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Endocrinology Vol. 145, No. 11 5259-5268
Copyright © 2004 by The Endocrine Society

Small-Molecule Insulin Mimetic Reduces Hyperglycemia and Obesity in a Nongenetic Mouse Model of Type 2 Diabetes

Mathias Z. Strowski, Zhihua Li, Deborah Szalkowski, Xiaolan Shen, Xiao-Ming Guan, Stefan Jüttner, David E. Moller and Bei B. Zhang

Medizinische Klinik mit Schwerpunkt Hepatologie, Gastroenterologie, Endokrinologie, und Stoffwechsel (M.Z.S., S.J.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; Departments of Metabolic Disorders-Diabetes (M.Z.S., Z.L., D.S., D.E.M., B.B.Z.), Comparative Medicine (X.S.), and Obesity Research (X.-M.G.), Merck Research Laboratories, Rahway, New Jersey 07065

Address all correspondence and requests for reprints to: Mathias Z. Strowski, M.D., Medizinische Klinik mit Schwerpunkt Hepatologie, Gastroenterologie, Endokrinologie, und Stoffwechsel Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: mathias.strowski{at}charite.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adiposity positively correlates with insulin resistance and is a major risk factor of type 2 diabetes. Administration of exogenous insulin, which acts as an anabolic factor, facilitates adipogenesis. Recently nonpeptidal insulin receptor (IR) activators have been discovered. Here we evaluate the effects of the orally bioavailable small-molecule IR activator (Compound-2) on metabolic abnormalities associated with type 2 diabetes using a nongenetic mouse model in comparison with the effects of a novel non-thiazolidinedione (nTZD) peroxisome proliferator-activated receptor-{gamma} agonist. Both Compound-2 and nTZD alleviated fasting and postprandial hyperglycemia; accelerated glucose clearance rate; and normalized plasma levels of nonesterified fatty acids, triglycerides, and leptin. Unlike nTZD, which increased body weight gain, and total fat mass, which is a common feature for PPAR{gamma} agonists, Compound-2 prevented body weight gain and hypertrophy of brown, and white adipose tissue depots and the development of hepatic steatosis in the mouse model of type 2 diabetes. The effect of the two compounds on proximal steps in insulin signal transduction pathway was analyzed in tissues. Compound-2 enhanced insulin-stimulated phosphorylation of IR tyrosine and/or Akt in the liver, skeletal muscle, and white adipose tissue, whereas nTZD potentiated the phosphorylation of IR and Akt in the adipose tissue only. In conclusion, small-molecule IR activators have unique features as insulin sensitizers and hold potential utility in the treatment of type 2 diabetes and obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HALLMARKS OF type 2 diabetes are hyperglycemia, peripheral insulin resistance, and pancreatic ß-cell dysfunction. Obesity is an important risk factor for the development of insulin resistance in type 2 diabetes (1). The majority of patients with type 2 diabetes are obese, and it has been demonstrated that weight gain correlates with deterioration of insulin resistance, whereas weight loss correlates with the improvement of insulin sensitivity (2, 3, 4, 5). Although the prevalence of obesity and type 2 diabetes is consistently increasing, an effective treatment is still lacking. Current pharmacotherapeutics insufficiently reverse hyperglycemia, have limited tolerability, and induce side effects. In addition oral antidiabetics often fail as the severity of the disease progresses, sooner or later requiring exogenous insulin administration. However, treatment with insulin, which is an anabolic hormone, further contributes to body weight gain of the obese type 2 diabetes patients, thereby increasing peripheral insulin resistance and the exogenous insulin requirements.

