Neuronal SH2B1 is essential for controlling energy and glucose homeostasis (original) (raw)
Neuron-specific restoration of SH2B1 rescues obesity and hyperlipidemia in SH2B1KO mice. To determine tissue distribution of SH2B1, tissue extracts were prepared from spleen, pancreas, heart, hypothalamus, skeletal muscle, liver, white adipose tissue, and brain of WT mice. Tissue extracts were immunoprecipitated with anti-SH2B1 antibody (α-SH2B1) and immunoblotted. α-SH2B1, which was raised against a fusion protein of glutathione-_S_-transferase full-length rat SH2B1β, specifically recognizes all isoforms of SH2B1 (α, β, γ, and δ); in contrast, previously reported anti-SH2B1 antibodies preferentially recognize SH2B1β (44). Multiple forms of SH2B1 were detected in spleen, pancreas, hypothalamus, and brain (Figure 1A, lanes 1, 2, 4, and 8). In contrast, the heart, muscle, liver and adipose tissues predominantly expressed the fastest-migrating form of SH2B1 (Figure 1A, lanes 3 and 5–7).
Neuron-specific restoration of SH2B1β rescues obesity and hyperlipidemia in SH2B1KO mice. (A) Tissue extracts (2 mg protein in hypothalamic and 3 mg protein in other tissue extracts) were prepared from WT mice, immunoprecipitated with α-SH2B1, and immunoblotted with α-SH2B1. Lanes 1–8 represent tissue extracts from spleen, pancreas, heart, hypothalamus, muscle, liver, white adipose tissue, and brain, respectively. (B) Schematic representation of the Myc-tagged _SH2B1_β transgene. (C) Brain extracts were prepared from an SH2B1Tg mouse and a WT littermate, immunoprecipitated with α-SH2B1, and immunoblotted with α-Myc. (D) Brain extracts were prepared from WT, SH2B1KO, and SH2B1TgKO-437 mice and immunoblotted with α-SH2B1. Each lane represents a sample from 1 mouse. (E) Tissue extracts were prepared from the hypothalamus (Hypo), brain, skeletal muscle, liver, pancreas (Pan), white adipose tissue (WAT), brown adipose tissue (BAT), heart, and lung from an SH2B1TgKO-437 female (16 weeks old); immunoprecipitated with α-SH2B1; and immunoblotted with α-Myc. (F) Growth curves for WT, SH2B1Tg, SH2B1KO, and SH2B1TgKO-437 mice. Number in parentheses indicates the number of mice per group. (G) Levels of plasma FFAs and TGs in males (17 weeks old) fasted overnight. (H) Liver weight and TG levels in mice (21–22 weeks old) fasted overnight. *P < 0.05.
To determine the role of SH2B1 in the brain, recombinant SH2B1β was specifically expressed in neural tissue under the control of the neuron-specific enolase (NSE) promoter/rat growth hormone (GH) enhancer. A similar NSE promoter/GH enhancer has been successfully used to drive the expression of LEPRb and proopiomelanocortin (POMC) transgenes in the hypothalamus, respectively, resulting in a reduction in both adiposity and body weight (48, 49). Rat SH2B1β cDNA was inserted into a transgenic vector 3′-end of the NSE promoter/GH enhancer sequences (Figure 1B). A Myc tag was engineered at the N terminus of SH2B1β to facilitate the detection of recombinant SH2B1β expression. The SH2B1β transgenic construct was used to generate heterozygous _SH2B1_-transgenic (SH2B1Tg) mice.
Eight independent SH2B1Tg founders were obtained. To examine the expression of the SH2B1 transgene, brain extracts were prepared from WT control and SH2B1Tg mice, immunoprecipitated with α-SH2B1 and immunoblotted with α-Myc. Recombinant SH2B1β was detected in SH2B1Tg but not WT mice (Figure 1C). All 8 SH2B1Tg lines expressed similar levels of recombinant SH2B1β (data not shown).
