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Molecular Endocrinology, doi:10.1210/me.2006-0224
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Molecular Endocrinology 20 (11): 2671-2681
Copyright © 2006 by The Endocrine Society

Orphan Receptor Small Heterodimer Partner Is an Important Mediator of Glucose Homeostasis

Li Wang, Jiansheng Huang, Pradip Saha1, Rohit N. Kulkarni1, Min Hu1, YongDeuk Kim1, KeunGyu Park, Lawrence Chan, Arun S. Rajan, Inkyu Lee and David D. Moore

Departments of Medicine and Pharmacology (L.W., J.H.), University of Kansas Medical Center, Kansas City, Kansas 66160; Departments of Molecular and Cellular Biology (P.S., L.C., D.D.M.) and Medicine (M.H., L.C., A.S.R.), Baylor College of Medicine, Houston, Texas 77030; Joslin Diabetes Center, Harvard Medical School (R.N.K.), Boston, Massachusetts 02215; and Department of Internal Medicine (Y.K., K.P., I.L.), Keimyung University School of Medicine, Daegu 700-712, Korea

Address all correspondence and requests for reprints to: Li Wang, Departments of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66160. E-mail: lwang2{at}kumc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The orphan receptor small heterodimer partner (SHP; NROB2) is a transcriptional repressor that inhibits nuclear receptor signaling in diverse metabolic pathways. Here, we report that SHP–/– mice exhibited hypoinsulinemia with age, which was associated with increased peripheral insulin sensitivity and increased response of isolated islets to glucose stimulation, yet maintain normal levels of blood glucose. Deficiency in SHP function resulted in up-regulation of glucose transporter 4 mRNA and glucose uptake in muscles, and overexpression of SHP in C2C12 cells inhibited both basal and peroxisomal proliferator-activated receptor {gamma} (PPAR{gamma}) coactivator-1{alpha}-stimulated glucose transporter 4 expression and glucose uptake. SHP–/– hepatocytes showed markedly decreased basal glucose production in cultures, and SHP–/– livers had increased glycogen stores and were more sensitive to insulin inhibition of glucose output, which were concomitant with decreased expression for PPAR{gamma}1, fatty acid translocase, glucose-6-phosphatase, and phosphoenol/pyruvate carboxykinase, and increased mRNAs for glucokinase and pyruvate kinase. In white fat, SHP deficiency resulted in up-regulation of genes involved in insulin sensitizing, including PPAR{gamma}2 and adiponectin. We show that, at the transcriptional level, SHP directly represses adiponectin promoter activity by PPAR{gamma}/liver receptor homolog-1. The results suggest that the increases in insulin sensitivity through multiple signaling pathways in muscle, liver, and fat, with an increase in islet secretory function, represent the complex mechanism whereby SHP deficiency leads to improvement in insulin sensitivity, secretion, and diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SMALL HETERODIMER PARTNER (SHP) is an unusual orphan nuclear receptor in that it lacks a DNA-binding domain (1). It is expressed in several tissues with relatively high expression in liver, heart, and pancreas (2). SHP interacts with a number of other nuclear receptors including estrogen receptor {alpha} (3, 4), retinoid X receptor {alpha} (RXR{alpha}), and hepatocyte nuclear factor-4{alpha} (HNF4{alpha}) (5), and especially liver receptor homolog-1 (LRH-1) (6) and estrogen receptor-related receptor {gamma} (7), and inhibits their transcriptional activity. Nuclear receptors are key regulators of metabolic pathways, and previous studies with SHP knockout mice have confirmed that SHP functions in bile acid, cholesterol, and triglyceride homeostasis (8, 9, 10, 11). In humans, loss of SHP function has been associated with mild obesity in Japanese subjects (12), but genetic variation in SHP is not commonly associated with obesity in United Kingdom Caucasians (13, 14). In contrast, we did not observe weight differences between wild-type and SHP–/– mice on a regular chow diet, but found that SHP–/– mice are resistant to high-fat diet-induced obesity, apparently as a consequence of increased peroxisomal proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) expression in brown adipose tissue (15).

