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Endocrinology Vol. 147, No. 5 2163-2170
Copyright © 2006 by The Endocrine Society

Differential Role of SH2-B and APS in Regulating Energy and Glucose Homeostasis

Minghua Li, Decheng Ren, Masanori Iseki, Satoshi Takaki and Liangyou Rui

Department of Molecular & Integrative Physiology (M.L., D.R., L.R.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0622; and Division of Immunology (M.I., S.T.), Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

Address all correspondence and requests for reprints to: Liangyou Rui, Ph.D., Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622. E-mail: ruily{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SH2-B and APS, two members of a pleckstrin homology and SH2 domain-containing adaptor family, promote both insulin and leptin signaling in a similar fashion in cultured cells. In addition, APS mediates insulin-stimulated activation of the c-Cbl/CAP/TC10 pathway in cultured adipocytes. Here we characterized genetically modified mice lacking SH2-B, APS, or both to determine the physiological roles of these two proteins in animals. Disruption of the SH2-B gene resulted in obesity, hyperglycemia, hyperinsulinemia, and glucose intolerance. Conversely, deletion of the APS gene did not alter adiposity, energy balance, and glucose metabolism. Energy intake, energy expenditure, fat content, body weight, and plasma insulin, leptin, glucose, and lipid levels were similar between APS–/– and WT littermates fed either normal chow or a high-fat diet. Moreover, deletion of APS failed to alter insulin and glucose tolerance. APS–/–/SH2-B–/– double knockout mice also developed energy imbalance, obesity, hyperleptinemia, hyperinsulinemia, hyperglycemia, and glucose intolerance; however, plasma leptin and insulin levels were significantly lower in APS–/–/SH2-B–/– than in SH2-B–/– mice. These results suggest that SH2-B, but not APS, is a key positive regulator of energy and glucose metabolism in mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN AND INSULIN are two primary hormones that regulate energy and glucose homeostasis, respectively. Leptin is produced and secreted by adipocytes in a proportion to total adipose mass and conveys peripheral information about energy storage and availability to the central nervous system (1, 2). Leptin inhibits feeding and promotes energy expenditure, thus reducing adiposity and body weight. Genetic deficiency in either leptin or leptin receptor LepRb results in severe obesity and type 2 diabetes (3, 4, 5). Insulin is produced and secreted by pancreatic ß-cells in response to glucose. Insulin inhibits hepatic glucose production and promotes glucose uptake in skeletal muscle and adipose tissue, reducing blood glucose levels. Defects in insulin secretion and/or response result in hyperglycemia, a hallmark of diabetes.

Both leptin and insulin exert their biological action via binding and activating their receptors on the plasma membrane. The leptin receptor LepRb interacts with Janus kinase (JAK) 2, a cytoplasmic tyrosine kinase. Leptin stimulates JAK2 activation, which subsequently tyrosyl phosphorylates and activates various downstream substrates, including signal transducer and activator of transcription 3, insulin receptor substrate (IRS) 1, and IRS2 (6). In contrast, the insulin receptor contains intrinsic tyrosine kinase activity that is stimulated by insulin. Activated insulin receptor tyrosyl phosphorylates various substrates, initiating multiple signaling pathways (7, 8).

The SH2-B family contains three members of SH2-B, APS, and Lnk, which share a conserved dimerization, pleckstrin homology, and SH2 domain (9, 10). Lnk is expressed restrictively in hematopoietic cells and negatively regulates the development of hematopoietic cells (11). In contrast, SH2-B and APS are widely expressed in multiple tissues including insulin and leptin targets (the brain, liver, muscle, and adipose tissue) (12, 13, 14). In cultured cells, both SH2-B and APS bind via their SH2 domain to JAK2, strongly potentiating JAK2 activation (15, 16, 17). SH2-B and APS also bind directly to the insulin receptor via their SH2 domain, promoting the insulin signaling pathway in cultured cells (18, 19, 20). Moreover, the insulin receptor tyrosyl phosphorylates APS, forming a binding site for c-Cbl (21, 22, 23, 24). APS mediates insulin-stimulated activation of the c-Cbl/CAP/TC10 pathway that appears to play an important role in regulating glucose uptake in cultured adipocytes (22, 23, 24, 25). These observations suggest that SH2-B and APS may positively regulate leptin and insulin sensitivity.