Two independent SH2B1Tg lines (SH2B1Tg-437 and SH2B1Tg-407) were crossed with heterozygous _SH2B1_-knockout mice to generate compound mutant mice (SH2B1TgKO-437 and SH2B1TgKO-407) that were heterozygous for the _SH2B1_β-transgenic allele and homozygous for the SH2B1KO allele. The expression of recombinant SH2B1β was similar in SH2B1Tg-437 and SH2B1Tg-407 mice and restricted to neural tissues in both SH2B1Tg-437 and SH2B1Tg-407 mice (data not shown). To compare the expression levels of the SH2B1 transgene and endogenous SH2B1 gene, brain extracts were immunoblotted with α-SH2B1. Multiple forms of endogenous SH2B1 were detected in WT but not homozygous _SH2B1_-knockout (SH2B1KO) mice (Figure 1D). Recombinant SH2B1β was the only form detected in SH2B1TgKO mice and was expressed at levels similar to those of endogenous SH2B1 in WT mice (Figure 1D). However, these data cannot exclude the possibility that the NSE promoter/GH enhancer may drive an overexpression of recombinant SH2Bβ in a subpopulation of neurons that either express extremely low levels of SH2B1 or do not express SH2B1 at all. To confirm specific expression of the _SH2B1_β transgene in neural tissue, multiple tissue extracts were prepared from SH2B1TgKO mice, immunoprecipitated with α-SH2B1, and immunoblotted with α-Myc. Recombinant SH2B1β was detected in both the hypothalamus and whole brain, but not in muscle, liver, pancreas, white adipose tissue, brown adipose tissue, heart, and lung (Figure 1E).
To determine the role of neuronal SH2B1 in regulating growth and body weight, SH2BKO, SH2BTgKO, SH2BTg, and WT mice were fed standard chow, and body weight was monitored. Systemic deletion of SH2B1 resulted in a marked increase in body weight in both male and female SH2B1KO mice, and neuron-specific restoration of SH2B1 (to endogenous levels) fully rescued the obese phenotype in SH2B1TgKO-437 mice (Figure 1F). Neuron-specific restoration of SH2B1 also markedly reduced body weight in another independent line (SH2BKO: 32.3 ± 1.6 g, n = 12; SH2B1TgKO-407: 25.5 ± 1.1 g, n = 5; 10 weeks). Neuron-specific restoration of recombinant SH2B1β alone was sufficient to rescue the obese phenotype observed in SH2BKO mice, suggesting that neuronal SH2B1 is required for maintaining normal body weight and that multiple isoforms of SH2B1 in the brain have similar functions in regulating body weight.
Obesity is commonly associated with hyperlipidemia. Systemic deletion of SH2B1 markedly increased both plasma FFA and triglyceride (TG) levels in SH2B1KO mice; neuron-specific restoration of SH2B1 completely corrected hyperlipidemia in SH2B1TgKO mice (Figure 1G). SH2B1KO mice had hepatic steatosis as revealed by an enlarged liver mass and significantly increased hepatic lipid content (Figure 1H). Neuron-specific restoration of SH2B1 largely reversed hepatic steatosis in SH2B1TgKO mice (Figure 1H).
Neuronal and adipose SH2B1 exert opposite effects on adiposity. To further analyze adiposity, individual white adipose depots were weighed, and whole body fat content was measured by the dual-energy x-ray absorptiometry (DEXA) method. Systemic deletion of SH2B1 markedly increased the mass of both epididymal and inguinal fat depots in SH2B1KO mice (Figure 2A). Whole body fat content was increased by approximately 2.6-fold in male and approximately 2.5-fold in female SH2B1KO mice (Figure 2B). Neuron-specific restoration of SH2B1 (to endogenous levels) completely reversed the elevation in fat mass in SH2B1TgKO mice (Figure 2, A and B). Histological examination of epididymal fat depots revealed that systemic deletion of SH2B1 markedly increased the size of individual adipocytes in SH2B1KO mice; neuron-specific restoration of SH2B1 completely reversed adipocyte hypertrophy in SH2B1TgKO mice (Figure 2C, upper panels). These data demonstrated that neuronal SH2B1 negatively controls adiposity in animals.
