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Am J Physiol Endocrinol Metab 288: E1074-E1081, 2005. First published January 18, 2005; doi:10.1152/ajpendo.00396.2004
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Defective insulin signaling in skeletal muscle of the hypertensive TG(mREN2)27 rat

Julie A. Sloniger, Vitoon Saengsirisuwan, Cody J. Diehl, Betsy B. Dokken, Narissara Lailerd, Andrew M. Lemieux, John S. Kim, and Erik J. Henriksen

Muscle Metabolism Laboratory, Department of Physiology, University of Arizona College of Medicine, Tucson, Arizona

Submitted 25 August 2004 ; accepted in final form 11 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Essential hypertension is frequently associated with insulin resistance of skeletal muscle glucose transport, with a potential role of angiotensin II in the pathogenesis of both conditions. The male heterozygous TG(mREN2)27 rat harbors the mouse transgene for renin, exhibits local elevations in angiotensin II, and is an excellent model of both hypertension and insulin resistance. The present study was designed to investigate the potential cellular mechanisms for insulin resistance in this hypertensive animal model, including an assessment of elements of the insulin-signaling pathway. Compared with nontransgenic, normotensive Sprague-Dawley control rats, male heterozygous TG(mREN2)27 rats displayed elevated (P < 0.05) fasting plasma insulin (74%), an exaggerated insulin response (108%) during an oral glucose tolerance test, and reduced whole body insulin sensitivity. TG(mREN2)27 rats also exhibited decreased insulin-mediated glucose transport and glycogen synthase activation in both the type IIb epitrochlearis (30 and 46%) and type I soleus (22 and 64%) muscles. Importantly, there were significant reductions (~30–50%) in insulin stimulation of tyrosine phosphorylation of the insulin receptor {beta}-subunit and insulin receptor substrate-1 (IRS-1), IRS-1 associated with the p85 subunit of phosphatidylinositol 3-kinase, Akt Ser473 phosphorylation, and Ser9 phosphorylation of glycogen synthase kinase-3{beta} in epitrochlearis and soleus muscles of TG(mREN2)27 rats. Soleus muscle triglyceride concentration was 25% greater in the transgenic group compared with nontransgenic animals. Collectively, these data provide the first evidence that the insulin resistance of the hypertensive male heterozygous TG(mREN2)27 rat can be attributed to specific defects in the insulin-signaling pathway in skeletal muscle.

insulin resistance; glucose transport; glycogen synthase; hypertension

THE "INSULIN RESISTANCE SYNDROME" is a condition characterized by insulin resistance of skeletal muscle glucose metabolism and is accompanied by additional metabolic and cardiovascular abnormalities, including essential hypertension, glucose intolerance, hyperinsulinemia, dyslipidemia, and central obesity (6). Increased cardiovascular mortality associated with this condition has been directly attributed to insulin resistance and compensatory hyperinsulinemia (8, 28). The relation between hyperinsulinemia and hypertension is supported by the observation that hyperinsulinemia can increase sympathetic nervous system activity (21, 28) and may result in proliferation of vascular smooth muscle (13).

The TG(mREN2)27 rat provides an excellent genetic model of both hypertension and insulin resistance. The TG(mREN2)27 rat is a transgenic animal that harbors the mouse Ren-2 renin gene (25) and develops severe fulminant hypertension, left ventricular hypertrophy, and cardiac failure with a clearly defined monogenetic origin (22–24, 31). Increases in the local renin-angiotensin system, leading to elevated tissue angiotensin II levels (2, 24), likely underlie these cardiovascular defects.

The male heterozygous TG(mREN2)27 rat is also characterized by insulin resistance at the whole body and skeletal muscle levels (18, 19). The TG(mREN2)27 rat displays an exaggerated insulin response during an oral glucose tolerance test and exhibits a significant decrease in whole body insulin sensitivity during this glucose challenge (18, 19). Moreover, insulin-mediated glucose transport activity is significantly decreased in isolated epitrochlearis and soleus muscles of male TG(mREN2)27 rats compared with that in nontransgenic, normotensive Sprague-Dawley controls (19).