We have generated SH2-B- and APS-deficient mice to examine physiological role of these two proteins in vivo. Disruption of the SH2-B gene results in severe leptin resistance, insulin resistance, obesity, and type 2 diabetes (13, 14). Leptin signaling is significantly impaired in the hypothalamus, whereas insulin signaling is dramatically attenuated in muscle, liver, and fat in SH2-B–/– mice (13, 14). Surprisingly, deletion of the APS gene results in a mild metabolic phenotype of slightly enhanced insulin sensitivity in young mice (12). It remains unclear whether APS plays a role in regulating leptin and/or insulin sensitivity in old animals fed a high-fat diet (HFD), and whether SH2-B compensates for the loss of APS to maintain leptin and insulin sensitivity in APS null mice. In this work, we have systemically examined energy and glucose metabolism in APS null mice fed either normal chow or HFD over a long period, and we compared insulin sensitivity between SH2-B–/– and APS–/–/SH2-B–/– mice. We conclude that SH2-B is a key positive regulator of both leptin regulation of adiposity and insulin regulation of blood glucose. APS may negatively regulate leptin and insulin sensitivity under certain conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
APS–/– and SH2-B–/– mice were generated by DNA homologous recombination as described previously (13, 26), and maintained in 129Sv/C57BL/6 mixed and/or C57BL/6 isogenic (backcross for six generations) genetic background. Mice were housed on a 12-h light, 12-h dark cycle in the Unit for Laboratory Animal Medicine at the University of Michigan, with free access to water and either standard rodent chow (9% kcal fat, 5058 chow of Picolab mouse diet 20; PMI Nutrition International LLC, Brentwood, MO) or HFD (45% kcal fat; Research Diets Inc., New Brunswick, NJ). Animal experiments were conducted following the protocols approved by the University Committee on the Use and Care of Animals.

Blood chemistry, insulin tolerance test (ITT), and glucose tolerance test (GTT)
Blood samples were collected from the tail vein and assayed for plasma insulin and leptin using rat insulin or mouse leptin Elisa kits (Crystal Chem Inc., Chicago, IL), respectively. Free fatty acids and triglycerides were measured using Wako NEFAC (Wako Chemicals USA, Inc., Richmond, VA) and Free Glycerol Reagent (Sigma, St. Louis, MO), respectively. For ITT, mice were fasted for 6 h, then injected ip with human insulin (0.8 IU/kg of body weight in mice fed a standard chow and 1.0 IU/kg of body weight in mice fed HFD). Blood glucose was monitored at 0, 15, 30, and 60 min after injection. For GTT, mice were fasted overnight (~16 h), then injected ip with D-glucose (2 g/kg of body weight). Blood glucose was monitored at 0, 15, 30, 60, and 120 min after glucose injection.

Body composition and energy balance
Metabolic rates were measured by indirect calorimetry (Windows Oxymax Equal Flow System, Columbus Instruments, Columbus, OH) in 16- to 19-wk-old mice. Mice were housed individually in air-tight cages through which room air was passed at a flow rate of 0.5 liter/min. Exhaust air was sampled at 27-min intervals for a period of 1 min; O2 and CO2 contents of exhaust were determined by comparison to O2 and CO2 contents of standardized sample air. Mice were acclimatized to the cages for 48 h before measurements. VO2, VCO2, and heat production were normalized to lean body mass.

To measure lean body mass and fat content, mice were briefly anesthetized with 4% isoflurane in O2 using a SurgiVet/ANESCO (SurgiVet Veterinary Surgical Products, Waukesha, WI), and subjected to analysis using a dual-energy x-ray absorptiometry method (Dexa Sabre Bone Densitometry, Norland Medical System Inc.).