Neuronal and adipose SH2B1 have opposite effects on adiposity. (A) Weight of epididymal (Epi) and inguinal fat depots (Ing) from SH2B1KO, SH2B1TgKO-437, and WT males at age 23–24 weeks. (B) Whole body fat content in SH2B1KO, SH2B1TgKO-437, and WT mice. (C) Representative H&E staining of epididymal fat depots from SH2B1KO, SH2B1TgKO-437, and WT males at age 23 weeks (upper panels) or from SH2B1TgKO-437 and WT males at age 10 weeks (lower panels). (D) 3T3-L1 preadipocytes were differentiated into adipocytes for 0, 3, 6, or 10 days. Cell extracts were immunoprecipitated with α-SH2B1 and immunoblotted with α-SH2B1 (upper panel). Cell extracts were also immunoblotted with anti–β-actin antibodies (lower panel). (E) WT and SH2B1KO MEF primary cultures were subjected to adipocyte differentiation for 10 days. Differentiated cells were stained with oil red O. *P < 0.05.
Compared with those in age-matched WT control mice, both total fat mass and the size of individual white adipocytes were significantly reduced in SH2B1TgKO mice, which lacked SH2B1 in adipose tissue (Figure 2, A–C). These results suggest that adipose SH2B1 may be involved in adipocyte growth and/or differentiation. To obtain additional evidence, we examined SH2B1 expression during 3T3-L1 adipocyte differentiation. Cell extracts were immunoprecipitated with α-SH2B1 and immunoblotted with α-SH2B1. 3T3-L1 preadipocytes expressed low levels of endogenous SH2B1. SH2B1 expression increased progressively during adipocyte differentiation, reaching maximal levels within 6 days after the induction of differentiation (Figure 2D). The effect of SH2B1 deficiency on adipocyte differentiation was examined using mouse embryonic fibroblasts (MEFs). Both WT and SH2B1KO MEF primary cultures were subjected to an in vitro adipocyte differentiation treatment and then stained with oil red O to identify differentiated adipocytes. Deletion of SH2B1 impaired the ability of MEFs to differentiate into adipocytes (Figure 2E). These results suggest that adipose SH2B1 is involved in adipogenesis in a cell-autonomous fashion. However, neuronal SH2B1 may play a dominant role over adipose SH2B1 in controlling adiposity in vivo; therefore, deletion of SH2B1 in both the brain and adipose tissue results in adipocyte hypertrophy and obesity in SH2B1KO mice.
Neuron-specific restoration of SH2B1 reverses energy imbalance in SH2B1KO mice. Systemic deletion of SH2B1 resulted in hyperphagia, markedly increasing food intake in SH2B1KO mice (Figure 3A). SH2B1KO mice also had significantly elevated energy expenditure, as revealed by significantly increased O2 consumption and CO2 production (Figure 3B). A previous report showed that energy intake still exceeds energy expenditure in this setting, resulting in obesity in SH2B1KO mice (45). Neuron-specific restoration of SH2B1 largely corrected hyperphagia and markedly reduced energy expenditure in SH2B1TgKO mice (Figure 3, A and B). These results suggest that SH2B1 in the brain controls body weight and adiposity by inhibiting both energy intake and expenditure.
Neuron-specific restoration of SH2B1 corrects energy imbalance in SH2B1KO mice. (A) Food intake in WT, SH2B1Tg, SH2B1KO, and SH2B1TgKO-437 mice at age 14–15 weeks. (B) O2 consumption and CO2 production in WT, SH2B1Tg, SH2B1KO, and SH2B1TgKO-437 mice at age 16–17 weeks. *P < 0.05.
