenesis, but it failed to improve insulin resistance in MKR mouse model of type 2 DM, suggesting that skeletal muscle may be the primary site of action of IGF-I. Materials and Methods Mice Generation and characterization of MKR mice have been described before (22). Homozygous MKR male mice (FVB/N background) used for the current study were subjected to Southern blot analysis for genotyping as previously described (22). Wild-type mice on FVB/N background were used as controls. Mice were maintained on a 12-h light, 12-h dark cycle, and all experiments were performed in agreement with National Institutes of Health guidelines and with the approval of the Animal Care and Use Committee of the National Institute of Diabetes and Digestive and Kidney Diseases.

Chronic administration of rhIGF-I Six-week-old male wild-type (WT) and MKR mice were injected ip with rhIGF-I (Genentech Inc. and Tercica, San Francisco, CA) [1 mg/kg body weight (BW)] or an equivalent volume of sterile saline twice a day (before 1000 and after 1700 h) for 3 wk. BW and food and water intake were measured every other day or weekly, respectively. A glucometer (Ascensia, ELITE XL; Bayer Corp., Mishawaka, IN) was used to measure glucose levels from the tail vein weekly between 0800 and 1000 h in the nonfasting state. Mice were killed on d 21 of treatment after anesthetization using 2.5% Avertin (15–17 ␮l/g BW) in the nonfasting state between 0800 and 1000 h. Tissues were harvested and carefully weighed. After the animals were killed, tissues were quickly removed and frozen in liquid nitrogen for RNA analysis.

Serum analysis Serum was obtained from the tail vein between 800 and 1000 h in the nonfasting state. Serum FA and triglyceride levels were measured using a fatty acid assay kit (Roche, Indianapolis, IN) and GPO-Trinder kit (Sigma, St. Louis, MO), respectively. Serum insulin and glucagon levels were determined using RIA (Linco Research, St. Charles, MO). GH levels were measured by RIA as described before (23).

Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

mice were given glucose (2 g/kg BW) by gavage, and glucose levels were measured during the GTT. A GRR was performed after 24 h of fasting. Mice were injected with glutamine (Sigma) (2 g/kg BW), and glucose appearance was measured at 0, 15, 30, 60, and 120 min after injection. In all cases, the last dose of rhIGF-I was injected the evening of the day before the test.

Hyperinsulinemic-euglycemic clamp The clamp studies were performed as developed by Kim et al. (24). Mice were treated with either rhIGF-I or saline for 2 wk, as described above. On d 14 of treatment (4 d before the clamp experiment), mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. A catheter was inserted into a lateral incision on the right side of the neck and advanced into the superior vena cava via the right internal jugular vein. The catheter was then sutured into place, according to the protocol of MacLeod and Shapiro (25). The day before the clamp analysis, mice received the final injection of rhIGF-I or saline at 1700 h and were then fasted overnight. To conduct experiments in awake animals with minimal stress, a tail-restrain method was used during the procedures. The basal rates of glucose turnover were measured by continuous infusion of [3-3H]glucose (0.02 ␮Ci/min) for 120 min, which followed a bolus of 2.5 ␮Ci, starting at 0900 h. Blood samples (20 ␮l) were taken at 90 and 115 min of the basal period for the determination of plasma [3H]glucose concentration. A 120-min hyperinsulinemic-euglycemic clamp was started at 1100 h. Insulin was infused at the rate of 2.5 mU/kg䡠min (Humulin R; Eli Lilly, Indianapolis, IN) to raise the plasma insulin concentration to approximately 2 ng/ml. During the clamp study, mice were restrained, and blood samples (20 ␮l) were collected via a small nick in the tail vein at 15-min intervals for the immediate measurement of plasma glucose concentration, and 20% glucose was infused at variable rates to maintain plasma glucose at approximately 160 mg/dl in WT mice [MKR mice were clamped at ⬃240 mg/dl (control) or ⬃200 mg/dl (rhIGF-I treated); because of severe insulin resistance, this was the lowest glucose level we can reach]. Insulin-stimulated whole-body glucose flux was estimated using a primed continuous infusion of HPLC-purified [3-3H]glucose (10 ␮Ci bolus, 0.1 ␮Ci/min; NEN Life Science Products, Boston, MA) throughout the clamps. To estimate insulin-stimulated glucose transport activity and metabolism in skeletal muscle, 2-deoxy-d-[1–14C]glucose (NEN Life Science Products) was administered as a bolus (10 ␮Ci) 45 min before the end of clamps. Blood samples (20 ␮l) were taken at 80, 85, 90, 100, 110, and 120 min after the start of clamps for the determination of plasma [3H]glucose, 2-deoxy-d-[1–14C]glucose and 3H2O concentrations. Additional blood samples (10 ␮l) were collected before the start and at the end of clamp studies for measurements of plasma insulin concentration. All infusions were performed using microdialysis pumps (CMA/Microdialysis, Acton, MA). On a separate occasion, MKR mice treated with saline (n ⫽ 6) or rhIGF-I (n ⫽ 6) underwent a clamp experiment designed to determine basal parameters. This second experiment, was performed as mentioned above but with no infusion of insulin during the second period of 120 min. At the end of the clamp period in both occasions, animals were anesthetized with ketamine and xylazine injection. Within 5 min, gastrocnemius muscle from hindlimbs, epididymal and brown adipose tissue, and liver were removed. Each tissue, once exposed, was dissected within 2 sec, frozen immediately in liquid nitrogen, and stored at ⫺70 C for later analysis.