The potential cellular mechanisms underlying the insulin-resistant state in skeletal muscle of the male heterozygous TG(mREN2)27 rat have not been determined in previous studies. Therefore, the purpose of the present investigation was to assess whether defects in the protein expression and functionality of specific elements of insulin signaling, including the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, and glycogen synthase kinase-3 (GSK-3), are associated with the insulin resistance of glucose transport in skeletal muscle of the male heterozygous TG(mREN2)27 rat. In addition, a secondary aim of this study was to determine whether activation of glycogen synthase activity by insulin is also defective in skeletal muscle of these hypertensive and insulin-resistant animals.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and treatments. Male heterozygous TG(mREN2)27 rats and male Hannover Sprague-Dawley normotensive control rats were purchased from the Hypertension and Vascular Disease Center of Bowman Gray School of Medicine at Wake Forest University (Winston-Salem, NC) at ~5–6 wk of age. The TG(mREN2)27 rats were derived from Hannover Sprague-Dawley rats housed in the breeding colony at this institute. All animals were housed in a temperature-controlled room (20–22°C) with a 12:12-h light-dark cycle (lights on from 7:00 AM to 7:00 PM) at the Central Animal Facility of the University of Arizona. The animals had free access to chow (Purina, St. Louis, MO) and water, and all of the procedures were approved by the University of Arizona Animal Care and Use Committee.

Animals were food restricted (4 g of chow given at 5:00 PM the previous evening) and at 8:00 AM underwent an oral glucose tolerance test (OGTT) using a 1 g/kg body wt glucose feeding by gavage. Blood (0.25 ml) was collected from a small cut at the tip of the tail immediately before and at 15, 30, 60, and 120 min after the glucose feeding. Whole blood was mixed thoroughly with EDTA (18 mM final concentration) and centrifuged at 13,000 g to isolate the plasma. The plasma was stored at –80°C and subsequently assayed for glucose (Sigma Chemical, St. Louis, MO), insulin (Linco Research, St. Charles, MO), and free fatty acids (FFA; Wako Chemicals, Richmond, VA). Immediately after completion of the OGTT, all animals received a 2.5-ml subcutaneous injection of 0.9% sterile saline to compensate for plasma loss.

Assessment of muscle glucose transport activity. Seventy-two hours after the OGTT, the animals were again food restricted as described above. At 8:00 AM, the animals were weighed and deeply anesthetized with pentobarbital sodium (50 mg/kg body wt ip). Soleus and epitrochlearis muscles were dissected and prepared for in vitro incubation. In one set of animals, the total amount of abdominal fat was assessed. Whereas the epitrochlearis muscles were incubated intact, the two soleus muscles were divided into three strips each. Four of the soleus strips (~30 mg) were incubated, and the other two were quickly frozen in liquid nitrogen for later use in biochemical assays. Each muscle was incubated for 30 min at 37°C in 3 ml of oxygenated (95% O2-5% CO2) Krebs-Henseleit buffer (KHB) supplemented with 8 mM glucose, 32 mM mannitol, and 0.1% BSA (radioimmunoassay grade, Sigma Chemical). One epitrochlearis muscle and two soleus strips were incubated in the absence of insulin, and the contralateral epitrochlearis muscle and soleus strips were incubated in the presence of a maximally effective concentration of insulin (2 mU/ml Humulin R; Eli Lilly, Indianapolis, IN). After this initial incubation period, two of the soleus strips (one incubated without insulin and one incubated with insulin) were removed, trimmed of excess fat and connective tissue, quickly frozen in liquid nitrogen, and weighed. These strips were subsequently used for insulin-signaling and other biochemical assays. The remaining incubated muscles were rinsed for 10 min at 37°C in 3 ml of oxygenated KHB containing 40 mM mannitol, 0.1% BSA, and insulin, if previously present. After the rinse period, the muscles were transferred to 2 ml of KHB containing 1 mM 2-deoxy-[1,2-3H]glucose (2-DG; 300 µCi/mmol, Sigma Chemical), 39 mM [U-14C]mannitol (0.8 µCi/mmol; ICN Radiochemicals, Irvine, CA), 0.1% BSA, and insulin, if previously present. At the end of this final 20-min incubation period at 37°C, the muscles were removed, trimmed of excess fat and connective tissue, quickly frozen in liquid nitrogen, and weighed. A small piece (~10–15 mg) was cut from each epitrochlearis and each soleus muscle and reweighed. These small pieces were dissolved in 0.5 ml of 0.5 N NaOH for assessment of glucose transport activity. After the muscles were completely solubilized, 5 ml of scintillation cocktail were added, and the specific intracellular accumulation of 2-[3H]DG was determined as described previously (16). This method for assessing glucose transport activity in isolated muscle has been validated (15).