Immunoprecipitation and immunoblotting
Mice were killed, and the brain and liver were isolated and homogenized in lysis buffer [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 250 mM sucrose, 1% Nonidet P-40]. Tissue extracts were immunoprecipitated and immunoblotted with indicated antibodies.

Statistical analysis
The data are presented as the mean ± SEM. Student’s t tests were used for comparisons between two groups. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SH2-B but not APS negatively regulates body weight and adiposity in mice
Because SH2-B and APS potentiate JAK2 activation in a similar fashion in cultured cells (17) (data not shown), we determined whether these two proteins regulate leptin sensitivity and body weight by a similar mechanism in animals. APS- and SH2-B-deficient mice were generated by DNA homologous recombination as described previously (13, 26). To determine whether SH2-B and APS act additively to enhance leptin responses, APS–/–/SH2-B–/– double knockout mice were generated by crossing APS+/– with SH2-B+/– heterozygous mice. To confirm successful deletion of APS or SH2-B, brown adipose tissues (BAT) was isolated from APS–/–, SH2-B–/–, APS–/–/SH2-B–/–, or wild-type (WT) mice, and homogenized in lysis buffer. SH2-B in BAT extracts was immunoprecipitated with {alpha}SH2-B and immunoblotted with {alpha}SH2-B. APS in BAT extracts was immunoprecipitated with {alpha}APS and immunoblotted with {alpha}APS. SH2-B was detected in both WT and APS–/– animals, but not in SH2-B–/– and APS–/–/SH2-B–/– animals as expected (Fig. 1AGo). Similarly, APS was detected in both WT and SH2-B–/– animals, but not in APS–/– and APS–/–/SH2-B–/– animals (Fig. 1AGo). All mice were fed a standard chow, and body weight and food intake were monitored.


Figure 1
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  		FIG. 1. Disruption of the SH2-B but not APS gene results in obesity. A, BAT was dissected from SH2-B–/–, APS–/–, APS–/–/SH2-B–/– and WT male mice (23 wk). BAT extracts (3 mg protein) were immunoprecipitated (IP) and immunoblotted (IB) with indicated antibodies. BAT extracts (60 µg protein) were also immunoblotted with {alpha}Actin. B–C, Growth curves of male (B) and female (C) APS–/–, SH2-B–/–, APS–/–/SH2-B–/– and WT littermates (in 129Sv/C57BL/6 mixed background). *, P < 0.01.

 
SH2-B–/– mice, both males and females, gained significantly more body weight than WT littermates after 6 wk of age (Fig. 1Go, B and C). At 16 wk of age, SH2-B–/– males were 1.7 times heavier than WT littermates (WT: 32.7 ± 1.8 g, n = 6; SH2-B–/–: 53.8 ± 2.3 g, n = 5), whereas SH2-B–/– females were 2.1 times heavier than WT littermates (WT: 25.2 ± 0.9 g, n = 11; SH2-B–/–: 52.8.0 ± 2.0 g, n = 11). In contrast, both male and female APS–/– mice gained a similar body weight as WT littermates (Fig. 1Go, B and C). Even at old age (> 25 wk), body weight was still similar between APS–/– and WT littermates (data not shown). These data indicate that SH2-B, but not APS, is required for maintaining normal body weight.

Both male and female APS–/–/SH2-B–/– mice also gained dramatically more body weight than WT littermates (Fig. 1Go, B and C). At 16 wk of age, APS–/–/SH2-B–/– males were 1.6 times heavier than WT littermates (WT: 32.7 ± 1.8 g, n = 6; APS–/–/SH2-B–/–: 52.7 ± 1.8 g, n = 5), whereas APS–/–/SH2-B–/– females were 2.1 times heavier than WT littermates (WT: 25.2 ± 0.9 g, n = 11; APS–/–/SH2-B–/–: 53.0 ± 6.0 g, n = 4). Importantly, both the onset and severity of obese phenotype were similar between SH2-B–/– and APS–/–/SH2-B–/– mice, and growth curves were indistinguishable between SH2-B–/– and APS–/–/SH2-B–/– mice (Fig. 1Go, B and C). These data suggest that APS and SH2-B are unable to compensate for each other in regulating body weight.