Body composition Body composition was measured in nonanesthetized mice using the Bruker minispec nuclear magnetic resonance (NMR) analyzer mq10 (Bruker Optics, Woodlands, TX).

Insulin (ITT), glucose (GTT), pyruvate (PTT), and glutamine (GlnTT) tolerance tests

Food and water intake

ITT, GTT, and PTTs were performed after overnight fasting. Mice were injected ip with insulin (0.75 U/kg BW), glucose (2 g/kg BW), or pyruvate (Sigma) (2 g/kg BW). Blood glucose levels were determined from the tail vein at 0, 30, and 60 min after insulin injection and at 0, 15, 30, 60, and 120 min after glucose and pyruvate injection. For oral GTT,

Mice were caged individually and treated with either rhIGF-I or vehicle (sterile saline), as described above. The amounts of food in the feeding container were measured at d 14 and 21 of treatment, normalize to the body weight and expressed as grams of food ⫻ grams BW⫺0.75 ⫻ d⫺1. To measure water intake, special bottles were used to avoid leaking,

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

and an exact volume was place in each bottle at d 14. Te remaining volume was measured at d 21, and water intake was calculated as the difference between initial and final volume of water.

Glycogen content For glycogen content, a piece of liver and muscle was dissected and immediately snap frozen in liquid nitrogen between 0700 and 0800 h before the animals were killed while the animals were under anesthesia. One hundred milligrams of each tissue was homogenized in 600 ␮l of 30% KOH and incubated at 97 C for 15 min. Cold 95% ethanol (3 ml) was added into each tube and incubated at ⫺30 C for 1 h. After centrifugation at 3300 rpm for 30 min at 4 C, pellets were washed with cold 95% ethanol three times and dissolved in 200 ␮l distilled water. Samples were then incubated in 100 ␮l of solution containing 1 U/ml glucokinase, 50 mm triethanolamine hydrochloride (pH 9.0), 2 mm MgCl2, 1 mg/ml BSA, and 40 ␮m [␥-32P]ATP at 30 C for 30 min, and then 100 ␮l of 2 n HClO4 with 0.2 mm H3PO4 were added and samples incubated at 90 C for 40 min. After adding 50 ␮l of 100 mm ammonium molybdate and 50 ␮l of 200 mm triethylamine, samples were centrifuged at 3000 rpm for 30 min. Tissue glucose was measured as incorporation of ␥-32P ATP and calculated using a standard curve with various glucose concentrations. All reagents were purchased from Sigma.

Histology The pancreas and left gonadal fat pad were removed after the animals were killed and fixed overnight in 4% paraformaldehyde in PBS. The tissues were then transferred to 70% ethanol and embedded in paraffin. Samples were cut into 5-␮m sections, and hematoxylin and eosin staining (H&E) was performed. To measure the average diameter of adipocytes, two samples of 20 –30 mg of adipose tissue were immediately fixed in osmium tetroxide as previously described (26) and incubated in a water bath at 37 C a for 48 h. Adipose cell size was determined using a Multisizer III with a 400-␮m aperture (Beckman Coulter, Fullerton, CA). After collection of pulse size, the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bin scale for the x-axis. Average area of adipocytes was calculated using MacBas version 2.52 software (Fuji, PhotoFilm, Tokyo, Japan), as previously described (27).

RNA analysis Total RNA was isolated using the TRIzol reagent (Life Technologies, Rockville, MD), and Northern blot analysis was performed as previously described (28).

Statistical analysis All data are expressed as means ⫾ se. One-way ANOVA followed by Student’s t test was used to determine statistically significant differences between groups.

Results BW, food and water intake, and GH levels after rhIGF-I or saline treatment

Six-week-old MKR and WT mice were injected with either rhIGF-I or an equivalent volume of sterile saline. BW was recorded at the beginning of the treatment and every other day during the following 3 wk. As reported before (22), MKR mice BW was lower than the BW of WT age-matched mice. There were no differences in basal BW within MKR (rhIGF-I vs. saline treated) or WT (rhIGF-I vs. saline treated) groups of mice (data not shown). At the end of the treatment, BW increased in both rhIGF-I- and saline-treated MKR and WT mice (data not shown). There was no difference in average BW between treatments in WT mice, whereas BW of rhIGFI-treated MKR mice was significantly higher than salinetreated MKR mice (Fig. 1A). However, there was no change