Recently, the potential role of SHP in glucose-stimulated insulin secretion of pancreatic ß-cell was investigated in an in vitro system (16), in which overexpression of SHP in islets by adenovirus enhanced secretion (16). However, the molecular basis for this observation is not clear. A potential role of SHP in regulating pancreatic function was further supported by the observation that SHP represses transactivation by neurogenic differentiation factor (NeuroD) (2) and forkhead box a 2 (Foxa2) (17), which both play important roles in regulating insulin secretion by pancreatic islets (18, 19, 20). This suggests that SHP may function as a novel negative regulator in controlling islet insulin secretion. Here, for the first time, we provide direct in vivo evidence to support this notion. We report that the loss of SHP function in SHP–/– mice results in a progressive decrease in circulating insulin levels that is associated with both an increased secretory response of islets to glucose and increased peripheral insulin sensitivity. We show that the increased insulin sensitivity is associated with increased expression of glucose-responsive genes in major insulin action tissues including muscle, fat, and liver. These results suggest that SHP is involved in diverse signaling pathways controlling islet insulin secretion and peripheral insulin sensitivity and that it may function as a negative modulator of type 2 diabetes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SHP–/– Mice Show Progressively Decreased Serum Insulin and Leptin Levels
Initial studies showed that the loss of SHP had no gross effects on glucose metabolism in young SHP–/– mice (8). To examine this in more detail, glucose and insulin levels were measured in random-fed and fasted states of young and older SHP+/+ and SHP–/– mice. At 2 and 6 months of age, SHP–/– mice had normal serum glucose and insulin levels. At 12 months, serum glucose levels of SHP–/– mice still remained no different from that of SHP+/+ mice under nonfasted and overnight-fasted conditions (Fig. 1AGo). However, fasting serum insulin levels were decreased by 50% in 6-month-old SHP–/– mice, and by 60% in 12-month-old mice lacking SHP (Fig. 1BGo). The adipokine leptin inhibits insulin biosynthesis and secretion in pancreatic ß-cells (21) and insulin stimulates leptin secretion from adipose tissue. This hormonal regulatory feedback loop, the "adipo-insular axis" (22), predicts that the hypoinsulinemia observed in SHP–/– mice should also result in reduced leptin secretion. As expected, serum leptin was decreased by 50% under nonfasted and fasted SHP–/– mice at 12 month of age, consistent with lower leptin mRNA in white fat (Fig. 1CGo). The hypoinsulinemia in SHP–/– mice could be due to increased insulin clearance, primarily by the liver, and/or decreased synthesis or secretion of insulin. To assess insulin clearance, we measured the ratio of the serum levels of insulin and C-peptide, which is produced in a 1:1 molar ratio with insulin but is cleared by a different mechanism than insulin (23, 24). SHP–/– mice showed a 65% higher C-peptide:insulin ratio than the SHP+/+ controls (Fig. 1DGo), indicating an increased rate of insulin clearance. We next assessed insulin synthesis by determining pancreatic insulin content and islet preproinsulin mRNA levels. SHP–/– mice had 30% less insulin than controls in acid-ethanol extracts of pancreas (Fig. 1EGo). In addition, the insulin mRNA in isolated islets from SHP–/– mice was much lower than in those from control mice under nonstimulatory conditions (3 mM glucose) (Fig. 1FGo). Although the decrease in basal insulin mRNA correlated with the decreased pancreatic insulin in SHP–/– mice, stimulation of insulin expression by incubating the islets in the presence of 25 mM glucose increased insulin mRNA in SHP–/– islets to a level similar to that of SHP+/+ mice, resulting in a greater glucose induced fold increase of insulin mRNA (2-fold in controls vs. 8-fold in SHP–/– mice). Thus, the islets of SHP–/– mice maintain a high capacity to synthesize insulin in response to increased glucose. This suggests that the reduction in pancreatic insulin and insulin mRNA in SHP–/– mice may be secondary to increased insulin sensitivity and a decreased demand for insulin to maintain glucose balance, rather than a decreased effect of SHP on insulin gene transcription.


Figure 1
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  		Fig. 1. SHP Deficiency Improves Glucose Homeostasis

A, Serum glucose levels remained unchanged under both nonfasting and fasted conditions. Results from 12-month-old mice are shown (n = 10–15). B, Fasting serum insulin levels were approximately 60% lower in SHP–/– mice of 12 months of age. C, Serum leptin levels were reduced by approximately 60% in 12-month-old SHP–/– mice under both fasting and nonfasting conditions, consistent with a decreased leptin mRNA in white fat (fasted state). D, The increased C-peptide:insulin ratio in the SHP–/– mice reflected increased insulin clearance. E, Pancreatic insulin content was decreased in SHP–/– mice of 12 months of age, as measured in acid-ethanol extracts of pancreas. Values are mean ± SEM in picograms/milligram wet weight of pancreas (n = 10–15). F, Northern blotting of RNA isolated from 200 freshly isolated islets from control and SHP–/– mice of the same age. The blot was hybridized with a rat insulin-1 cDNA as probe. Insulin, leptin, and C-peptide were determined by RIA. Values are means ± SEM. *, P < 0.01. O/N, Overnight.

 
Morphological Analyses of Islets in SHP–/– Mice
Immunohistochemical studies of the pancreas were performed to determine morphological changes in islets. ß-Galactosidase expression directed by the ß-gal/GK-neo cassette from the SHP targeting construct was observed only in SHP–/– islets (Fig. 2AGo), demonstrating SHP expression in islets. Consistently, earlier reports also demonstrated SHP expression in pancreas by Northern blotting (7) and SHP expression in islet cells (2). In addition, analysis of pancreas sections using a cocktail of antibodies to non-ß-cell hormones showed no apparent differences in the size of islets at 2 and 6 months of age (data not shown). Assessment of ß-cell morphology was further carried out by immunofluorescence with insulin (red) and glucagon (green) antibodies specific in 12-month-old mice. Well-preserved architecture was observed with no apparent differences in the average size of most islets (Fig. 2Go, B and C). However, detailed quantitative analyses revealed that there were significant decreases in the number of islets per pancreatic section and islet area per pancreatic section (Fig. 2Go, D and E), but not in islet size (Fig. 2FGo). Because the pancreas weights were similar between the SHP–/– and SHP+/+ mice, these results suggest that the decrease in total islet number occurs due to long-term SHP deficiency, which may be associated with a decrease in total pancreatic insulin content (Fig. 1EGo).


Figure 2
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  		Fig. 2. SHP Deficiency Increases Pancreatic Islet Function

A, ß-Galactosidase staining detected SHP expression in SHP–/– islets. B and C, Immunohistologic analysis of insulin (red) and glucagon (green) in islets. SHP–/– islets (C) showed normal morphology compared with SHP+/+ islets (B) (x25 magnification for both SHP+/+ and SHP–/– islets). Representative images are shown. D–F, Quantitative analysis of islet mass. Detailed methods are described in Materials and Methods. SHP–/– mice showed decreased islet number, but not islet size. G and H, Islet perfusion. Islets of 12-month-old mice were isolated and used for in vitro perfusion in response to glucose (G) stimulation (in millimoles per liter). Insulin secretion in two successive glucose stimulation, i.e. phase I (G) and phase II (H), was significantly higher (~3-fold) in SHP–/– islets (red lines) than of SHP+/+ islets (black lines). Each time point represented an average of four groups (200 islets/group, each group from 15–20 mice). Values are means ± SEM. *, P < 0.01.