To determine whether deletion of APS, SH2-B, or both alters adiposity in mice, fat content was measured by dual-energy x-ray absorptiometry. Deletion of either SH2-B alone or both SH2-B and APS dramatically increased fat content (Fig. 2AGo), demonstrating that an increase in body weight of SH2-B–/– and APS–/–/SH2-B–/– mice is primarily caused by an elevation of adiposity. The size of individual white adipocytes is significantly enlarged in SH2-B–/– mice, suggesting that adipose hypertrophy may contribute to obesity in SH2-B–/– and APS–/–/SH2-B–/– mice (Fig. 2BGo). Fat content was similar between SH2-B–/– and APS–/–/SH2-B–/– mice (Fig. 2AGo). In contrast, APS–/– mice had similar fat content as WT littermates (Fig. 2AGo). These results indicate that SH2-B, but not APS, negatively regulates adiposity in mice.


Figure 2
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  		FIG. 2. Disruption of the SH2-B but not APS gene increases adiposity. A, Fat content of female APS–/–, SH2-B–/–, APS–/–/SH2-B–/–, and WT littermates (in 129Sv/C57BL/6 mixed background). B, Hematoxylin and eosin staining of inguinal fat in female mice (3 months). *, P < 0.01.

 
Deletion of SH2-B but not APS disrupts energy balance
Obesity is commonly associated with elevated energy intake and reduced energy expenditure. To determine whether deletion of SH2-B or APS disrupts energy balance, we monitored food intake, oxygen consumption, and carbon dioxide production in APS–/–, SH2-B–/–, or APS–/–/SH2-B–/– mice. Both male and female SH2-B–/– mice were hyperphagic (Fig. 3AGo and data not shown), confirming our recent report (14). APS–/–/SH2-B–/– mice were also extremely hyperphagic (Fig. 3AGo). After normalization to body weight, the average values of food intake were higher in SH2-B–/– mice than WT control but not statistically different (Fig. 3AGo). In contrast, food intake was similar between APS–/– and WT littermates (see Fig. 6BGo and data not shown).


Figure 3
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  		FIG. 3. Deletion of the SH2-B but not APS gene disrupts energy balance. A, Food intake of SH2-B–/–, APS–/–/SH2-B–/–, and WT littermates fed normal chow (17–20 wk old, and in 129Sv/C57BL/6 mixed background). In right panel, food intake was normalized to body weight. B–C, O2 consumption (B) and CO2 production (C) of female APS–/–, SH2-B–/–, APS–/–/SH2-B–/–, and WT littermates in 129Sv/C57BL/6 mixed background. *, P < 0.01.

 

Figure 6
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  		FIG. 6. Disruption of the APS gene does not affect HFD-induced energy imbalance and obesity in mice in C57BL/6 background. A, SH2-B levels in the brain and liver from APS–/– and WT mice. Tissues extracts were immunoprecipitated (IP) with {alpha}SH2-B or {alpha}APS and immunoblotted (IB) with {alpha}SH2-B or {alpha}APS as indicated. B, Food intake in APS–/– and WT mice fed HFD for 8 wk (16 wk old). C, O2 consumption and CO2 production of APS–/– and WT littermates fed HFD for 12 wk (20 wk old). D, Growth curves of male APS–/– and WT control (APS+/+) fed a HFD (45% fat content) after wk 8. E, Fat content of APS–/– and WT males (in C57BL/6 background) fed either normal chow (18 wk old) or HFD for 15 wk (24 wk old). V, Volume.