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in food intake (Fig. 1B) during rhIGF-I treatment, compared with saline treatment in MKR mice that could account for this difference. Water intake was also monitored (Fig. 1C). For that purpose, mice were single caged and special bottles were used. The exact volume of water was measured at the beginning and end of the third week of treatment. As expected, MKR saline-treated mice drank more water than WT salinetreated mice, and water intake decreased with rhIGF-I treatment in MKR diabetic mice, whereas there was no effect on water intake in WT mice. After 3 wk of treatment with rhIGF-I, WT mice showed a significant reduction in basal levels of GH, whereas MKR mice showed a similar tendency that did not reach statistical significance (Fig. 1D). Serum biochemistry and changes in body composition after 3 wk of rhIGF-I treatment in MKR and WT mice

rhIGF-I therapy has been shown to decrease glucagon secretion (29, 30). Nevertheless, 3 wk of treatment with rhIGF-I did not reduce serum levels of glucagon (66 ⫾ 13 vs. 68 ⫾ 3; 69 ⫾ 7 vs. 95 ⫾ 2 ng/ml, WT or MKR saline vs. rhIGF-I-treated mice, respectively); free fatty acids (0.56 ⫾ 0.08 vs. 0.66 ⫾ 0.05; 0.68 ⫾ 0.02 vs. 0.57 ⫾ 0.06 mm, WT or MKR saline vs. rhIGF-I treated mice, respectively); or triglycerides (181 ⫾ 15.6 vs. 164 ⫾ 7.4; 404 ⫾ 29 vs. 351 ⫾ 17 mg/dl, WT or MKR saline vs. rhIGF-I treated mice, respectively) in fed mice. Body composition was measured by NMR or manual dissection. MKR or WT mice body composition was analyzed using a NMR machine at the beginning and end of the treatment with either saline or rhIGF-I. As shown in Table 1, there was an increase in lean mass in both MKR and WT mice as well as a decrease in fat mass after rhIGF-I in MKR, in absolute (grams) or relative to BW values. A similar tendency was observed in WT mice but did not reach statistical significance. To assess which organs accounted for such modifications, MKR and WT mice were manually dissected and several organs harvested and precisely weighed. As expected, there was no significant change in skeletal muscle weight in MKR mice (31). However, there was a significant increase in spleen weight, kidney (data not shown), and a decrease in liver and epididymal fat pad. Accordingly, H&E staining of epididymal fat tissue showed a comparable reduction in adipocyte average size after rhIGF-I treatment in both MKR and WT mice (Fig. 2, A and B). rhIGF-I treatment decreased blood glucose levels without improvement in overall insulin sensitivity in MKR mice

With the purpose of studying the effect of rhIGF-I treatment in a type 2 diabetes mouse model with extreme insulin resistance in skeletal muscle, we treated MKR mice with rhIGF-I ip for 3 wk. Random glucose levels were measured weekly, in the fed state, during morning hours. Glucose levels in MKR mice decreased after 1 wk of treatment (data not shown) and reached statistical significance after 2 wk of treatment (Fig. 3A), remaining low until the end of the study. There were no changes in blood glucose levels during either rhIGF-I or saline treatment in WT mice (Fig. 3B). Urine glucose levels were significantly higher in MKR mice, compared

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

FIG. 1. Effect of rhIGF-I on BW (A), food intake (B), water intake (C), and serum GH levels (D). Six-week-old male MKR or FVB (WT) mice were treated with either rhIGF-I (1 mg/kg, ip, twice a day) or an identical volume of sterile saline for 3 wk. A, BW was recorded every other day in the morning before rhIGF-I injection. Open bars, BW of MKR/WT mice at 3 wk of saline treatment; closed bars, BW at 3 wk of rhIGF-I injection. B, After 2 wk of treatment, mice were single caged to measure food and water intake (C, after saline (䡺) or rhIGF-I (f) treatment, as described in Materials and Methods. D, At the end of the study, mice were killed and bled in the morning (12–14 h after the last injection of rhIGF-I). Total levels of GH were measured by RIA as indicated in Materials and Methods. Results are expressed as mean ⫾ SEM (n ⫽ 6 – 8 in each group). *, P ⬍ 0.05 vs. saline treated; ##, P ⬍ 0.05 MKR vs. WT, saline-treated groups; #, P ⬍ 0.05 MKR vs. WT, rhIGF-I-treated groups.

with WT, and decreased dramatically after rhIGF-I treatment (Fig. 3, C and D) in MKR mice, even as early as 10 d of treatment (data not shown). These results suggested that the decrease in blood glucose was not due to an increase in renal glucose excretion. Clamp studies.It has been previously reported that rhIGF-I treatment in human subjects with type 2 DM reduces serum insulin levels and improves insulin sensitivity (10, 11, 18). Interestingly, basal levels of insulin (fed state) remained unchanged in MKR mice as well as in WT mice (Fig. 3E) after 3 wk of IGF-I treatment. Consistent with our previous studies (22), MKR mice showed severe insulin resistance, compared with WT mice. Chronic treatment with rhIGF-I did not improve insulin sensitivity in MKR mice as assessed by the ITT