Neuron-specific restoration of SH2B1 corrects leptin resistance and hypothalamic neuropeptide expression in SH2B1KO mice. Systemic deletion of SH2B1 dramatically increased plasma leptin levels (hyperleptinemia) in both fasted and fed SH2B1KO mice, a hallmark of leptin resistance (Figure 4A). Neuron-specific restoration of SH2B1 expression completely reversed hyperleptinemia in SH2B1TgKO mice (Figure 4A). Acute exogenous leptin treatment markedly reduced body weight and food intake in WT mice, as predicted (Figure 4B and data not shown). Systemic deletion of SH2B1 abolished these physiological responses to leptin in SH2B1KO mice, including leptin-induced reduction in body weight (Figure 4B). Neuron-specific restoration of SH2B1 fully rescued the ability of leptin to reduce body weight in SH2B1TgKO mice (Figure 4B). These data indicate that neuron-specific restoration of SH2B1 (to endogenous levels) is sufficient to rescue leptin resistance in SH2B1KO mice.
Neuron-specific restoration of SH2B1 reverses leptin resistance in SH2B1KO mice. (A) Plasma leptin levels in fasted (10 weeks old) and fed ad libitum (13 weeks old) mice. (B) Male mice (9 weeks old) were housed individually and injected intraperitoneally with leptin (2 mg/kg body weight) or PBS (control) twice a day (6:00 pm and 12:00 am). Body weight was monitored both before and after the injection. Changes in body weight were calculated as a percentage of the initial values prior to the injection. (C) Female (right panel: 6 weeks old) and male (middle panel: 12 weeks old; right panel: 9 weeks old) mice were fasted for 24 hours and injected intraperitoneally with leptin (1 mg/kg of body weight) or PBS as control. Hypothalamic extracts were prepared 45 minutes after injection and immunoblotted with α–p-STAT3 or α-STAT3. Each lane represents a combination of 2 hypothalami. (D) Hypothalamic RNA was prepared from males (22 weeks old, fasted overnight). NPY, AgRP, and POMC mRNA levels were measured using quantitative real-time PCR and normalized to the expression of β-actin. *P < 0.05.
To examine leptin signaling in the hypothalamus, mice were fasted overnight and injected intraperitoneally with leptin (1 mg/kg body weight). Hypothalamic extracts were immunoblotted with anti–phospho-STAT3 antibodies that specifically recognize phosphorylated and active STAT3. Systemic deletion of SH2B1 significantly impaired leptin-stimulated phosphorylation of STAT3 (Figure 4C). Neuron-specific restoration of SH2B1 increased leptin-stimulated phosphorylation of hypothalamic STAT3 in SH2B1TgKO mice to levels similar to those in age-matched WT controls, suggesting that neuronal SH2B1 may cell-autonomously enhance leptin signaling in the hypothalamus (Figure 4C).
Leptin inhibits the expression of orexigenic neuropeptide Y (NPY) and agouti-related protein (AgRP) and stimulates the expression of anorexigenic POMC in the arcuate nucleus of the hypothalamus (1, 3, 50). NPY and AgRP promote positive energy imbalance, whereas α–melanocyte-stimulating hormone (α-MSH), a proteolytic product of POMC, promotes negative energy imbalance (2, 3, 51). The abundance of hypothalamic NPY, AgRP, and POMC mRNA was measured using quantitative real-time PCR assays and normalized to the expression of β-actin. Systemic deletion of SH2B1 markedly increased NPY and AgRP but not POMC expression in SH2B1KO mice (Figure 4D). Neuron-specific restoration of SH2B1 dramatically reduced NPY expression in SH2B1TgKO mice to levels similar to those in WT controls (Figure 4D). AgRP expression was also significantly reduced in SH2B1TgKO mice, but to a lesser extent (Figure 4D). Neuronal SH2B1 may inhibit the expression of orexigenic NPY and AgRP presumably by enhancing leptin signaling in the hypothalamus.
Neuron-specific overexpression of SH2B1 protects against HFD-induced leptin resistance and obesity. To determine whether a modest increase in neuronal SH2B1 expression protects against leptin resistance and obesity, SH2B1Tg and WT littermates were fed an HFD. Body weight and fat content were similar in SH2B1Tg and WT littermates (Figure 5, A and B). However, blood leptin levels were significantly reduced, by 58%, in SH2B1Tg mice, suggesting an increase in leptin sensitivity in SH2B1Tg mice (Figure 5C). These results suggest that while a modest increase in neuronal SH2B1 expression mildly increases leptin sensitivity, this is insufficient to protect against HFD-induced obesity in SH2B1Tg mice.