 
Energy expenditure was estimated by measuring O2 consumption and CO2 production by indirect calorimetry. Both O2 consumption and CO2 production were significantly increased in SH2-B–/– mice (Fig. 3Go, B and C), confirming our previous report (14). However, energy intake still exceeded energy expenditure, resulting in obesity in SH2-B–/– mice (14). Similarly, energy expenditure was also dramatically increased in APS–/–/SH2-B–/– mice (Fig. 3Go, B and C). In contrast, deletion of APS did not alter energy expenditure (Fig. 3Go, B and C). Together, these results suggest that SH2-B but not APS negatively regulates both feeding and energy expenditure.

SH2-B but not APS positively regulates leptin sensitivity in mice
Both SH2-B and APS promote JAK2-activated pathways in cultured cells, suggesting that these two proteins may enhance leptin sensitivity in vivo. To determine whether deletion of SH2-B or APS impairs leptin sensitivity, we measured plasma leptin levels in APS–/–, SH2-B–/–, or APS–/–/SH2-B–/– mice. Plasma leptin was increased by 7.2 times in fasted SH2-B–/– mice (6 wk) and by 5.6 times in randomly fed SH2-B–/– mice (15 wk) (Fig. 4Go). Consistent with these data, we reported recently that SH2-B–/– mice are severely leptin resistant as demonstrated by both inability of exogenous leptin to inhibit feeding and a marked reduction of leptin-stimulated activation of hypothalamic JAK2, signal transducer and activator of transcription 3 and IRS2 in SH2-B–/– mice (14). In contrast, plasma leptin levels were similar between APS–/– mice and WT littermates (Fig. 4Go). These data suggest that SH2-B but not APS is an endogenous positive regulator of leptin sensitivity.


Figure 4
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  		FIG. 4. Disruption of the SH2-B but not APS gene results in hyperleptinemia. Fasted (6 wk old) (A) or randomly fed (15 wk old) (B) plasma leptin levels in APS–/–, SH2-B–/–, APS–/–/SH2-B–/– and WT littermates (in 129Sv/C57BL/6 mixed background). *, P < 0.01.

 
APS–/–/SH2-B–/– mice also developed hyperleptinemia; surprisingly, plasma leptin levels were significantly lower in APS–/–/SH2-B–/– mice than in SH2-B–/– mice (Fig. 4Go). A decrease in plasma leptin levels is commonly associated with enhanced leptin sensitivity. These results suggest that APS may negatively regulate leptin sensitivity under certain conditions in animals.

APS is not required for insulin regulation of glucose homeostasis in mice
The role of APS in insulin action remains controversial. APS recruits c-Cbl to the insulin receptor, which mediates the activation of the phosphatidylinositol 3-kinase-independent pathway required for insulin to stimulate glucose uptake in cultured adipocytes (22, 23, 24, 25, 27). On the other hand, c-Cbl promotes ubiquitination of the insulin receptor, down-regulating the insulin receptor on the plasma membrane (21). In addition, APS binds to Asb6 that is associated with an ubiquitin ligase, and insulin stimulates APS-mediated recruitment of the ubiquitin ligase to the insulin receptor complex, thus facilitating degradation of components of the insulin pathway (28). To clarify the role of APS in insulin-regulated glucose metabolism, we examined blood glucose and insulin levels and glucose and insulin tolerance in APS–/– mice at both young and old ages, in two different genetic backgrounds and fed either normal chow or HFD.

Both blood glucose and insulin levels were similar between APS–/– and WT littermates in 129Sv/C57BL/6 mixed genetic background (Fig. 5Go, A and B). In contrast, SH2-B–/– mice developed age-dependent hyperglycemia and hyperinsulinemia (Fig. 5Go, A and B). At 15 wk of age, randomly fed blood glucose levels increased by approximately 2.4 times in SH2-B–/– than in WT littermates (WT: 127.0 ± 6.0 mg/dl, n = 7; SH2-B–/–: 300.6 ± 60.7 mg/dl, n = 5). These results indicate that SH2-B but not APS is a key positive regulator of insulin sensitivity.