Insulin reduces hyperglycemia by acting on heterotetrameric ({alpha}, {alpha}, ß, ß) insulin receptor (IR). Through binding to the extracellular {alpha}-subunits of the IR insulin induces conformational changes of IR and stimulates the autophosphorylation of the ß-transmembrane IR subunit as well as the receptor’s intrinsic tyrosine kinase activity (6, 7, 8). The activated insulin receptor tyrosine kinase (IRTK) leads to transphosphorylation of a number of downstream cascade proteins including phosphatidylinositol 3-kinase, protein kinase B (Akt), a serine threonine kinase, which are involved in insulin-mediated regulation of glucose transport and production and glycogen synthesis (9). The ability of insulin to increase cellular glucose uptake and inhibit endogenous glucose production in type 2 diabetes is reduced, which manifests as fasting and/or postprandial hyperglycemia. The molecular basis for an ineffective insulin action is not entirely understood. Whereas some studies demonstrated a decrease in the density of IR in type 2 diabetes, others have reported a decrease in IRTK and phosphatidylinositol 3-kinase activation as well as Akt phosphorylation (10, 11, 12). Recently orally bioavailable nonpeptidal small-molecule natural-product derivatives with insulin-like activity have been discovered (13, 14). These agents (Compound-1, CPD1; Compound-2, CPD2) interact specifically with IR and activate IRTK, thereby leading to improved insulin sensitivity with concomitant reduction of blood glucose levels in rodent models of type 2 diabetes (13, 14, 15, 16). In studies with pancreatic ß-cells, insulin mimetics also have been shown to have effects on insulin secretion (17). We previously demonstrated that central as well as oral administrations of these agents induce anorexigenic and antiobesity effects in rodent models of diet-induced obesity (DIO) (18). Because obesity is associated with insulin resistance in both diabetic and nondiabetic status, we questioned whether small-molecule insulin mimetic (CPD2) could affect body weight control in type 2 diabetes. We therefore characterized the effects of CPD2, which is a potent activator of IRTK (14, 15), on metabolic abnormalities associated with type 2 diabetes and on obesity in a nongenetic mouse model of type 2 diabetes. Furthermore, we compared the effects of CPD2 with a novel nonthiazolidinedione (nTZD) peroxisome proliferator-activated receptor (PPAR){gamma} agonist, a recently described agent exerting antidiabetic activity in rodent models of type 2 diabetes (19, 20).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human insulin and streptozotocin (STZ) were purchased from Sigma-Aldrich, St. Louis, MO). Mouse monoclonal antibody (clone 4G10) specifically raised against tyrosine phosphorylated proteins were purchased from Upstate, Inc. (Waltham, MA). Rabbit polyclonal antibodies against phosphorylated protein kinase B [phospho-Akt (Thr308)] were obtained from Cell Signaling Technology, Inc. (Beverly, MA).

Diets and compounds
Chow diet (7012) was purchased from Harlan Teklad (Madison, WI), high-fat diet (HFD) was obtained from Research Diets Inc. (New Brunswick, NJ; D00031501). A nTZD PPAR{gamma} agonist [2-(2-[4-phenoxy-2propylphenoxy]ethyl)indole-5-acetic acid] was kindly provided by Drs. Derek von Langen and Michael Kress (Merck Research Laboratories, Rahway, NJ) (20). Nonpeptidal small-molecule insulin mimetic is 2,5-dihydroxy-3-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (14, 15). Chemical structures of both compounds are shown in Fig. 1Go.



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  		FIG. 1. Temporal changes in PP (A) blood glucose levels in mice with HFD and STZ-induced diabetes (HFD/STZ/Veh) between the onset (d 0) and the 20th day of therapy with a small-molecule insulin mimetic (HFD/STZ/CPD2) or nonthiazolidinedione PPAR{gamma} agonist (HFD/STZ/nTZD). Intraperitoneal glucose tolerance test (IGTT) (B) in mice with HFD/STZ-induced diabetes after 4 wk of treatment with either CPD2 (HFD/STZ/CPD2) or nTZD (HFD/STZ/CPD2). Nondiabetic chow-fed and vehicle-treated animals (Chow/Veh/Veh) served as controls. Data are expressed as means ± SEM obtained from n = 30 (A) or n = 8 (B) animals. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. HFD/STZ/Veh group by Student’s t test or two-way ANOVA.

 
Animal husbandry
All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Merck Research Laboratories. Male Institute of Cancer Research mice at the age of 3 wk were obtained from Taconic Farms (Germantown, NY). Unless otherwise stated, all animals were maintained under controlled conditions of 25 C and 12-h light, 12-h dark cycle with food and water available ad libitum.

Experimental induction of diabetes
A nongenetic mouse model of diabetes (21) was generated as follows. Four-week-old male Institute of Cancer Research mice were fed HFD (36% fat content by weight, and 58.4% kcal) for 3 wk to generate peripheral insulin resistance followed by a single injection of 90 mg/kg–1 body weight (BW) of freshly prepared STZ to induce partial ß-cell dysfunction. Animals were then divided into three groups receiving HFD containing small-molecule insulin mimetic (HFD/STZ/CPD2) at 0.02% (wt/wt), nTZD PPAR{gamma} agonist (HFD/STZ/nTZD) at 0.02% (wt/wt), and HFD only (HFD/STZ/Veh), for an additional 4 wk, ad libitum. A group of mice was fed chow diet and injected with vehicle (Chow/Veh/Veh) served as controls. Two other groups consisting of animals fed chow diet and injected with STZ (Chow/STZ/Veh) or HFD-fed animals and injected with vehicle (HFD/Veh/Veh) were used to monitor the contribution of HFD or STZ to the development of diabetes. The amount of consumed food was monitored every 72 h, and body weights of each animal were determined at the indicated time points.