(Fig. 3F). To further understand the mechanism underlying the decrease of glucose levels after rhIGF-I treatment on MKR mice, we performed clamp experiments in two separate occasions: one set of mice was used to determine basal parameters, by performing the last 120 min of the clamp experiment (see Materials and Methods) with no infusion of insulin (Table 2). In the second experiment, mice were subjected to the hyperinsulinemic-euglycemic clamp (Table 3 and Fig. 4). As shown in Table 2, basal endogenous glucose production (EGP) was elevated in MKR mice, compared with WT mice, and decreased in MKR mice after rhIGF-I treatment, whereas there was no effect in WT mice. Glucose infusion rate (GIR) was dramatically reduced in MKR compared with WT mice (22, 28) as reported before. There was

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TABLE 1. Body composition of WT and MKR mice treated with saline or IGF-I

NMR Fat mass Lean mass Dissection Gonadal fat Spleen Liver Gastroc ⫹ soleus Quadriceps

WT saline

WT IGF-I

MKR saline

MKR IGF-I

3.17 ⫾ 0.25 11.9 ⫾ 1.1 20.6 ⫾ 0.9 76.9 ⫾ 1.1

2.49 ⫾ 0.21 9.3 ⫾ 0.8 21.5 ⫾ 0.8 79.9 ⫾ 0.8a

1.89 ⫾ 0.13 9.2 ⫾ 0.7 15.9 ⫾ 0.6 77.6 ⫾ 0.6

1.61 ⫾ 0.18a 6.9 ⫾ 0.8a 18.3 ⫾ 0.4a 79.7 ⫾ 0.6a

0.444 ⫾ 0.043 1.55 ⫾ 0.11 0.102 ⫾ 0.004 0.36 ⫾ 0.01 1.43 ⫾ 0.03 5.03 ⫾ 0.12 0.162 ⫾ 0.004 0.57 ⫾ 0.01 0.194 ⫾ 0.008 0.68 ⫾ 0.01

0.389 ⫾ 0.020 1.38 ⫾ 0.05 0.119 ⫾ 0.009 0.42 ⫾ 0.05a 1.47 ⫾ 0.11 5.20 ⫾ 0.14 0.168 ⫾ 0.005 0.60 ⫾ 0.02 0.198 ⫾ 0.007 0.71 ⫾ 0.02

0.354 ⫾ 0.026 1.82 ⫾ 0.13 0.093 ⫾ 0.006 0.48 ⫾ 0.04 1.16 ⫾ 0.04 5.9 ⫾ 0.1 0.093 ⫾ 0.010 0.48 ⫾ 0.12 0.095 ⫾ 0.010 0.49 ⫾ 0.13

0.265 ⫾ 0.031a 1.21 ⫾ 0.16a 0.128 ⫾ 0.005a 0.58 ⫾ 0.02a 1.22 ⫾ 0.04 5.5 ⫾ 0.1a 0.102 ⫾ 0.008 0.47 ⫾ 0.01 0.116 ⫾ 0.010 0.52 ⫾ 0.13

Body composition in MKR and WT mice during rhIGF-I or saline treatment. Six-week-old MKR and WT mice were injected with either rhIGF-I or saline during 3 wk. At the end of the treatment, body composition was measured by NMR and manual dissection after the animals were killed during the morning hours. Tissues were carefully dissected and weighed. Results are expressed as mean ⫾ SEM of absolute values (grams) (above) or the percentage of BW (below). a P ⬍ 0.05 vs. saline.

a very modest but significant increase in GIR after rhIGF-I treatment, although overall it remained very low, compared with WT mice. There was also an increase in brown adipose tissue glucose uptake in both WT and MKR mice. During the second experiment, hyperinsulinemic-euglycemic clamp studies confirmed the small changes in GIR MKR mice after rh IGF-I treatment. Furthermore, the increased EGP did not return to normal levels (Fig. 4, A and B) after insulin infusion. Whole-body glucose uptake (Fig. 4C) was reduced in MKR mice as reported previously (22, 28) and did not change after 3 wk of rhIGF-I treatment. Whole body glycolysis (Fig. 4D) and insulin levels reached during clamp (Fig. 4E) were similar in WT and MKR mice, regardless of the treatment Liver and skeletal muscles are the main tissues where glycogen is synthesized and stored. We therefore measured the glycogen content in muscle and liver of fed MKR and WT mice after 3 wk of either rhIGF-I or saline treatment. Liver glycogen content was significantly decreased in MKR, whereas no changes were found in WT mice at the end of 3 wk of treatment with rhIGF-I [51 ⫾ 4 vs. 35 ⫾ 5 (P ⬍ 0.05), 42 ⫾ 4 vs. 33 ⫾ 5 pmol glucose per milligram tissue, MKR and WT mice, respectively]. There were no changes in skeletal muscle glycogen content in saline- or rhIGF-I-treated MKR or WT mice [44 ⫾ 5 vs. 40 ⫾ 2 (P ⫽ NS), 46 ⫾ 6 vs. 50 ⫾ 8 (P ⫽ NS) pmol glucose per milligram tissue, MKR and WT mice, respectively]. Taken together, these results suggest that rhIGF-I treatment reduces serum glucose levels without improving serum insulin levels or whole-body insulin insensitivity in MKR mice. rhIGF-I treatment decreases gluconeogenesis occurring in liver, kidney and small intestine in type 2 diabetic MKR mice