Figure 5
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  		FIG. 5. Disruption of the SH2-B but not APS gene results in insulin resistance and hyperglycemia. A, Fasted blood glucose in male APS–/–, SH2-B–/–, APS–/–/SH2-B–/– and WT littermates in 129Sv/C57BL/6 mixed background. B, Fasted blood insulin in male APS–/–, SH2-B–/–, APS–/–/SH2-B–/– and WT littermates in 129Sv/C57BL/6 mixed background at 11 wk of age. C, GTT in APS–/–, SH2-B–/–, APS–/–/SH2-B–/–, and WT littermates in 129Sv/C57BL/6 mixed background at 13 wk of age. D, ITT in APS–/–, SH2-B–/–, APS–/–/SH2-B–/–, and WT littermates in 129Sv/C57BL/6 mixed background at 14 wk of age. *, P < 0.05.

 
APS–/–/SH2-B–/– mice also developed severe hyperglycemia and hyperinsulinemia (Fig. 5Go, A and B). Randomly fed blood glucose increased by approximately 2.5 times in APS–/–/SH2-B–/– mice (15 wk) (WT: 127.0 ± 6.0 mg/dl, n = 7; APS–/–/SH2-B–/–: 318.8 ± 83.6 mg/dl, n = 5). At 10 wk of age, fasted blood glucose levels were slightly higher in APS–/–/SH2-B–/– than in SH2-B–/– mice (Fig. 5AGo); however, fasted plasma insulin levels were disproportionally approximately 50% lower in APS–/–/SH2-B–/– than in SH2-B–/– mice (Fig. 5BGo). A reduction of plasma insulin without a concomitant increase of blood glucose is commonly associated with an increase of insulin sensitivity. These data suggest that APS may negatively regulate insulin sensitivity under certain conditions in animals.

To provide additional evidence of insulin resistance in SH2-B–/– and APS–/–/SH2-B–/– mice, GTT and ITT were performed in APS–/–, SH2-B–/–, APS–/–/SH2-B–/– and WT littermates. Intraperitoneal injection of exogenous glucose increased blood glucose levels to a much greater extent in SH2-B–/– than in WT littermates (Fig. 5CGo). APS–/–/SH2-B–/– mice exhibited similar glucose intolerance as SH2-B–/– mice (Fig. 5CGo). Intraperitoneal injection of exogenous insulin significantly decreased blood glucose in WT littermates, whereas the reduction of blood glucose was dramatically attenuated in both SH2-B–/– and APS–/–/SH2-B–/– mice (Fig. 5DGo). In contrast, blood glucose was similar between APS–/– and WT littermates in both GTT and ITT (Fig. 5Go, C and D). Together, these data indicate that SH2-B but not APS is required for insulin regulation of glucose homeostasis in mice.

APS deficiency does not alter susceptibility to HFD-induced obesity and insulin resistance
The experiments described above were carried out on mice in 129Sv/C57BL/6 mixed genetic background fed standard mouse chow. Because both genetic background and dietary fat content affect leptin and insulin sensitivity, we characterized APS–/– mice in C57BL/6 background and fed HFD. Deletion of the APS gene did not alter the expression of SH2-B in both leptin and insulin target tissues, including the brain and liver (Fig. 6AGo). The body weight was still similar between APS–/– and WT control (in C57BL/6 background) fed standard chow over a period of 24 wk (data not shown). Food intake, O2 consumption and CO2 production were similar between APS–/– and age-matched WT control (Fig. 6CGo, and data not shown).