Determination of plasma and blood parameters
All parameters were determined from individual animals. Overnight fasting and postprandial (PP) blood glucose levels were detected by One Touch glucometer (LifeScan, Inc., Milpitas, CA). Blood was collected by a tail nick. Plasma levels of insulin and leptin were measured by ELISA (Alpco, Windham, NH). Plasma levels of triglycerides (TGs) and nonesterified fatty acids (NEFAs) were quantified by the colorimetric method, using wet reagent diagnostic kits (Roche, Nutley, NJ). For determination of plasma levels of hormones and triglycerides, blood was collected from nonanesthetized animals from the retroorbital vein plexus within 20 sec using heparin-coated capillaries.

Intraperitoneal glucose tolerance test
Overnight fasted mice were challenged with glucose (1.5 g/kg–1 BW, ip), and blood glucose levels of every animal were measured at the indicated time points.

Determination of body composition
Animals were anesthetized by ketamine, and the whole-body composition of each animal was determined by a dual-energy x-ray absorption assay (DEXA)-scan (QDR 4500, Hologic, Waltham, MA) using the QDR 4500 small animal studies software version 9.0, as described (22). The differential attenuation of low- and high-energy x-rays by the tissues stands in proportion to tissue density, and this information is used by the detector and associated software in conjunction with tissue calibration phantoms to assess body composition. Lean mass consists primarily of skeletal muscle, blood, bones, organs, tendons, cartilage, and body fluids, and fat mass consists primarily of adipose tissue.

Histological examination
At the end of the study, epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT), and livers were dissected out and fixed in 10% (vol/vol) buffered formaldehyde and embedded in paraffin. Sections (8 µm) were cut and stained with standard hematoxylin/eosin procedures. Images were captured by the Optronics DEI-750 camera (Goleta, CA) at 200-fold magnification. Tissue slides were examined by two independent pathologists, and representative images were taken.

Induction of insulin-dependent phosphorylation of Akt and IR tyrosine
At the end of the study, animals received either saline or insulin (2 U/kg–1 BW) via tail vein, and 5 min later animals were killed by CO2. Tissues [liver, EWAT, and leg muscle (M. soleus)] were immediately removed and frozen in liquid N2.

Extraction of proteins
Animals were euthanized by CO2, and tissues were quickly removed and immediately frozen in liquid nitrogen. Frozen tissues were homogenized with a polytron homogenizer (Fisher, Pittsburgh, PA) in ice-cold lysis buffer [20 mM HEPES buffer (pH 7.4), 2 mM EGTA, 2 mM dithiothreitol, 50 mM b-glycerol phosphate, 1 mM sodium-vanadate, 50 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 x Sigma protease inhibitor mix]. Lysates were allowed to solubilize on ice for 30 min, and particulate mass was removed by centrifugation (20,000 x g) for 45 min at 4 C. Supernatant was stored at –80 C.

Western blot analysis
Tissue lysates were resuspended in SDS-loading buffer (Invitrogen, Carlsbad, CA) and denatured proteins (75 µg per lane) were resolved on 4–12 or 4–20% precast gradient NuPAGE SDS-PAGE gels (Invitrogen) and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were then incubated overnight with the primary antibody. Blots were washed extensively, incubated with fluorescein-conjugated secondary antibodies, and processed for enhanced chemifluorescence using an ECF Western blotting kit (Amersham Pharmacia Biotech, Piscataway, NJ). Duplicate blots of samples from seven animals of each treatment group were processed. The signal intensity of the immunoreactive bands was quantified using a Storm 830 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (version 4.0, Amersham Pharmacia Biotech).

Calculations
Data are expressed as means ± SEM. Statistical analysis was conducted using Student’s t test or ANOVA. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Small-molecule insulin mimetic (CPD2) and nTZD reduce hyperglycemia
We induced peripheral insulin resistance and partial pancreatic ß-cell dysfunction in mice by HFD feeding that was combined with a single ip administration of a moderate dose of STZ (21). This nongenetic mouse model, which closely mimics the late stage of type 2 diabetes with a (relative) deficiency of insulin at increased peripheral insulin resistance, was used to evaluate the effects of a small molecule insulin mimetic (CPD2) and a PPAR{gamma} agonist (nTZD) on metabolic abnormalities associated with type 2 diabetes (chemical structures of both agents are shown in Fig. 1Go). Both agents were mixed with HFD and given to mice ad libitum starting 3 wk after HFD induction of insulin resistance and on the day of STZ administration. PP blood glucose of mice on the HFD and injected with STZ (HFD/STZ/Veh) increased by approximately 3-fold (20 d post STZ), compared with animals fed only chow diet or by 2.5-fold, compared with mice fed HFD only (Fig. 1AGo and data not shown). After 3 wk of therapy with CPD2 (11.5 mg/kg BW per day) or with nTZD (14 mg/kg BW per day) the increase of postprandial blood glucose levels was attenuated by approximately 50 and 58%, respectively, compared with nontreated diabetic mice (n = 30, P < 0.001 vs. HFD/STZ/Veh) (Fig. 1AGo).