 
SHP–/– Pancreatic Islets Have Increased Insulin Secretion
Based on the results in Fig. 1FGo, we predicted that the pancreatic islets of SHP–/– mice would show an increased insulin secretory response to glucose. Thus, we examined insulin secretion in cultured islets. Size-matched pancreatic islets were isolated from 12-month-old control and SHP–/– mice, calibrated overnight, and were perfused with varying concentrations of glucose the next day. No significant differences in basal insulin secretion were observed between the two genotypes. Insulin secretion revealed significant differences in response to high glucose concentrations or potassium chloride (KCl). In the first-phase perfusion, islet insulin secretion was 3-fold higher upon 16.7 mM glucose stimulation in SHP–/– islets than in SHP+/+ islets (Fig. 2GGo). A similar elevated response was observed upon two successive restimulations with 16.7 mM glucose and KCl (Fig. 2HGo). The results suggest that there is no impaired coupling of stimulus and secretion in SHP–/– ß-cells. In contrast, the increase in glucose-induced insulin secretion in SHP–/– islets suggests that the islets of SHP–/– mice maintain a high capacity to secrete insulin, consistent with the marked up-regulation of insulin mRNA by glucose (Fig. 1FGo). On the other hand, the insulin content appeared to be similar in size-matched SHP–/– and SHP+/+ islets (data not shown), suggesting normal insulin synthesis and stores. Thus, the decreased basal insulin mRNA (Fig. 1FGo) is suggestive of a secondary effect of a decreased need for insulin that may result from increased insulin sensitivity.

SHP–/– Mice Have Increased Insulin Sensitivity
The results described above suggest that the progressive hypoinsulinemia in aged SHP–/– mice is not due to decreased islet function, but rather the result of increased insulin sensitivity. Glucose and insulin tolerance tests were first performed and we observed increased glucose tolerance and insulin sensitivity in 12-month-old SHP–/– mice (data not shown). Next, the hyperinsulinemic euglycemic clamp was used to more critically test this. Basal glucose production was comparable between the SHP+/+ and SHP–/– mice. Low-dosage insulin infusion decreased glucose production more profoundly in SHP–/– mice than in SHP+/+ mice (6.48 ± 2.65 vs. 11.54 ± 2.82 mg·kg–1·min–1, respectively; P = 0.02). The rate of glucose infusion necessary to maintain euglycemia during low dose insulin infusion was nearly twice as high in SHP–/– as in SHP+/+ mice (Fig. 3AGo), and slightly more than 2-fold higher in the high-dose clamp (Fig. 3BGo). Under basal conditions, glucose disposal rate was similar in SHP+/+ and SHP–/– vehicle-infused mice. Hyperinsulinemia increased glucose disposal in both groups, but the disposal rate was elevated in SHP–/– animals compared with SHP+/+ controls under both low-dose (Fig. 3CGo) and high-dosage insulin infusion (Fig. 3DGo). In addition, insulin-mediated whole-body glucose uptake was greater in both low- and high-dosage insulin-infused SHP–/– animals than controls (Fig. 3Go, E and F), which reached statistical significance (P = 0.01). These results demonstrate that the loss of SHP function results in an increase in peripheral insulin sensitivity.


Figure 3
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  		Fig. 3. SHP Deficiency Increases Whole-Body Insulin Sensitivity

Increased whole-body glucose infusion rate (A and B), glucose disposal rate (C and D), and glucose uptake (E and F) in SHP–/– mice compared with SHP+/+ mice. Hyperinsulinemic euglycemic clamp was performed on 12-month-old mice (n = 4). Left panel, Low dosage insulin perfusion. Right panel, High dosage insulin perfusion. Values are means ± SEM. *, P < 0.01.

 
SHP–/– Mice Show Increased Glucose Transporter 4 (Glut4) and Muscle Glucose Uptake
To determine which tissue is responsible for the increased insulin sensitivity, the expression of critical genes in insulin target tissues in 6-month-old mice were determined, in which alterations in insulin levels and gene expression become more evident. The glucose transporter Glut4 is primarily responsible for insulin-dependent glucose uptake in skeletal muscle (25), Glut4 mRNA expression was approximately 2-fold greater in fasting SHP–/– mice than in SHP+/+ mice, whereas the cytochrome c oxidase subunit II and cytochrome oxidase subunit IV were not altered by the loss of SHP (Fig. 4AGo). Consistent with the up-regulation of Glut4 mRNA, the glucose uptake in soleus muscles was also markedly increased in the same aged SHP–/– mice (Fig. 4BGo). To further determine the cell-autonomous effect of SHP on Glut4, an in vitro cell system was used with murine muscle-derived C2C12 cells, because C2C12 cells have been widely used to study glucose uptake (26), even though basal Glut4 level is relatively low in this cell line. Overexpressing PGC-1{alpha} markedly induced Glut4 mRNA, whereas overexpressing SHP decreased it by 90% (Fig. 4CGo). When PGC-1{alpha} and SHP were coexpressed, SHP mRNA was up-regulated by approximately 50%, whereas PGC-1{alpha} mRNA was down-regulated by approximately 50% (Fig. 4CGo). This suggests a dual regulatory loop, in which PGC-1{alpha} activates SHP, and SHP in turn inhibits PGC-1{alpha} expression. The more sensitive semiquantitative RT-PCR analysis further revealed direct repression of Glut4 mRNA by SHP (Fig. 4CGo). Consistently, the basal and insulin-stimulated glucose uptake in C2C12 cells was significantly increased by PGC-1{alpha}, which was further inhibited by SHP (Fig. 4DGo). These results demonstrate that SHP represses basal and PGC-1{alpha}-stimulated glucose uptake in muscle by inhibiting Glut4. However, it does not rule out the possibility for SHP repression of basal glucose uptake through other glucose transporter.