Neuronal SH2B1 increases leptin sensitivity. (A) SH2B1Tg-437 males and WT littermates were fed an HFD at age 7 weeks, and body weight was monitored weekly. (B) Fat content of SH2B1Tg-437 males and WT littermates fed an HFD for 9 weeks. (C) Plasma leptin levels in SH2B1Tg-437 males and WT littermates fed an HFD for 10 weeks. *P < 0.05.
To increase neuronal SH2B1 expression, SH2B1Tg mice were bred to generate homozygous _SH2B1_-transgenic mice (SH2B1Tg/Tg). Two independent SH2B1Tg/Tg lines (SH2B1Tg/Tg-437 and SH2B1Tg/Tg-407) were obtained. To examine the SH2B1 transgene expression, brain extracts were immunoprecipitated with α-SH2B1 and immunoblotted with α-Myc to detect Myc-tagged recombinant SH2B1β. The expression of the _SH2B1_β transgene was significantly higher in SH2B1Tg/Tg than in SH2B1TgKO-437 and SH2B1Tg-407 mice (heterozygous for the _SH2B1_β transgene) (Figure 6A). Neuron-specific overexpression of SH2B1 did not have an obviously deleterious effect on the overall health of SH2B1Tg/Tg mice.
Neuron-specific overexpression of SH2B1 dose-dependently reduces HFD-induced leptin resistance and obesity. (A) Brain extracts were prepared from WT, SH2B1TgKO-437, SH2B1Tg/Tg-437, SH2B1Tg-407, SH2B1Tg/Tg-407, and SH2B1Tg-407/437 males, immunoprecipitated with α-SH2B1, and immunoblotted with α-Myc. Extracts were also immunoblotted with anti–β-actin. Each lane represents a sample from 1 mouse. (B) Growth curves for male mice (WT, SH2B1Tg-407, SH2B1Tg/Tg-407, and SH2B1Tg-407/437) fed normal chow or HFD as indicated. (C) Fat content in male mice (16 weeks old) fed an HFD for 9 weeks. (D) Fasting plasma leptin levels in male mice fed standard chow (WT and SH2B1Tg/Tg-437; 17 weeks old) or HFD for 7 weeks (WT, SH2B1Tg/Tg-407, SH2B1Tg/Tg-437, and SH2B1Tg-407/437; 14 weeks old). *P < 0.05.
To determine the dosage effect of neuronal SH2B1 on leptin sensitivity and adiposity, body weight and blood leptin levels were measured. Body weight markedly decreased in both SH2B1Tg/Tg-437 and SH2B1Tg/Tg-407 compared with WT control mice fed normal chow (Figure 6B, left panel). Fasting plasma leptin levels were significantly lower in SH2B1Tg/Tg than in WT control mice (Figure 6D).
Mice were fed an HFD at 7 weeks of age. HFD markedly increased both body weight and fat content in WT mice, whereas both SH2B1Tg/Tg-407 and SH2B1Tg/Tg-437 mice were resistant to HFD-induced obesity (Figure 6, B, right panel, and C). HFD induced severe hyperleptinemia (a hallmark of leptin resistance) in WT mice, increasing fasting blood leptin levels by 26-fold; in contrast, plasma leptin levels were only mildly elevated in HFD-fed SH2B1Tg/Tg mice. Neuronal overexpression of SH2B1 reduced blood leptin levels by 98% in SH2B1Tg/Tg-407 and 92% in SH2B1Tg/Tg-437 compared with WT control mice (Figure 6D, right panel). Since the 2 lines of SH2B1Tg/Tg mice were similarly protected against HFD-induced leptin resistance and obesity, overexpression of neuronal SH2B1, rather than other mutations derived from the random insertion of the SH2B1 transgene, enhances leptin sensitivity in SH2B1Tg/Tg mice.