A GTT was performed in MKR and WT mice treated with either saline or rhIGF-I for 3 wk. After an overnight fast, MKR and WT mice were challenged with ip administration of 2 g/kg BW of glucose. As previously reported (22, 27, 28),

MKR mice show impaired GTT, compared with WT mice (Fig. 5A). After 3 wk of rhIGF-I, GTT was significantly improved in MKR mice, compared with saline-treated MKR. Insulin levels during GTT (data not shown) confirmed that despite the decrease in glucose levels, rhIGF did not decrease insulin levels (fed or fasted state). Because blood glucose levels are mainly the result of the equilibrium between EGP and glucose uptake in peripheral tissues, we tested the possibility that the reduction in glucose levels after rhIGF-I therapy in MKR was due to a reduction in gluconeogenesis. The major contributors to the EGP are liver, kidney, and small intestine (32–34). Using different gluconeogenic substrates, it is possible to detect changes in the gluconeogenic rate that may occur during treatment in these organs. Pyruvate is preferentially used by the liver as a substrate for gluconeogenesis (35), whereas glutamine is used mostly by the kidney and small intestine (33, 34). We performed a PTT and GlnTT and measured glucose appearance in both MKR and WT mice treated with either rhIGF-I or saline. As shown in Fig. 5, B and C, both PTT and GlnTT showed a significantly reduced glucose appearance in MKR mice after rhIGF-I treatment, whereas no changes were observed in WT mice. These results suggest that rhIGF-I is suppressing gluconeogenesis in liver, kidney, and small intestine in MKR mice. Expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) in liver, kidney and small intestine of MKR mice

To further investigate the mechanism by which IGF-I decreased gluconeogenesis in MKR mice, we analyzed the mRNA levels of two key enzymes involved in this process, PEPCK and G-6-Pase, in liver, small intestine, and kidney from MKR mice (Fig. 6). Membranes were probed for PEPCK, G-6-Pase, and 18S as loading control. There were no differences between levels of expression of PEPCK in liver or small intestine from MKR mice treated with rhIGF-I, compared with saline-treated MKR mice (data not shown), al-

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

FIG. 2. Effect of rhIGF-I treatment on epididymal adipose tissue histology. After 3 wk of treatment, left epididymal adipose pad was harvested, fixed, and stained for histological analysis (H&E) (A) and size average measurement (B and C) (see Materials and Methods). Results are expressed as mean ⫾ SEM. *, P ⬍ 0.05 vs. basal; ##, MKR vs. WT.

though there was a tendency to a decrease in the expression of PEPCK in kidney after rhIGF-I treatment (P ⫽ 0.058, data not shown).There were no differences in G-6-Pase expression levels in liver, small intestine, or kidney after rhIGF-I treatment, compared with saline treatment in MKR mice. Discussion

rhIGF-I has been shown to improve glucose homeostasis and insulin sensitivity in patients with insulin resistance such as type A syndrome of severe insulin resistance (19, 20) and both type 1 (9, 12) and type 2 DM (8, 10, 18). The mechanisms responsible for the improved metabolic control are still unclear In the present study, we demonstrate that rhIGF-I treatment in MKR mice improves hyperglycemia by decreasing gluconeogenesis, without improvement in whole-body in-

sulin sensitivity, whereas no effect was observed in control mice. Acute injection of rhIGF-I reduced glucose levels in WT mice [this study, data not shown, and Yakar et al. (36)]; however, this effect was not seen after 3 wk of rhIGF-I injection. These observations are in accordance with several reports using rhIGF-I in healthy subjects (13, 37, 38). Mauras and Beaufrere (39) have shown that 5–7 d of rhIGF-I therapy in normal volunteers did not change glucose levels, but insulin sensitivity was not tested in this study. In MKR mice, glucose levels were decreased acutely (data not shown) and after 2 wk of treatment, remaining low until the end of the study. rhIGF-I administration to patients representing severe insulin resistance improved fasting and 24-h mean glucose levels (20). In patients with type 1 DM, rhIGF-I therapy reduced both glucose levels and insulin requirements (9, 12). Several studies (8, 10, 11) showed that patients

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

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FIG. 3. Serum and urine glucose levels, basal insulin levels, and ITT in MKR and WT mice during saline or rhIGF-I treatment. Six-week-old male MKR or FVB (WT) mice were treated with either rhIGF-I (1 mg/kg BW ip, twice a day) or sterile saline for 3 wk. Serum glucose levels in the nonfasted state were measured weekly during the morning period in MKR (A) and WT (B) mice treated with rhIGF-I or saline. Urine glucose levels were determined in parallel, in spontaneous samples obtained from MKR (C) or WT (D) mice at the time of the blood measurements. B (open squares), Basal levels at the beginning of the treatment; W 2 (gray squares), levels at the end of 2 wk of treatment; W 3 (black squares), levels after 3 wk of treatment. Results are expressed as mean ⫾ SEM (n ⫽ 10 –12 in each group). E, Insulin levels in nonfasting state, during the morning, were measured at the beginning (open squares) and end of 3 wk of treatment (black squares) with either saline (⫺) or rhIGF-I (⫹) (n ⫽ 6 – 8 in each group). After overnight fasting, ITT (F) was performed by ip injection of 0.75 U insulin per kilogram BW, and blood glucose was measured at the indicated time points (n ⫽ 10 –12 in each group). 䡺, rhIGF-I-treated MKR mice; f, saline-treated MKR; Œ, rhIGF-I-treated WT mice; ‚, saline-treated WT mice.*, P ⬍ 0.05 vs. basal; ##, P ⬍ 0.05 WT basal vs. MKR basal.