Figure 4
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  		Fig. 4. SHP Deficiency Increases Glut4 and Muscle Glucose Uptake

A, Glut4 mRNA was up-regulated about 2-fold in SHP–/– muscles compared with SHP+/+ mice (n = 5–7; 6 months of age). Similar results were obtained in 12-month-old mice. B, SHP deficiency increased glucose uptake in soleus muscles (n = 5; 12 months of age). C, Overexpression of SHP inhibited basal and PGC-1{alpha} stimulated Glut4 mRNA in C2C12 myotubes. Top, Northern blot analysis of gene expression. Bottom, Semiquantitative RT-PCR analysis of Glut4 mRNA. D, Overexpression of SHP inhibited basal, PGC-1{alpha}, and insulin (INS)-stimulated glucose uptake in C2C12. Values are means ± SEM. *, P < 0.01. CON, Control; COX II, cytochrome oxidase subunit II; COX IV, cytochrome oxidase subunit IV; G, GFP; P, PGC-1{alpha}; S, SHP; UCP-2, uncoupling protein 2.

 
SHP–/– Mice Show Decreased Hepatic Gluconeogenesis
Hepatic expression of peroxisomal proliferator-activated receptor {gamma}1 (PPAR{gamma}1), which is elevated in fatty liver (27) and in livers of obese ob/ob and db/db mice (28), was completely diminished in SHP–/– mice (Fig. 5AGo). This was evident with no basal expression in 2-month-old livers, and was more striking in 6- and 12-month-old mice and high-sucrose (HS)-fed mice (15). Consistent with this, the fatty acid translocase CD36, a PPAR{gamma} target, was also decreased in SHP–/– livers (Fig. 5AGo). These results are consistent with recent studies in which overexpressing the human SHP gene markedly increased PPAR{gamma} and CD36 mRNAs in mouse liver (29). Consistent with the role of insulin in inhibiting hepatic gluconeogenesis and stimulating glycogen synthesis, mRNAs for the gluconeogenic enzymes glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) were decreased, whereas mRNAs of glycolytic enzymes glucokinase (GK) and pyruvate kinase (PK) were increased (Fig. 5BGo), concomitant with increased total glycogen stores in SHP–/– livers of mice fed chow diet, as assayed by both hepatic periodic acid-Schiff (PAS) staining and quantitative analysis of liver glycogen content (Fig. 5CGo).


Figure 5
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  		Fig. 5. SHP Deficiency Increases Hepatic Sensitivity to Insulin in Glucose Production

A, Liver mRNAs for PPAR{gamma}1 and CD36 were diminished in SHP–/– mice, but markedly increased in SHP+/+ mice with age and HS diet. Both the SHP+/+ and SHP–/– mice of 3 months of age were fed HS diet for 3 months. B, Liver mRNAs for G6P and PEPCK were decreased, whereas for GK, mRNA increased in SHP–/– mice (n = 5–7; 6 months of age). Similar results were obtained in 12-month-old mice. C, PAS histochemistry and quantification for glycogen in liver. High levels of glycogen were observed in SHP–/– livers with chow diet (n = 5; 6 months of age). Similar results were obtained in 12-month-old mice. D, Glucose output from livers of SHP–/– or SHP+/+ mice of the same age. Livers were perfused with buffer containing high (H; 20 ng ml–1) or low (L; 0.5 ng ml–1) concentrations of insulin. Low dosage insulin efficiently inhibited glucose output in SHP–/– mice. E, Basal glucose production from SHP–/– hepatocytes was markedly lower than controls. All values are means ± SEM (n =3). *, P < 0.01.

 
The physiological relevance of SHP regulation of glucose production was examined in vivo in mice by liver perfusion (30). At high insulin concentrations (20 ng ml–1), the glucose output was similar in SHP+/+ and SHP–/– mice (Fig. 5DGo). At low dose of insulin (0.5 ng ml–1), hepatic glucose production was significantly increased in SHP+/+ mice, but not in SHP–/– mice. The effect of SHP on gluconeogenesis was further examined in cultured hepatocytes. Basal glucose production was markedly lower in SHP-deficient cells (Fig. 5EGo). The addition of insulin significantly decreased glucose output in SHP+/+ cells to a level comparable to SHP–/– cells but did not further reduce glucose production in SHP–/– cells, suggesting a maximal effect of the loss of SHP. Taken together, the results suggest increased sensitivity of SHP–/– livers to insulin inhibition of gluconeogenesis.

SHP–/– Mice Show Increased Expression of Insulin-Sensitizing Genes in White Adipocyte
PPAR{gamma}2 plays a crucial role in adipogenesis and insulin sensitization in adipose tissue (31). In striking contrast to the elimination of PPAR{gamma}1 expression in liver (Fig. 5AGo), a robust increase in PPAR{gamma}2 expression was observed in SHP–/– white fat (Fig. 6AGo), as were lipoprotein lipase (LPL) (32), long-chain acyl-CoA dehydrogenase (LCAD) (33), hormone-sensitive lipase (HSL), perilipin (34), and stearoyl-CoA desaturase-1 (SCD-1) (35). Surprisingly, CD36 was not altered in SHP–/– white fat (Fig. 6AGo), in contrast to its decreased expression in liver (Fig. 5AGo). No changes for resistin and aP2 (data not shown) mRNA were observed.