Next the effects of both agents on fasting blood glucose levels were determined. After an overnight fast, blood glucose levels of nontreated animals with HFD/STZ-diabetes were more than 2.5-fold higher when compared with chow-fed control animals (Table 1Go). CPD2 and nTZD effectively attenuated the rise in overnight fasting blood glucose levels (Table 1Go).


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  		TABLE 1. Effects of CPD2 and nTZD on glucose, insulin, triglyceride and non-esterified fatty acid levels in mice with of high fat diet/streptozotocin-induced diabetes

 
Furthermore, we investigated the effects of both agents on glucose changes in ip glucose tolerance test. After application of 1.5 g/kg–1 BW of glucose, blood glucose levels of HFD/STZ group increased up to 25.2 mmol/dl–1 at the 60-min time point and decreased only slightly (20.3 mmol/dl–1) at the 180-min time point (Fig. 1BGo). Blood glucose levels in CPD2- and nTZD-treated mice increased maximally up to 19.6 (30 min) and 19.4 mmol/dl–1 (60 min) and decreased at 180-min time point to 10.1 or 8.3 mmol/dl–1 (Fig. 1BGo), respectively, suggesting that both agents improve glucose clearance. Mice treated with nTZD had PP plasma insulin levels that were reduced by 60%, compared with nontreated diabetic animals (Table 1Go). In contrast, CPD2 had no effect on insulin levels (Table 1Go). Thus, nTZD and CPD2 effectively abolish hyperglycemia and improve insulin sensitivity in this nongenetic mouse model of type 2 diabetes.

CPD2 and nTZD reduce plasma levels of triglycerides and NEFAs
Type 2 diabetes is associated with high levels of TGs and NEFAs; both of which play a role in development of peripheral insulin resistance. We evaluated whether CPD2 and nTZD affect plasma TG and NEFA levels in mice with HFD/STZ-diabetes. Compared with chow-fed and nondiabetic animals, induction of HFD/STZ-diabetes led to a 2.8- and 2.3-fold elevation in PP TG or NEFA levels, respectively (Table 1Go). CPD2 completely prevented the increase of TG levels, whereas nTZD diminished the rise of TG levels by 79%, compared with nontreated mice with HFD/STZ-diabetes (Table 1Go). CPD2 and nTZD effectively attenuated the increase in NEFA levels in diabetic mice by 90 or 72%, respectively (Table 1Go).

CPD2 and nTZD differentially regulate body weights through modulation of adipose tissue content
Beginning at the day of STZ administration until the end of the study, vehicle-treated diabetic mice gained approximately 1.47 g BW within 3 wk of administration of HFD (Fig. 2AGo). In contrast, diabetic mice consuming CPD2 lost 4.22 g BW after 3 wk of therapy, whereas diabetic mice treated with the PPAR{gamma} agonist gained significantly more body weight, compared with vehicle-treated diabetic mice. (Fig. 2AGo). CPD2-treated mice had cumulative food intake of 2.12 ± 0.05 (grams x mouse–1 x 24 h–1), which was significantly lower, compared with nontreated diabetic mice (Fig. 2BGo). Cumulative food intake of mice fed HFD mixed with nTZD was 2.45 ± 0.07 and similar to that observed in the nontreated diabetic animals (2.65 ± 0.01) (Fig. 2BGo).



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  		FIG. 2. Temporal changes in body weights (A) over a period of 3 wk in mice with HFD and STZ-induced induced diabetes (HFD/STZ/Veh) treated with either a small-molecule insulin mimetic (HFD/STZ/CPD2) or nonthiazolidinedione PPAR{gamma} agonist (HFD/STZ/nTZD). Cumulative food intake (B) of mice with HFD/STZ-diabetes, treated with either CPD2 or nTZD. After induction of HFD/STZ-diabetes, mice were fed HFD containing the indicated agents ad libitum, and food intake was determined on a 72-h basis between the onset (d 0) and the 18th day of therapy, and body weight changes were monitored. Each data point represents mean ± SEM obtained from n = 30–40 animals. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. HFD/STZ/Veh group in Student’s t test or two-way ANOVA.