To determine whether APS deficiency predisposes to HFD-induced obesity, APS–/– and WT males (8 wk) were fed HFD (45% fat content). Food intake was similar between APS–/– and WT control (Fig. 6BGo), consistent with previous data. HFD-fed APS–/– and WT control (in C57BL/6 background) gained similar body weight (Fig. 6DGo). Even after they were fed a HFD for 18 wk, APS–/– mice still had similar body weight as WT control at 26 wk of age (data not shown). Adiposity was dramatically increased in both APS–/– and age-matched WT control fed HFD; however, fat content was similar between APS–/– and WT control fed either normal chow or HFD (Fig. 6EGo). These results further support the conclusion that APS is not required for regulation of adiposity and body weight, regardless of genetic background and dietary fat content.

To determine whether APS deficiency alters susceptibility to HFD-induced insulin resistance, APS–/– mice and WT control (in C57BL/6 background, 8 wk) were fed HFD for additional 20 wk. Glucose tolerance was significantly impaired in mice fed HFD as revealed by GTT (Fig. 7AGo). Fasted blood glucose levels were similar between APS–/– and WT control fed either normal chow or HFD (Fig. 7BGo). Fasted plasma insulin levels were higher in males than in females, but similar between APS–/– and age-matched WT control in both males and females (Fig. 7CGo). GTT and ITT were performed on mice fed HFD for 11 and 13 wk, respectively, and also revealed similar insulin sensitivity between APS–/– and WT control (Fig. 7DGo). Together, these results indicate that APS is not required for insulin to regulate blood glucose in mice regardless of genetic background and dietary fat content.


Figure 7
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  		FIG. 7. Disruption of the APS gene does not affect HFD-induced hyperglycemia and insulin resistance in mice in C57BL/6 background. A, WT male mice (8 wk) were fed either normal chow or HFD for 9 wk, and GTTs were performed. B, Fasted blood glucose in male APS–/– and WT control fed either normal chow or HFD as indicated. C, Fasted plasma insulin in APS–/– and age-matched WT mice fed HFD for 17 wk for males (25 wk old) and 19 wk for females (27 wk old). D, GTT and ITT in male APS–/– and age-matched WT control. GTT was performed in mice fed HFD for 10 wk (18 wk old), whereas ITT in mice fed HFD for 13 wk (21 wk old). *, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SH2-B is widely expressed in multiple tissues, and binds via its SH2 domain to various tyrosine kinases in cultured cells, raising a possibility that this protein may play pleiotrophic functions in animals. SH2-B binds directly to both JAK2 and the insulin receptor via its SH2 domain, enhancing the activation of these two tyrosine kinases in cultured cells (13, 15, 16, 29). In addition, SH2-B also binds to IRS1 and IRS2, promoting IRS protein-mediated activation of the phosphatidylinositol 3-kinase pathway (30). We reported recently that disruption of the SH2-B gene results in severe resistance to both leptin and insulin, obesity, and type 2 diabetes (13, 14). Hypothalamic leptin signaling is significantly impaired in SH2-B–/– mice; similarly, insulin signaling is dramatically attenuated in skeletal muscle, liver, and adipose tissues (13, 14). In the current work, we confirmed our earlier findings by showing that SH2-B–/– mice had extremely increased body weight and adiposity, hyperleptinemia, hyperglycemia, hyperinsulinemia, and glucose intolerance. We conclude that SH2-B is a key endogenous positive regulator of leptin and insulin sensitivity in mice. SH2-B deficiency results in resistance to both leptin and insulin, contributing to the development of obesity and type 2 diabetes. SH2-B may enhance insulin sensitivity both indirectly by reducing adiposity and directly by enhancing insulin signaling in target tissues, including the liver, skeletal muscle, adipose tissue, and the brain.