Figure 6
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  		Fig. 6. SHP Deficiency Increases Insulin-Sensitizing Gene Expression in White Fat

A, mRNAs for PPAR{gamma}2, LPL, LCAD, HSL, perilipin, and SCD-1 in white fat were increased in SHP–/– mice (6 months, n = 5–7). Similar results were obtained in 12-month-old mice. B, SHP inhibited ADP promoter transactivation by PPAR{gamma}/RXR{alpha}/LRH-1 in transient transfection, and SHP was recruited to the ADP promoter as examined by ChIP assay. P, PPAR{gamma}; L, LRH-1; S, SHP. Similar results were obtained in both NIH-3T3 and HeLa cells in transfection assays, and representative results from HeLa cells were shown. ChIP assay was performed in 3T3-L1 cells. Values are means ± SEM (n = 3). **, P < 0.001. C, Both ADP mRNA (top) and plasma protein (bottom) levels were increased in SHP–/– mice with age. Data from pooled (6 month; n = 3) or individual (12 month; n = 3/genotype) animals are shown.

 
Adiponectin (ADP) is a fat-derived hormone with antidiabetic properties, which improves insulin sensitivity and glucose use and is also a PPAR{gamma} target (36). The potential interaction of SHP with PPAR{gamma} and LRH-1 on ADP promoter activity was tested by transient transfection (Fig. 6BGo). Coexpression of PPAR{gamma}/RXR{alpha} increased ADP promoter activity (lane 2), which was significantly augmented by cotransfection of LRH-1 (lane 3). Coexpression of SHP dose-dependently repressed the activation of the ADP promoter by PPAR{gamma}/RXR{alpha}/LRH-1 (lanes 4–6). Moreover, the inhibitory effect of SHP on PPAR{gamma}/RXR{alpha} transactivation of the ADP promoter by rosiglitazone (Rosi) was more profound in the presence of LRH-1 (lanes 4–6 vs. 7). These results demonstrate that SHP directly represses the ADP promoter activity by PPAR{gamma}/RXR{alpha}/LRH-1. We further performed chromatin immunoprecipitation (ChIP) assay with specific SHP antibody in 3T3-L1 cells and SHP was recruited to the ADP promoter (Fig. 6BGo). To determine the in vivo physiological relevance for SHP regulation, ADP mRNA in white fat was examined and we observed only marginal elevation of ADP in SHP–/– 6-month-old mice, but a more clear elevation at 12 months of age (Fig. 6CGo). Similar results were observed when serum levels of ADP were measured by Western blots. This result was somewhat unexpected based on the marked increase of PPAR{gamma} in SHP–/– fat of 6-month-old mice (Fig. 6AGo) and suggests that factors other than PPAR{gamma} may play a more dominant role on ADP expression in young SHP–/– mice. Nevertheless, the increased ADP mRNA in 12-month-old SHP–/– mice may at least in part account for the increased insulin sensitivity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SHP inhibits the function of a variety of nuclear receptors that function as metabolic regulators (37). Previous studies of SHP–/– mice have identified functions for SHP in the negative regulation of bile acid production, lipogenesis, and energy production (8, 9, 10, 11, 15). Recent results using SHP overexpression in liver suggest an important function in control of triglyceride metabolism (29). The results described here demonstrate that the loss of SHP function increases both insulin sensitivity in older SHP–/– mice and insulin secretion in isolated perfused islets, and provide in vivo evidence for a role of SHP in regulating whole-body glucose homeostasis (Fig. 7Go).


Figure 7
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  		Fig. 7. Coordinate Regulation of Glucose Homeostasis by SHP in Pancreas, Muscle, Liver, and Fat

Activation of SHP decreases pancreatic islet insulin secretion, likely by inhibiting Foxa2, NeuroD, and HNF4{alpha}; decreases muscle glucose uptake by inhibiting Glut4 and PGC1{alpha}; increases hepatic lipid content by activating PPAR{gamma}1 and CD36, and increases gluconeogenesis and decreases glycogen storage by inhibiting GK; and decreases fat insulin sensitivity by inhibiting PPAR{gamma}2, LPL, LCAD, HSL, perilipin, SCD-1, and ADP. Loss of SHP function may coordinately increase islet insulin secretion and insulin sensitivity in muscle, liver, and fat.

 
The increased insulin sensitivity of the older SHP–/– mice is supported by the combination of decreased circulation insulin levels while still maintaining normal glucose levels, and was confirmed by the "clamp" results. The islets of aged SHP–/– mice appeared generally normal in size and morphology, indicating that the hypoinsulinemia is not due to decreased islet function. Studies with isolated islets showed a marked increase in glucose stimulated insulin secretion, demonstrating that the hypoinsulinemia is not due to impaired coupling of stimulus and secretion in SHP–/– ß-cells. The isolated islets show lower basal levels of insulin mRNA, and this reduction of insulin mRNA is unlikely due to the direct loss of SHP regulatory functions, because SHP was shown to be a negative regulator of insulin gene transcription (1). In contrast, it may be in part due to increased insulin sensitivity and the subsequent decreased need for insulin to maintain normal glucose levels in SHP–/– mice. In fact, glucose administration results in a more profound induction of insulin gene transcription in the SHP–/– islets. The existence of lower serum insulin levels in the face of normal blood glucose concentrations, and an increased capacity for insulin clearance, demonstrates that SHP–/– mice have a greatly improved ß-cell function. The lower pancreas insulin content could also be contributed in part by decreased islet numbers in SHP–/– mice, although the cause for this is currently unknown. The pattern of normal glucose levels in the hypoinsulinemia state also reflects the preservation of insulin-dependent glucose uptake in muscle in SHP–/– mice.