 
Leptin decreases food intake, and circulating leptin levels have been shown to positively correlate with body fat mass (23, 24). In our study, induction of HFD/STZ diabetes resulted in a 2.7-fold increased plasma leptin levels, compared with chow-fed mice, suggesting an increase of adipose tissue content in the mouse model of diabetes (Fig. 3AGo). CPD2 fully prevented the rise in plasma leptin levels, suggesting that animals in the CPD2 group had decreased adipose tissue content (Fig. 3AGo). Mice treated with nTZD displayed markedly reduced plasma leptin levels as compared with nontreated diabetic mice (Fig. 3AGo), consistent with the notion that PPAR{gamma} agonists can reduce circulating leptin levels. To better quantify the changes of total adipose tissue contents, we performed a DEXA scan. Mice with HFD/STZ-diabetes had 70% increased total adipose tissue content, compared with nontreated chow-fed animals (Fig. 3BGo). Mice consuming nTZD had further increased total adipose tissue contents by 30%, whereas the fat content of CPD2-consuming animals were reduced to that observed in chow-fed mice (Fig. 3BGo). Of note, in contrast to the changes in total fat mass, we did not observe any changes in the lean body mass (data not shown), consistent with the lack of general toxicity or wasting of the animals due to the compound treatment.



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  		FIG. 3. Plasma leptin levels (A) and total fat mass (B) of mice with HFD/STZ-induced diabetes (HFD/STZ/Veh) after 4 wk of treatment with either a small-molecule insulin mimetic (HFD/STZ/CPD2) or nonthiazolidinedione PPAR{gamma} agonist (HFD/STZ/nTZD). Nondiabetic, chow-fed, and vehicle-treated animals (Chow/Veh/Veh) served as controls. Each bar represents the mean ± SEM obtained from n = 8–10 animals. ***, P < 0.001; **, P < 0.01 vs. HFD/STZ/Veh group by one-way ANOVA.

 
We next characterized the influence of different treatment regimens on the morphological appearance of two adipose tissue compartments, EWAT and IBAT. Histological analysis of EWAT revealed a marked hyperplasia of adipocytes in the HFD/STZ group, which was similar to that observed in nTZD-treated mice (Fig. 4Go). In contrast, EWAT of CPD2-treated diabetic mice consisted of adipocytes that had similar morphology as adipocytes in the chow-fed control mice (Fig. 4Go). Size of adipocytes of IBAT of diabetic HFD/STZ-mice increased markedly, compared with the size observed in the chow-fed control mice. Microscopically, IBAT-adipocytes of the HFD/STZ mice closely resembled adipocytes of EWAT. Treatment with nTZD partially reduced the morphological changes of IBAT-adipocytes that were observed in HFD/STZ-diabetes. (Fig. 4Go). In contrast, IBAT-adipocytes of CPD2-treated mice were similar to that observed in control nondiabetic mice (Fig. 4Go). Fatty liver disease is commonly observed in type 2 diabetes and obesity, and we therefore investigated the morphology of hepatocytes by light microscopy. Compared with liver cells of chow-fed control mice, hepatocytes of diabetic HFD/STZ-animals displayed marked morphological changes with coalescing cytoplasmic vacuoles, which is strongly suggestive for accumulation of fat droplets (Fig. 4Go). The increase of vacuoles in response to induction of HFD/STZ-diabetes was alleviated by nTZD as well as CPD2 (Fig. 4Go). Thus, these data suggest that small-molecule insulin mimetic (CPD2) and PPAR{gamma} agonist (nTZD) differentially regulate body weight in a nongenetic mouse model of type 2 diabetes by affecting the content and morphology of brown and white adipose tissue compartments. In addition, both agents decrease lipid accumulation associated with HFD/STZ-diabetes in nonadipose tissues such as liver.



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  		FIG. 4. Morphology of epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT) and livers isolated from healthy control mice (Chow/Veh/Veh), from nontreated diabetic mice (HFD/STZ/Veh), or from diabetic mice treated for 4 wk either with CPD2 (HFD/STZ/CPD2) or with nTZD. (HFD/STZ/nTZD). Representative HE-images (200-fold magnification) of all four different treatment groups are shown.