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

TABLE 2. Basal clamp parameters in MKR mice treated with saline or IGF-I

Insulin Glucose Basal EGP Basal GIR RD Basal WBglycolysis Basal MGU Basal WATGU Basal BATGU

MKR saline

MKR IGF-I

2.2 ⫾ 0.3 358 ⫾ 36 249 ⫾ 73 2⫾1 251 ⫾ 73 138 ⫾ 49 114 ⫾ 14 12 ⫾ 5 109 ⫾ 26

2.5 ⫾ 0.3 279 ⫾ 29 175 ⫾ 39a 16 ⫾ 8a 191 ⫾ 35 119 ⫾ 28 105 ⫾ 27 8⫾1 190 ⫾ 45a

Basal parameters during clamp in MKR mice treated with saline or rhIGF-I injection. Six-week-old MKR mice were injected with either rhIGF-I or saline during 3 wk (n ⫽ 6 in each group). At the end of the treatment and after 12 h fasting, mice were subjected to clamp to measure basal parameters. Results are expressed as mean ⫾ SEM. EGP, whole-body glycolysis (WBglycolisis), and GIR are expressed in millimoles per kilogram BW per minute; muscle glucose uptake (MGU), BAT glucose uptake (BATGU), and WAT glucose uptake (WATGU) are expressed in micromoles per kilogram tissue per minute. a P ⬍ 0.05 vs. saline.

with type 2 DM get also a substantial reduction in glucose levels when treated with rhIGF-I. More recently, using euglycemic clamp techniques, Cusi and Defronzo (10) reported that 1 wk of rhIGF-I treatment in a cohort of eight patients with type 2 DM suppressed basal EGP and enhanced insulinmediated glucose uptake, resulting in improved insulin sensitivity in both liver and skeletal muscle. In contrast, MKR mice treated with rhIGF-I for 3 wk showed no improvement in insulin sensitivity, despite the reduction in glucose levels. Insulin levels remained high and ITT did not improve. Hyperinsulinemic-euglycemic clamp studies showed that, despite the modest decrease of EGP in the basal state, EGP was not suppressed by insulin infusion after rhIGF-I treatment, nor was the whole-body glucose uptake improved. Several possible mechanisms by which rhIGF-I could improve insulin sensitivity have been proposed: 1) direct effects of rhIGF-I through its binding to IGR-IR, IR, or hybrid IGFI/IRs mainly in liver and skeletal muscle; 2) decrease in blood glucose levels and therefore improvement in glucotoxicity; 3) indirect effects due to an improved lipid profile; 4) inhibition of insulin secretion; and 5) reduction in plasma glucagon or GH concentrations. Direct effects on the liver are unlikely because IGF-IRs are scarce (40) or absent (41) and because the affinity of IGF-I for

the IR is approximately 5% of that of insulin (1, 17, 42). Also, although hybrid IGF/IRs have been found in adipocytes (3) and muscle (43), such receptors have not been reported in liver. Both lipotoxicity and glucotoxicity impair ␤-cell function and increase insulin resistance in liver, muscle, and adipose tissue (44, 45). When given acutely, rhIGF-I has been reported to reduce (46, 47) FA levels. When administered chronically to patients with type 2 DM, plasma FA concentration remained unchanged (10). rhIGF-I treatment in MKR mice did not improve triglyceride or FA levels and had no effect in WT mice. In a previous study (28), we have shown that treatment with the peroxisomal proliferator-activated receptor-␣ agonist WY14,463 decreased serum FAs and triglycerides and triglyceride stores in liver and muscle with a subsequent normalization in glucose, insulin levels, and insulin sensitivity. These findings suggested that abrogation of lipotoxicity improves insulin sensitivity in MKR mice. In this study, the lack of effect of rhIGF-I on serum levels of FAs and triglycerides could be one of the factors contributing to its inability to improve insulin resistance in this model. However, Cusi and DeFronzo (10) and Clemmons (9) have shown that rhIGF-I treatment improved insulin sensitivity despite the unchanged plasma FA or triglyceride levels in type 2 DM and type 1 DM patients, respectively. This discrepancy may suggest that direct effects of rhIGF-I in skeletal muscle may be the key component of rhIGF-I’s ability to improve insulin sensitivity in type 2 DM. Treatment of MKR mice with phloridzin, an inhibitor of intestinal glucose uptake and renal glucose reabsorption, revealed a decrease in blood glucose with no effect on the levels of insulin resistance, suggesting that glucotoxicity contributes only partially to secondary insulin resistance in these mice. Therefore, it is not surprising that insulin resistance did not improve in MKR mice despite the decrease in glucose levels after rhIGF-I treatment (27). IGF-I administration to diabetic animals and healthy subjects has been related to glucagon suppression (29, 30). However, a recent publication (10) showed no changes in glucagon levels in patients with type 2 DM after infusion of rhIGF-I. In agreement with this finding, glucagon levels remained unchanged after 3 wk of treatment with rhIGF-I in both WT and MKR mice. GH levels are also known to decrease after IGF-I administration (7, 29). Accordingly, we found a reduction in basal GH levels in WT mice and a