The results with isolated SHP–/– islets is in contrast with a recent report in which overexpression of SHP by adenovirus transduction enhanced glucose-stimulated insulin secretion in INS-1 cells, especially those overexpressing the mitochondrial uncoupling protein UCP-2 (16). It should be noted that the acute overexpression of SHP in a cultured cell line is very different from the lifelong loss of function in the whole animal, and it is not clear that these contrasting results are in conflict. Several possible mechanisms may explain why increased insulin secretion was observed after loss of SHP function in mice. SHP was shown to repress the transactivation of several important regulators in islet insulin secretion, including HNF4{alpha} (5), NeuroD (2), and Foxa2 (17). The loss of SHP function should lead to their increased activity, resulting in increased insulin secretion. In addition, our recent study demonstrates that SHP acts as a corepressor for BETA2 by competing with coactivator p300 for binding to BETA2 on insulin promoter (Park, K. G., K. M. Lee, D. D. Moore, L. Wang, K. C. Won, J. Y. Park, K. U. Lee, H. S. Choi, I. K. Lee, submitted for publication). Collectively, the results reported indicate that multiple regulatory pathways exist for controlling pancreatic islet insulin secretion, and that SHP can play a major role in regulating these pathways by altering transcription of critical genes. It would be of great interest to explore these possible regulatory mechanisms in future in vivo studies.

Our gene expression studies identified several critical genes in skeletal muscle, liver, and fat that may contribute to the increased insulin sensitivity seen in SHP–/– mice (Fig. 7Go). Increased skeletal muscle mRNA for Glut4 contributes to the increased muscle glucose uptake in SHP–/– mice, because SHP represses Glut4 and its associated glucose uptake. The activation of Glut4 expression and glucose uptake by PGC-1{alpha}, and a potential dual regulatory loop between PGC-1{alpha} and SHP, represent a new hypothesis regarding mechanisms for SHP regulation of muscle insulin sensitivity. Detailed transcriptional analysis will be required to characterize these regulatory mechanisms. Another striking observation is the diminished hepatic PPAR{gamma}1 in SHP–/– mice, which provides an explanation for the lower lipid content in SHP–/– livers (8, 15) that will result in improved hepatic insulin sensitivity (28). This result is in agreement with the up-regulation of PPAR{gamma}1 in SHP-overexpressed liver (29). On the other hand, the strong up-regulation of GK and down-regulation of G6P and PEPCK is consistent with the decreased gluconeogenesis in SHP–/– livers and in cultured hepatocytes. However, this result is in contrast to the reported inhibitory role of SHP on G6P and PEPCK by bile acids (38). Thus, decreased expression of G6P and PEPCK in SHP–/– livers may occur as a secondary effect of other metabolic changes.

Similarly, increased white adipose tissue expression of PPAR{gamma}2 is consistent with increased insulin sensitivity, because adipose-specific PPAR{gamma}–/– mice exhibit insulin resistance in fat (39). The induction of both LPL/SCD-1 and HSL/perilipin in adipose tissue of SHP–/– mice is in agreement with the role of PPAR{gamma} in promoting both lipid uptake (LPL) and triglyceride synthesis (SCD-1), as well as lipolysis (HSL, perilipin), and increases in expression of these genes are associated with increased insulin sensitivity (34, 35, 40). Surprisingly, no increase in CD36 mRNA was observed, indicating fatty acid uptake may not be increased in SHP–/– fat, because we do not know whether fatty acid transporter protein is up-regulated to increase fatty acid uptake (34). No increase in ADP expression in young SHP–/– mice is unexpected and suggest that ADP expression in young SHP–/– mice is regulated by factors other than PPAR{gamma}. In addition, no changes of ADP receptors (26) are observed in both liver and muscle of SHP–/– mice (data not shown), suggesting that the increased insulin sensitivity is not due to a secondary effect through ADP receptors. The marked improvement in hepatic function and obese phenotype in SHP–/– mice becomes more evident when the mice are older or challenged with an excess fat load (8, 15), which may explain the lack of apparent glucose or insulin metabolism phenotypes in 2-month-old SHP–/– mice. We anticipate that a more pronounced phenotype could be obtained if the mice are subjected to an appropriate stress, such as streptozotocin, to induce diabetes. It is also possible that chronic and progressive alterations in multiple overlapping systems contribute to the emergence of the improved insulin sensitivity in SHP–/– mice. Moreover, age-dependent changes in glucose homeostasis are also seen in other mouse models (41), and in SH-2B–/– mice (42) and Pten–/– mice (43); thus, it is not surprising to see them in SHP–/– mice. Lastly, the striking differential expression pattern for PPAR{gamma}2 in white fat and PPAR{gamma}1 in liver of SHP–/– mice is extremely intriguing, which suggests a differential regulation by SHP in cell- and tissue context-dependent manner. Future studies will be required to provide more mechanistic insights into SHP regulation.

In conclusion, our results identify a multifunctional and apparent inhibitory role of SHP in pancreas, muscle, liver, and fat in the regulation of glucose homeostasis and whole-body insulin sensitivity. These are complex processes, but the transcriptional regulatory consequences of the loss of SHP provides a firm ground for detailed studies regarding the mechanisms for changes in insulin sensitivity. By using tissue-specific loss or gain of SHP mouse models, we should be able to elucidate tissue-specific functions of SHP in glucose homeostasis and their impact on the dysregulation of these processes in type 2 diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
SHP-deficient mice were generated by gene targeting as previously described (8), and were initially maintained with a C57BL/129sv hybrid background, and then backcrossed with C57BL mice to the 10th generation. Age-matched groups of 12-month-old SHP+/+ and SHP–/– male mice were used in the experiments, unless otherwise indicated. They were fed a standard rodent chow and water ad libitum. All protocols for animal use were approved by the Animal Care Committee at University of Kansas Medical Center and Baylor College of Medicine.

Serum Chemistry
Blood glucose concentration was determined with a Hemocue blood glucose analyzer. Serum glucose was determined using enzymatic assay (Sigma-Aldrich, St. Louis, MO). Serum glucagon, leptin, and C-peptide were determined by RIA using each RIA kit from Linco Research (St. Charles, MO). Serum insulin was measured using ELISA kit (Crystal Chem, Chicago, IL) or RIA (Linco). Pancreas insulin content was measured by RIA after acid/ethanol extraction from homogenized whole pancreas that had been dissected, removed from the animal, and weighed.