 
CPD2 and nTZD tissue specifically enhance insulin-induced phosphorylation of IR tyrosine and Akt
We evaluated the effects of both antidiabetic agents on basal and insulin-induced phosphorylation of IRTK and Akt in liver, skeletal (soleus) muscle, and EWAT. After a 4-wk treatment period, animals of all groups received either vehicle (0.9% NaCl) or insulin (2 U/kg BW iv). Five minutes later mice were killed; liver, skeletal muscle, and EWAT were quickly removed; and phosphorylated IRTK and Akt were analyzed by Western blots. Basal levels of phosphorylation of Akt or IRTK in livers, skeletal muscle, and EWAT were comparable in all animal groups (data not shown). On infusion of insulin, the phosphorylation of IRTK and Akt increased in all animal groups, compared with mice injected with a vehicle only (Fig. 5Go, A–F). The potentiation of insulin-induced hepatic phosphorylation of Akt and IRTK in CPD2-treated diabetic mice was significantly increased, compared with that observed in nontreated diabetic mice (Fig. 5Go, A and B). Insulin more potently increased phosphorylation of IRTK in vehicle-treated healthy (control) mice, compared with diabetic mice (Fig. 5Go, A and B). In contrast, insulin-induced potentiation of hepatic Akt or IRTK phosphorylation was not affected by nTZD (Fig. 5Go, A and B). As compared with nontreated diabetic mice, CPD2 potentiated insulin-induced phosphorylation of IRTK in skeletal muscle to a significantly higher degree; however, CPD2 failed to influence the phosphorylation of Akt (Fig. 5Go, C and D). Insulin markedly potentiated skeletal muscle IRTK phosphorylation in healthy mice, whereas treatment with nTZD did not affect the insulin-dependent phosphorylation of Akt or IRTK (Fig. 5Go, C and D). As compared with nontreated diabetic animals, administration of nTZD potently increased insulin-induced phosphorylation of Akt and IRTK in EWAT (Fig. 5Go, E and F), consistent with the notion that white fat is the major target tissue of PPAR{gamma} agonists. CPD2 potentiated insulin-induced phosphorylation of Akt in EWAT, without affecting IRTK (Fig. 5Go, E and F). In summary, these data suggest that small-molecule insulin mimetic CPD2 and nTZD PPAR{gamma} agonist target different tissues to exert their effects on insulin sensitivity and differentially regulate body weights.



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  		FIG. 5. Insulin-stimulated Akt and IRTK phosphorylation in liver (A and B), skeletal muscle (C and D), and EWAT (E and F) in mice with HFD/STZ-induced diabetes (HFD/STZ/Veh), treated for 4 wk with either a small-molecule insulin mimetic (HFD/STZ/CPD2) or nonthiazolidinedione PPAR{gamma} agonist (HFD/STZ/nTZD). At the end of the fourth week of therapy with CPD2 or nTZD, mice were injected with insulin (2 U/kg BW, iv) or with vehicle. Five minutes later animals were killed, and tissues were collected for quantitative analysis of phosphorylated Akt and IRTK by Western blots. Nondiabetic, chow-fed, and vehicle-treated animals (Chow/Veh/Veh) served as controls. Results are expressed as fold of increase of Akt/TK phosphorylation in response to insulin, compared with Akt/TK phosphorylation (basal levels) in response to an injection with a vehicle (0.9% NaCl). Each bar of the figure represents a mean ± SEM obtained from n = 7 animals of each group. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. HFD/STZ/Veh group using Student’s t test. At the bottom of each figure, representative Western blots are shown; +, Signals obtained from insulin injected animals; –, Signals obtained from vehicle injected animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently small-molecule compounds have been discovered that specifically bind to IR and augment IR-dependent signal transduction cascade, thereby mimicking the action of insulin (14). The principle of enhancing insulin sensitivity by small-molecule insulin mimetics has been demonstrated in animal models of types 1 and 2 diabetes in vivo and in vitro (13, 14, 15). These agents are capable of increasing the hypothalamic expression of proopiomelanocortin and reducing neuropeptide Y expression in a rat model of DIO, thereby leading to a reduction of body weight and appetite suppression (18). The anorexigenic effects of these substances clearly did not result from interference with the taste aversion or induction of nausea (18). Our current study extends the earlier observations in DIO model by demonstrating that oral feeding with CPD2 induces antiobesity effects in a nongenetic mouse model of type 2 diabetes, in addition to alleviating hyperglycemia and peripheral insulin resistance. This animal model generated by a combination of a HFD-induced insulin resistance and STZ-induced partial ß-cell dysfunction has been reported to closely mimic metabolic abnormalities encountered in the majority of individuals with the advanced stage of type 2 diabetes (21). Consistent with the previous reports in other animal models of type 2 diabetes using either CPD1 (13) or its deriviate CPD2, a more potent activator of IRTK (14, 15), we found that CPD2 reduced fasting and PP hyperglycemia and improved glucose clearance in diabetic mice. Although these findings are fully compatible with that observed in the earlier studies, there are several differences in the experimental settings. In our study CPD2 was mixed with food, and animals were fed ad libitum for a period of at least 3 wk instead of short-term administration. Furthermore, the treatment was started 3 wk after the peripheral insulin resistance has been generated. Alleviation of hyperglycemia by CPD2 in our current study was not associated with changes of insulin levels. This contrasts to the previously reported decrease of insulin levels in ob/ob mice in response to treatment with CPD2 (15). Possible explanation for that apparent discrepancy is that ob/ob mice have extreme degrees of hyperinsulinemia, whereas HFD/STZ-model of diabetes shows only moderately increased insulin levels due to a STZ-dependent disruption of a fraction of pancreatic ß-cells.