TABLE 3. Hyperinsulinemic-euglycemic clamp parameters in WT and MKR mice

BW Basal insulin Basal glucose Clamp glucose MGU WATGU BATGU

WT saline

WT IGF-I

MKR saline

MKR IGF-I

21.5 ⫾ 0.4 0.48 ⫾ 0.19 159.8 ⫾ 9.2 159.3 ⫾ 9.1 173.3 ⫾ 40 8.55 ⫾ 0.82 346.9 ⫾ 61.4

22.0 ⫾ 0.6 0.35 ⫾ 0.03 182.5 ⫾ 8.7 162.5 ⫾ 8.8 132 ⫾ 20 10.85 ⫾ 3.25 754.7 ⫾ 90b

17.8 ⫾ 0.8a 2.29 ⫾ 0.87a 296.3 ⫾ 9.3a 239.4 ⫾ 32.8a 84.9 ⫾ 2.8 9.6 ⫾ 2.1 271.7 ⫾ 92.8

19.3 ⫾ 0.3a,b 2.18 ⫾ 0.44a 300.6 ⫾ 36a 205.7 ⫾ 22 193.4 ⫾ 61 9.0 ⫾ 1.6 225 ⫾ 88.7a

Hyperinsulinemic-euglycemic clamp parameters in WT and MKR mice treated with saline or rhIGF-I. Six-week-old WT or MKR mice were injected with either rhIGF-I or saline during 3 wk (n ⫽ 4 in each group). At the end of the treatment and after 12 h fasting, mice were subjected to hyperinsulinemic-euglycemic clamp (see Materials and Methods). Results are expressed as mean ⫾ SEM. Muscle glucose uptake (MGU), BAT glucose uptake (BATGU), and WAT glucose uptake (WATGU) are expressed in micromoles per kilogram tissue per minute. a P ⬍ 0.05, MKR vs. WT same treatment. b P ⬍ 0.05 vs. saline.

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

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FIG. 4. Effect of rhIGF-I or saline treatment on glucose infusion rate (A), clamp EGP (B), whole-body glucose uptake (C), and whole-body glycolysis (D) during hyperinsulinemic-euglycemic clamp analysis. E, Clamp insulin levels. Six-week-old male MKR and WT mice were injected with rhIGF-I (black bars) or saline (white bars) for 3 wk. After a period of 12 h of fasting, hyperinsulinemic-euglycemic clamp was performed. Results are expressed as mean ⫾ SEM (n ⫽ 4 in each group). *, P ⬍ 0.05 vs. basal; ##, P ⬍ 0.05 vs. WT.

similar tendency in MKR mice after rhIGF-I treatment. Suppression of GH and its counterregulatory effects on EGP and gluconeogenesis have been proposed to play a role in the reduction in insulin requirements in type 1 DM patients after rhIGF-I therapy (9). Therefore, it is possible that a reduction in GH levels plays a role in the modest decrease in the basal EGP observed in MKR mice but was insufficient to improve insulin sensitivity in these mice. In accordance with previous findings (11), rhIGF-I treatment decreased fat content in WT and MKR mice as measured by NMR or by dissection. Histological changes in adipocytes from white adipose tissue revealed a similar decrease in average size in WT and MKR mice after treatment, compared with vehicle-treated mice. The changes in fat content were mirrored by changes in lean mass in MKR mice with a similar trend in WT mice that did not reach significance. Increases in spleen and kidney weight could partially account for the changes in lean mass. On the other hand, the compensatory hyperplasia in skeletal muscle that occurs in MKR mice between 5 and 8 wk of age (48) might be an additional cause for the increase in lean mass as well as the increase in BW without changes in food intake observed in MKR mice.

GTT improved in MKR mice after rhIGF-I treatment. However, basal fasting glucose was already significantly reduced in rhIGF-I-treated MKR, and the increments in glucose levels observed during the test were similar to those observed in saline-treated MKR mice. This observation is consistent with previous findings in patients with type 2 DM after 1 wk of treatment with rhIGF-I (10) and suggests that the major effect of rhIGF-I was on EGP and not on insulin-mediated glucose disposal. Because skeletal muscle accounts for more than 80% of postprandial glucose uptake, these results are in agreement with the fact that there are no functional IGF-IRs and very few IRs in skeletal muscle in MKR mice that could mediate such an effect on glucose disposal. To rule out the possibility that the lowering effects of rhIGF-I in glucose levels were related to increased renal excretion of glucose, we monitored urine glucose levels in WT and MKR treated mice. Urine glucose levels paralleled changes in blood glucose levels in MKR mice, suggesting that the decrease in serum glucose levels was not due to an increase in renal glucose excretion. Liver (49), kidney (32), and small intestine (34) hold sufficient gluconeogenic enzyme activity and G-6-Pase activity to allow them to release glucose into the circulation as a result