In Vivo Glucose Kinetics
Hyperinsulinemic euglycemic clamp study was performed using chronically cannulated, conscious mice as described (44). All groups of mice were fasted 6–8 h before the experimentation. Body weight, hematocrit, and general appearance were used as indices of health. A variable infusion of 50% glucose was used to raise blood glucose levels to approximately 300 mg/dl. A 4-µCi bolus of tracer ([3-3H]glucose; NEN Life Science Products, Boston, MA) was given at –100 min, followed by a constant 0.04 µCi/min for the duration of the study. The glucose turnover rate (in milligrams/kilogram·minute) was calculated as the rate of tracer infusion (in disintegrations per minute/minute) divided by the blood glucose specific activity (in disintegrations per minute/milligram) corrected to the body weight of the mouse. Glycogen synthesis via the direct pathway was determined by measuring incorporation of 3H into glycogen (in disintegrations per minute/gram of liver)/3H specific activity in plasma glucose (in disintegrations per minute/milligram).

Islet Isolation, Culture, and Insulin Secretion
The isolation of islets was described elsewhere (45). Briefly, pancreatic islets were isolated by collagenase P digestion (Roche, Basel, Switzerland), hand-picked, and transferred to DMEM supplemented with 10% FBS, glucose, glutamine, sodium pyruvate, and antibiotics. Freshly isolated islets were cultured overnight at 37 C, size-matched, divided into groups, and used for perfusion experiments (200 islets). In perfusion experiments, Krebs-Ringer bicarbonate buffer was pumped at a rate of 1 ml/min around islets that were loaded into temperature- and CO2-controlled 300-µl plastic chambers. Islets were washed under basal conditions for 1 h before the experiment. The amount of insulin released into the medium was determined by ELISA. For islet insulin content assay, isolated islets were suspended in 100 µl of acid ethanol, and cellular insulin was extracted and assayed by RIA.

Islet Staining
Generation of the SHP–/– mice was described elsewhere (8). In brief, the first exon of the SHP gene was disrupted by homologous recombination with a LacZ-neo cassette. This insertion provides an endogenous marker of SHP expression by quantification of LacZ expression. Because LacZ is not expressed in tissues of SHP+/+ mice, the background signal is low and therefore is very sensitive to defining SHP expression. Pancreas harvested from animals were infused with paraformaldehyde, isolated, and frozen. The tissue was cryosectioned (5-µm sections were acquired). Pancreatic sections were stained for ß-galactosidase activity using standard techniques. For islet insulin and glucagon staining, immunohistochemistry was performed following standard procedures as described (46). Briefly, pancreas was immersion-fixed in 4.0% (wt/vol) paraformaldehyde, 0.1 M sodium phosphate buffer at 4 C overnight. Diluted guinea pig antiporcine insulin (A564; DakoCytomation, Carpinteria, CA) (1:200), or rabbit antiporcine glucagon (A565; DakoCytomation) (1:200), or rabbit antihuman somatostatin (A566; DakoCytomation) (1:200) was applied to the sections for 45 min at room temperature. The sections were then rinsed with Tris-buffered saline, and then treated with a second antibody followed by visualization using a confocal microscope.

Point Count for Quantification of Islet
The relative area of islet in the pancreas was counted by the point counting method using a microscope (Carl Zeiss, Oberkochen, Germany) connected to a personal computer monitor with a 90-point transparent overlay. In brief, immunostained pancreatic sections were visualized under x200 magnification and positioned a regular lattice overlay on personal computer monitor. The islets were counted simultaneously in insulin-stained slides. Average of 104 fields and 14,210 points in nonoverlapping fields was counted from each tissue; three sections were counted in each tissue block. The number of islets per each field was recorded and the area of each islet was measured with NIH Image analysis program equipped with WACOM Intuos Tablet System (Wacom, Saitama, Japan). Relative area of islet in pancreatic tissue was represented as follows: number of points corresponding to insulin antibody-stained area/number of points corresponding to remaining pancreatic area (pancreatic area). Average size of islet and islet appearance rate were represented as follows: summation of total area of islet/islet number and total number of islet/pancreatic area, respectively.

Adenoviral Transduction
Recombinant adenoviruses for expression of PGC-1{alpha}, SHP, and the GFP control were described (15). Cells were plated at 2 x 106 per 10-cm dish, cultured to 70–80% confluence, and infected the next day with viral supernatant at different multiplicities of infection for 2 h. Virus-containing media were removed and cells were continuously cultured for 2 d. Gene expression was analyzed by Northern blotting.

Glucose Uptake Assay
C2C12 mouse muscle myoblasts (American Type Culture Collection, Manassas, VA) were grown in six-well plates to confluence in DMEM with 10% FBS and antibiotics. Cells were differentiated for 1 d in DMEM with 2% horse serum, and then were infected with adenoviruses for GFP, PGC-1, and/or SHP for 2 h. The cells were continuously differentiated for 2 d to myotubes. For the last 5 h on the fourth day, the cells were serum-starved in serum-free DMEM with 25 mM glucose and 0.2% BSA, and insulin was added at 100 nM for the last 20 min. Cells were then rinsed twice in Krebs-Ringer-phosphate (KRPH) buffer, and glucose uptake was assessed with 100 µM 2-deoxy-D-glucose (1 µCi/ml [1,2-3H]-2-deoxy-D-glucose) with or without 10 µM cytochalasin B in KRPH for 10 min at room temperature. Uptake was terminated by adding cold stop medium (50 mM [SCAP];d-glucose in KRPH). Cells were then washed, and radioactivity associated with the cells was determined by cell lysis in NaOH/sodium dodecyl sulfate, followed by scintillation counting. Aliquots of cell lysates were used for protein content determination. 2-Deoxy-D-[1,2-3H]glucose (2-DG) uptake was expressed as picomoles per minute per milligram of protein. For insulin-stimulated glucose uptake in muscles, soleus muscles were isolated and incubated for 30 min in Krebs-Henseleit buffer, with or without insulin (100 nM). The muscles were then rinsed for 10 min at 29 C and incubated for 20 min at 29 C in Krebs-Henseleit buffer containing 8 mM 2-DG (2.25 µCi/ml) and 32 mM [14C]mannitol (0.3 µCi/ml). After incubation, the muscles were rapidly solubilized. Radioactivity in the resultant samples was counted, and 2-DG uptake rates were corrected for extracellular trapping with mannitol counts. The protein concentration was determined by the Bradford method.