In the current study, we also evaluated a novel PPAR{gamma} agonist nTZD (20) that has been reported to effectively reduce hyperglycemia in a nongenetic mouse model of type 2 diabetes (26) and lower lipid levels in obese Zucker fa/fa rats and rat models of DIO (19, 27). In our study nTZD markedly lowered hyperglycemia; however, in contrast to CPD2, it also diminished levels of insulin, consistent with the potent insulin-sensitizing activity of PPAR{gamma} agonists.

Metabolic abnormalities observed in obese patients with type 2 diabetes include increased levels of NEFAs and TGs. In addition to the atherogenic potential of lipids, NEFAs contribute to insulin resistance through inhibition of insulin-dependent glucose transport and/or phosphorylation through increasing hepatic glucose production, decreasing glycogen synthesis, and affecting pancreatic ß-cell-secretion (28, 29, 30). Reduction of increased plasma levels of NEFAs and TGs improves glycemic control in obese individuals with and without type 2 diabetes, enhances insulin sensitivity, and restores pancreatic ß-cell dysfunction (28, 30). Our animal model of diabetes displayed increased NEFAs and TG levels, and administration of CPD2 and nTZD potently reduced PP plasma levels of NEFAs and TGs. Lowering of lipids in a nongenetic animal model of type 2 diabetes, an effect that has not yet been demonstrated by small-molecule insulin mimetic, could contribute to the improvement of insulin sensitivity. Mechanisms underlying PPAR{gamma} agonists-induced alleviation of insulin resistance may include increased flux of NEFAs from liver and muscle into white adipose tissue, reduction of NEFA release from adipocytes, and promotion of adipocyte differentiation (31, 32).

Additional observation in our study was a reduction of body weight increase by CPD2, and this effect was associated with diminution of food intake, expressed in amount of ingested food per mouse. Normalization of food intake against body weight (ingested calories divided by the body weight of each group) revealed that there was a statistically significant decrease in CPD2-treated mice in comparison with vehicle-treated and nTZD-treated diabetic animals (data not shown). Thus, the reduction of body weight of animals fed CPD2 results, at least partly, from diminished food intake. Evaluation of the body composition revealed that CPD2-treated animals induced a marked decrease in total adipose tissue as detected by DEXA scan, which correlated with the reduced weight of freshly removed EWAT and IBAT. We demonstrated similar anorexigenic and antiadipogenic effects of small-molecule insulin mimetics (CPD1 and CPD2) in a rat and mouse model of DIO (18). In that study, CPD2 also prevented hypertrophy of adipocytes in the EWAT as well as a transformation of IBAT-adipocytes into metabolically less active white adipose tissue-like adipocytes. Similar to the effects observed in DIO model, CPD2 prevented the transformation of IBAT-adipocytes into white adipose tissue-like adipocytes, an effect that was observed in nontreated mice with HFD/STZ-diabetes. Because white adipose tissue adipocytes are metabolically less active, compared with IBAT-adipocytes, it is possible that CPD2-treated mice may exhibit a higher energy expenditure, thereby curtailing the development of obesity in those animals.

In contrast to the CPD2-group, nTZD-treated mice with type 2 diabetes displayed a marked weight gain, compared with CPD2-treated and vehicle-treated diabetic mice, despite lack of effect on food intake. Furthermore, the nTZD group had increased total adipose tissue content, compared with nontreated mice. Adipocytes of both fat compartments appeared bigger as compared with chow-fed and CPD2-treated diabetic mice. This observation is consistent with the role of PPAR{gamma} agonists at promoting adipocyte differentiation and fat accumulation, effects that contribute to increased body weight gain. Another mechanism through which PPAR{gamma} induces adiposity could be due to down-regulation of leptin levels (33). Because leptin inhibits feeding and augments catabolic lipid metabolism, this effect of nTZD might explain the increased caloric storage observed in diabetic mice. Our data agree with observations made in obese individuals with type 2 diabetes treated with a PPAR{gamma} agonist troglitazone and corroborate the adipogenic effects reported in a mouse model of DIO that was treated with nTZD PPAR{gamma} agonist (34).