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

FIG. 6. PEPCK and G-6-Pase gene expression in liver, small intestine, and kidney of MKR mice. Six-week-old MKR mice were treated with either rhIGF-I (1 mg/kg BW ip, twice a day) or sterile saline for 3 wk. Frozen tissues harvested from MKR mice at the end of the treatment were extracted for RNA. Membranes were probed for PEPCK, G-6-Pase, and 18S as a loading control. Results are expressed as arbitrary units (AU) corrected for 18S. Open bars represent salinetreated MKR mice; closed bars represent rhIGF-I-treated MKR mice (n ⫽ 7– 8 mice per group).

FIG. 5. GTT, PTT, and GlnTT after rhIGF-I treatment. Six-week-old male MKR or FVB (WT) mice were treated with either rhIGF-I (1 mg/kg, ip, twice a day) or sterile saline for 3 wk. After overnight fasting, GTT (A) was performed by ip of 2 g/kg BW of glucose (n ⫽ 10 –12 in each group). B and C, After an overnight or 24-h-long fasting period, PTTs and GlnTTs were performed in MKR mice by ip injection of 2 g/kg BW of pyruvate or 2 g/kg BW of glutamine, respectively, and glucose appearance was measured at the indicated time points (n ⫽ 7 in each group). f, rhIGF-I-treated MKR mice; 䡺, saline-treated MKR; Œ, rhIGF-I-treated WT mice; ‚, saline-treated WT mice. Results are expressed as mean ⫾ SEM. *, P ⬍ 0.05 MKR (⫹) vs. MKR (⫺); #, MKR(⫹) vs. WT (⫹) or (⫺).

of gluconeogenesis. Nonetheless, until recently the liver was considered the sole source of gluconeogenesis in normal states, whereas the kidney became important in acidotic con-

ditions or prolonged fasting (32). This concept has been challenged, and it is becoming an accepted view that the kidney and small intestine play significant roles in glucose balance in various situations (32, 34). Current evidence (50, 51) indicates that in normal subjects after overnight fasting, renal gluconeogenesis accounts for about 40% of all gluconeogenesis, suggesting that the kidney is as important a gluconeogenic organ as the liver. Studies in diabetic animals (52) have demonstrated increased renal gluconeogenic enzyme activity and increased renal glucose release. Additionally, in humans with type 1 and type 2 DM, it has been shown that renal glucose release was as increased as hepatic glucose release (32, 53). Even though lactate, glutamine, alanine, and glycerol are the main glucogenic precursors, it appears that glutamine is the preferential gluconeogenic substrate for the kidney (70%) (32) and small intestine (80%) (34), whereas alanine is preferentially used by the liver (35). Taking advantage of these differences in substrate use, we performed glutamine and PTTs to assess the effect of rhIGF-I on renal/small intestine and hepatic gluconeogenic potential. We found that glucose appearance after injection of both glutamine and pyruvate were significantly reduced in MKR mice after treatment, compared with saline-treated mice. These results show for

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Pennisi et al. • rhIGF Decreases Gluconeogenesis in MKR Mice

the first time the ability of rhIGF-I to decrease gluconeogenesis in a mouse model of type 2 DM. The mechanisms underlying these effects are still unclear. We found no changes in expression of two major gluconeogenic enzymes such us PEPCK and G-6-Pase in liver, kidney, or small intestine except for a tendency to decrease expression levels of PEPCK in kidney that did not reach significance, which could account for the decrease in gluconeogenesis. Nevertheless, we cannot exclude the possibility of a reduction in activity of these enzymes after rhIGF-I therapy. To summarize, in a mouse model of type 2 DM lacking in functional IGF-IRs and with few functional IRs in skeletal muscle, rhIGF-I treatment decreased glucose levels not by improving insulin sensitivity but by decreasing gluconeogenesis. These results suggest that rhIGF-I effects in regard to enhancement of insulin sensitivity require a functional IGF-IR in skeletal muscle and that skeletal muscle may be the primary site of rhIGF-I action. In addition, using the MKR model allowed us to unveil a novel effect of rhIGF-I in the regulation of glucose homeostasis. Acknowledgments Received December 7, 2005. Accepted February 23, 2006. Address all correspondence and requests for reprints to: Derek LeRoith, M.D., Ph.D., Director of the Division of Endocrinology and Diabetes, Mt. Sinai School of Medicine, 1 Gustave Levy Place-Box 1055, Annenberg 23-66, New York, New York 10029-6574. E-mail: [email protected]. This work was supported in part by funds from the intramural National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, and from a grant (to D.L.) from the American Diabetes Association. Disclosures: P.P., O.G., J.S.-P., W.J., S.S., and S.Y. have nothing to declare. D.C. has received consulting fees from Pfizer and Eli Lilly and lecture fees from Pfizer. D.L. has received consultant fees from Imclone, Pfizer, Sanofi-Aventis, and Merck.

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