APS shares high homology with SH2-B, including a conserved dimerization, pleckstrin homology, and SH2 domain (9, 31, 32). APS binds to both JAK2 and the insulin receptor via its SH2 domain, and enhances the activation of these two tyrosine kinases in a similar fashion as SH2-B in cultured cells (13, 17, 29). Moreover, the insulin receptor phosphorylates APS at Tyr618, which binds directly to c-Cbl (24). APS is required for tyrosine phosphorylation of c-Cbl by the insulin receptor and subsequent activation of the c-Cbl/CAP/TC10 pathway (22, 23, 24, 33). This pathway has been reported to be required for insulin-stimulated Glut4 translocation to the plasma membrane and glucose uptake in adipocytes (25, 27). These findings raise a possibility that APS may positively regulate leptin and insulin sensitivity in animals as SH2-B. On the other hand, APS recruits an ubiquitin ligase to the insulin receptor complex, facilitating ubiquitination and degradation of components of the insulin signaling pathway (21, 28). Consistent with this idea, Minami et al. (12) reported that deletion of the APS gene increases insulin sensitivity in young mice at 8–11 wk of age. In the current work, we have systemically examined energy and glucose metabolism in APS–/– mice at both young and old ages, in two different genetic backgrounds, and fed either normal chow or HFD. We found that deletion of APS alone did not alter feeding, energy expenditure, body weight, adiposity, and plasma leptin levels regardless of age, genetic background, and dietary fat content. These observations indicate that APS is unlikely to play an important role in leptin regulation of energy homeostasis and adiposity. Moreover, the onset and severity of obese phenotype in SH2-B–/– mice were not exacerbated by additionally deleting the APS gene, further demonstrating that APS may not play an important role in regulating adiposity and body weight. Surprisingly, we did not detect enhanced insulin sensitivity in APS–/– mice as reported by Minami et al. (12). The reasons for this discrepancy are unclear. Moreover, we demonstrated that fasted blood glucose and insulin levels and glucose and insulin tolerance were comparable between APS–/– and WT controls at both young and old ages, in two different genetic backgrounds, and fed either normal chow or HFD. We conclude that, in these experimental conditions, APS appears not to modulate, either positively or negatively, insulin regulation of glucose metabolism in mice.

Interestingly, both plasma leptin and insulin levels were significantly lower in APS–/–/SH2-B–/– than in SH2-B–/– mice. Because reduced plasma leptin or insulin levels are commonly associated with improved leptin or insulin sensitivity, both leptin and insulin sensitivity may be higher in APS–/–/SH2-B–/– than in SH2-B–/– mice. It is likely that APS negatively regulates leptin and/or insulin sensitivity under certain conditions, including absence or reduced expression of SH2-B. APS has been shown to negatively regulate cell signaling which promotes B cell proliferation (34). However, SH2-B may exert a dominant stimulatory effect over an APS inhibitory effect on leptin and insulin sensitivity; therefore, deletion of APS alone does not alter leptin and insulin sensitivity, adiposity and glucose metabolism in the presence of SH2-B.

In summary, both SH2-B–/– and APS–/–/SH2-B–/– mice developed severe leptin resistance, insulin resistance, obesity, and type 2 diabetes; however, plasma leptin and insulin levels were significantly lower in APS–/–/SH2-B–/– than in SH2-B–/– mice. Deletion of the APS gene alone did not alter either energy or glucose metabolism regardless of age, genetic background, and dietary fat content. These observations suggest that SH2-B positively regulates leptin and insulin sensitivity, whereas APS may negatively regulate leptin and insulin sensitivity under certain conditions.


   Footnotes
 
This study was supported by Career Development Award (7-03-CD-11) from the American Diabetes Association and RO1 DK 065122 from National Institutes of Health (NIH) (both to L.R.). This work used the cores supported by the Michigan Diabetes Research and Training Center (funded by NIH 5P60 DK20572), University of Michigan’s Cancer Center (funded by NIH 5 P30 CA46592), and University of Michigan Center for Integrative Genomics.

Disclosure Statement: All authors have nothing to declare.

First Published Online February 2, 2006

Abbreviations: BAT, Brown adipose tissue; GTT, glucose tolerance test; HFD, high-fat diet; IRS, insulin receptor substrate; ITT, insulin tolerance test; JAK, Janus kinase; WT, wild type.

Received October 17, 2005.

Accepted for publication January 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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