The majority of obese type 2 diabetics have hepatomegaly and fatty liver disease (35), and there are data indicating that hepatic steatosis contributes to insulin resistance (36). Mechanisms underlying fatty liver development are not fully understood; however, increased hepatic fatty acid flux has been postulated to play a role in this process (37). Both CPD2 and nTZD reduced hepatic vacuoles accumulation induced by HFD/STZ-diabetes, thereby probably contributing to improved insulin sensitivity in both treatment groups. Because there is a marked correlation between the severity of fatty liver and circulating fatty acids and triglycerides in type 2 diabetes (38), potential mechanisms underlying the reversal of hepatic morphology by CPD2 and nTZD in mice with HFD/STZ-diabetes may involve reduction of circulating NEFAs and TGs. Whereas there are no data on the effects of insulin mimetics on hepatic morphology, a number of studies that evaluated the effects of PPAR{gamma} agents on hepatic lipid accumulation have been reported with inconsistent results. Whereas some studies demonstrated an increase in hepatic lipid content in response to PPAR{gamma} administration (39, 40), others observed a decrease of hepatomegaly, which correlated with the reduced lipid content in animal models of obesity or type 2 diabetes (41, 42). Reasons for the apparent discrepancy are currently unknown; however, it has been reported that rodent models of DIO and genetic models of type 2 diabetes display quantitative differences in the expression levels of PPAR{gamma}, compared with healthy counterparts (41). Whether changes in the hepatic expression status of PPAR{gamma} are responsible for the discrepancies remains to be determined.

The insulin signaling pathway involves a phosphorylation of a serine threonine kinase Akt, also referred to as protein kinase B, which is also activated by various growth and survival factors and functions in a wortmannin-sensitive pathway involving PI-3 kinase (9). Akt plays a role at maintaining glucose homeostasis; it can induce glucose transporter (GLUT4) translocation to the cell membrane, stimulates glucose uptake in muscle and adipocytes and reduces glucose output from the liver (16, 43, 44, 45). Insulin-stimulated activation of Akt in diabetic states in humans and rodents is markedly impaired, and normalization of hyperglycemia correlates with restoration of insulin-stimulated Akt activity (46, 47). In our study CPD2 enhanced insulin-stimulated phosphorylation of hepatic IR tyrosine and Akt. In muscle CPD2 increased insulin-stimulated phosphorylation of IR tyrosine, whereas in EWAT it phosphorylated Akt. These data suggest that CPD2 exhibits insulin-sensitizing activity in the liver and muscle as well as in EWAT, thereby potentiating insulin-dependent signal transduction cascade in type 2 diabetes. In contrast, PPAR{gamma} agonist phosphorylated tyrosine and Akt in EWAT only, a tissue with the highest level of PPAR{gamma} expression. We did not observe any additional enhancement of insulin-dependent phosphorylation of IR tyrosine and Akt in mice with HFD/STZ diabetes. This finding is consistent with the notion that PPAR{gamma} agonists enhance insulin action primarily by interaction with adipose tissue. Lack of the effects in the liver and muscle may be explained by low levels of PPAR{gamma} expression in both tissues, compared with adipocytes (48). Our findings agree with the recently reported study in genetically obese Zucker fa/fa rats, in which non-TZD PPAR{gamma} agonist potently increased the phosphorylation of Akt and IR tyrosine residues in EWAT (19). These findings are also consistent with the facts that in vitro PPAR{gamma} agonists can induce an increase in adipose tissue insulin action, whereas no such effects on isolated skeletal muscle have been observed (49, 50).

In summary, our study demonstrates that small-molecule insulin mimetic (CPD2) and non-TZD PPAR{gamma} agonists (nTZD) exert antidiabetogenic effects in a nongenetic animal model of type 2 diabetes. CPD2 displays beneficial anorexigenic effects; it reduces adiposity and decreases hepatic lipid accumulation. nTZD increases body weight without affecting visceral adipose tissue compartment and reduces hepatic lipid accumulation. Both agents improve insulin sensitivity by potentiating insulin-dependent signaling and decreasing plasma lipid levels. In conclusion, these data suggest that CPD2 has a suitable profile as therapeutic agent in type 2 diabetes.


   Footnotes
 
This work was supported by Grant STR 558/2-1 from the Deutsche Forschungsgemeinschaft (to M.Z.S.) and by the Deutsche Diabetes Gesellschaft.

Abbreviations: BW, Body weight; CPD1 and 2, compound-1 and -2 (small molecule insulin mimetic); DEXA, dual-energy x-ray absorption; DIO, diet-induced obesity; EWAT, epididymal white adipose tissue; HFD, high fat diet; IBAT, interscapular brown adipose tissue; IR, insulin receptor; IRTK, insulin receptor tyrosine kinase; NEFA, nonesterified fatty acid; nTZD, nonthiazolidinedione; PP, postprandial; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; STZ, streptozotocin; TG, triglyceride.

Received May 12, 2004.

Accepted for publication July 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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