Liver Glycogen Staining and Content
Briefly, livers were fixed in 4% formaldehyde, dehydrated, embedded in paraffin, and sectioned (9). Sections were stained with PAS to examine glycogen content in liver. For determination of glycogen, 50-mg fragments of liver were homogenized in KOH and incubated at 97 C for 15 min. The glycogen was precipitated by Na2SO4 and EtOH at –20 C for 16 h. After centrifugation, the pellet was collected and washed with KOH and EtOH and hydrolyzed in H2SO4 at 100 C for 1 h. The solution was neutralized, and glucose was determined by the glucose oxidase method.

Hepatocyte Culture and Glucose Production
Hepatocytes were isolated (8) with cell viability assessed by the trypan blue exclusion at greater than 80%. Cells were serum-starved overnight and stimulated with insulin (100 nM) for 24 h. Cells were washed with DMEM and incubated with glucose production medium (without glucose) containing 20 mM lactate and 2 mM pyruvate for 3 h. Culture medium was collected and centrifuged, and glucose levels were determined (Sigma-Aldrich), normalized to protein content of the cells.

To measure glucose production in vivo, the liver perfusion was performed on starved SHP+/+ and SHP–/– mice (30). After anesthesia with pentobarbitone sodium (60 mg/kg of body weight, ip), the portal vein and the inferior vena cava were cannulated. The liver was perfused with oxygenated Krebs-Henseleit buffer with varying amounts of insulin at 37 C in a single-pass mode with a total flow rate of 1.5–2 ml min–1. The outflow was collected, and glucose concentrations were measured. Glucose output was calculated by subtracting the amount of glucose contained in the perfusion buffer from that measured in the outflow.

Transient Transfection and ChIP Assay
NIH 3T3 cells and HeLa cells were maintained in DMEM with 10% FBS. For luciferase assays, cells were plated in 24-well plates 1 d before transfection and transfections were carried out using calcium phosphate (15). ADP promoter Luc was cotransfected with PPAR{gamma} (P; 25 ng), LRH-1 (L; 100 ng), and/or SHP (S; 100, 200, 300 ng) expression vectors, respectively. Forty-eight hours posttransfection, cells were harvested, and luciferase activity was measured and normalized against ß-galactosidase activity as an internal control with a Dual-Luciferase Reporter System (Promega, Madison, WI). The transfection experiments were carried out independently three times with similar efficiency. For ChIP assay, 3T3-L1 cells were used and chromatin cross-linked, immunoprecipitated with anti-SHP antibodies (29), with IgG as negative control.

Northern and Western Blotting
Northern blotting and semiquantitative PCR analysis were performed as previously described (9). The primer sequences of each gene were from the PubMed GenBank database and were designed and used to synthesize various probes for Northern blot analysis. Primer sequences are available upon request. Western blotting was followed by standard procedures.

Statistical Analysis
Data are expressed as mean ± SD. Statistical analyses were carried out using Student’s unpaired t test; P < 0.01 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Dr. Curt Hagedorn for critical reading of the manuscript. We thank Dr. Iannis Talianidi for the SHP antibody.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant P20 RR016475 from the IDeA Networks of Biomedical Research Excellence Program of National Center for Research Resources and funds from Kansas Masonic Cancer Research Institute (to L.W.), R01 DK53366 and P01 DK57743 (to D.D.M.), R01 DK67536 (to R.N.K.), and R01 HL51586 and DK68037 (to L.C.). L.W. is also supported by the Liver Scholar Award from American Liver Foundation/American Association for the Study of Liver Diseases, and Junior Faculty Award from American Diabetes Association.

L.W., J.H., P.S., R.N.K., M.H., Y.K., K.P., L.C., A.S.R., I.L., D.D.M. have nothing to declare.

First Published Online June 27, 2006

1 P.S., R.N.K., M.H., and Y.K. contributed equally to this work. Back

Abbreviations: ADP, Adiponectin; CD36, fatty acid translocase; ChIP, chromatin immunoprecipitation; 2-DG, 2-deoxy-D-[1,2-3H]glucose; Foxa2, forkhead box a 2; GK, glucokinase; Glut4, glucose transporter 4; G6P, glucose-6-phosphatase; HNF4{alpha}, hepatocyte nuclear factor-4{alpha}; HS, high sucrose; HSL, hormone-sensitive lipase; KRPH, Krebs-Ringer-phosphate; LCAD, long-chain acyl-CoA dehydrogenase; LPL, lipoprotein lipase; LRH-1, liver receptor homolog-1; NeuroD, neurogenic differentiation factor; PAS, periodic acid-Schiff; PEPCK, phosphoenol/pyruvate carboxykinase; PGC-1{alpha}, peroxisomal proliferator-activated receptor {gamma} coactivator-1{alpha}; PPAR{gamma}, peroxisomal proliferator-activated receptor {gamma}; RXR{alpha}, retinoid X receptor {alpha}; SCD-1, stearoyl-CoA desaturase-1; SHP, small heterodimer partner.

Received for publication May 24, 2006. Accepted for publication June 23, 2006.


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