To avoid the complete disruption of a gene by the transgenic insertion, SH2B1Tg/Tg-437 and SH2B1Tg/Tg-407 mice were crossed to generate SH2B1Tg-407/437 mice heterozygous for both SH2B1Tg-437 and SH2B1Tg-407 alleles. The expression levels of recombinant SH2B1β were similar in SH2B1Tg-407/437 and SH2B1Tg/Tg-437 or SH2B1Tg/Tg-407 mice but higher in SH2B1Tg-407/437 than in heterozygous SH2B1Tg mice (Figure 6A). Importantly, SH2B1Tg-407/437 mice were protected against HFD-induced obesity to a similar extent as were SH2B1Tg/Tg-407 and SH2B1Tg/Tg-437 mice (Figure 6, B, right panel, and C). Blood leptin levels were also reduced by 97% in SH2B1Tg-407/437 mice compared with WT mice (Figure 6D, right panel).
Neuron-specific restoration of SH2B1 reverses peripheral insulin resistance and glucose intolerance in SH2B1KO mice. SH2B1 binds directly to the insulin receptor, thereby enhancing the activation of the insulin receptor and multiple downstream pathways in cultured cells (44, 52). Systemic deletion of SH2B1 results in severe insulin resistance and type 2 diabetes (44–46). However, it is unclear whether SH2B1 enhances insulin sensitivity directly by promoting insulin signaling in the liver, skeletal muscle, and/or adipose tissue or indirectly by reducing adiposity through its action in the brain.
Insulin sensitivity and glucose metabolism were compared in SH2B1KO mice (which completely lack SH2B1 in all tissues) and SH2B1TgKO mice (which only express SH2B1 in neural tissue). SH2B1KO mice developed severe hyperglycemia and hyperinsulinemia, hallmarks of insulin resistance (Figure 7A). Fasting plasma insulin levels increased by more than 26-fold in SH2B1KO compared with age-matched WT controls. Neuron-specific restoration of SH2B1 corrected both hyperglycemia and hyperinsulinemia in SH2B1TgKO mice (Figure 7A).
Neuron-specific restoration of SH2B1 rescues insulin resistance and glucose intolerance in SH2B1KO mice. (A) Blood glucose in fasted males at age 17 weeks and plasma insulin in fasted males at age 21 weeks. GTTs (B) and ITTs (C) in males at age of 21 weeks. The area under the curve (AUC) was calculated in both GTTs and ITTs using the trapezoidal rule. *P < 0.05.
To further examine peripheral insulin sensitivity, glucose and insulin tolerance tests (GTTs and ITTs) were performed. In GTTs, mice were fasted overnight and injected intraperitoneally with d-glucose (2 g/kg body weight), and blood glucose levels were measured at various time points after glucose injection. Compared with WT controls, SH2B1KO mice were severely intolerant to exogenous glucose as revealed by a marked increase in both the magnitude and duration of the elevation of blood glucose in response to glucose injection (Figure 7B). Neuron-specific restoration of SH2B1 (to endogenous levels) fully rescued glucose intolerance in SH2B1TgKO mice (Figure 7B). In ITTs, exogenous insulin (1 U/kg body weight) failed to decrease blood glucose levels in SH2B1KO mice; in contrast, insulin-induced reductions in blood glucose levels were comparable in SH2B1TgKO and WT mice (Figure 7C). The area under the curve (AUC) significantly increased in SH2BKO mice in both GTTs and ITTs, and this was reversed by neuron-specific restoration of SH2B1 in SH2B1TgKO mice (Figure 7, B and C). Blood glucose and plasma insulin levels and glucose tolerance were similar in SH2B1Tg and WT mice (Figure 7, A and B). Therefore, neuronal SH2B1 rather than SH2B1 expressed in the liver, muscle, and adipose tissue controls peripheral insulin sensitivity and glucose metabolism under these experimental conditions.