Glycogen synthase activity. Glycogen synthase activity was assessed as the activity ratio (activity in the absence of glucose 6-phosphate divided by the activity in the presence of 5 mmol/l glucose 6-phosphate) using the filter paper assay of Thomas et al. (35), as modified by Henriksen et al. (17).

Biochemical assays. Pieces (~15 mg) of epitrochlearis and soleus muscle were reweighed and homogenized in 30 volumes of ice-cold 20 mM HEPES (pH 7.4) containing 1 mM EDTA and 250 mM sucrose. These homogenates were used for the determination of total protein content using the bicinchoninic acid (BCA) method (Sigma Chemical), GLUT4 protein level (16), total hexokinase activity (37), and citrate synthase activity (33). The triglyceride concentration was assessed in the remaining piece of soleus muscle by use of the chloroform-methanol extraction described by Folch et al. (9), followed by the processing method of Frayn and Maycock (12), as modified by Denton and Randle (7). Glycerol was ultimately assayed spectrophotometrically using a commercially available kit (Sigma Chemical).

Insulin signaling. The remaining pieces of muscles were homogenized in 8 volumes of ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 20 mM Na pyrophosphate, 20 mM {beta}-glycerophosphate, 10 mM NaF, 2 mM Na3VO4, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1 mM CaCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 µg/ml pepstatin, and 2 mM PMSF). Homogenates were incubated on ice for 20 min and then centrifuged at 13,000 g for 20 min at 4°C. Protein concentration was determined using the BCA method (Sigma Chemical). Insulin-signaling proteins were separated by SDS-PAGE on 7.5 or 12% polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) and transferred to nitrocellulose membranes. To determine protein expression of insulin-signaling factors, the blots were incubated with the appropriate dilution of commercially available antibodies against IR {beta}-subunit (IR{beta}), IRS-1, the p85 regulatory subunit of PI 3-kinase, GSK-3{alpha}/{beta} (Upstate Biotechnology, Lake Placid, NY), and Akt1/2 (Cell Signaling Technology, Beverly, MA). Activation of protein kinase C (PKC) in lysates prepared from frozen, unincubated soleus muscle was accessed by determining phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS), a substrate of PKC (30), using a commercially available antibody against ser152/156 of MARCKS (Cell Signaling Technology).

Muscle pieces incubated in the absence or presence of insulin were used for evaluation of Akt and GSK-3 serine phosphorylation. Blots were incubated with antibodies against Akt Ser473 and GSK-3 Ser21/9 (Cell Signaling Technology). It should be noted that, in our hands, the protein expression and Ser21 phosphorylation of GSK-3{alpha} are very low (Sloniger JA and Henriksen EJ, unpublished data), and all GSK-3 data in this study are restricted to GSK-3{beta} Ser9 phosphorylation. Membranes were then incubated with secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP; Chemicon, Temecula, CA). The proteins were visualized on Kodak X-Omat AR film (Kodak, Rochester, NY) using an enhanced chemiluminescence detection system (Amersham Pharmacia, Piscataway, NJ). Band intensities on the autoradiographs were quantified using an imaging densitometer (Bio-Rad model GS-800) and Quantity One software.

For measurement of tyrosine-phosphorylated IR{beta} (IR/pY) and IRS-1 (IRS-1/pY) and for IRS-1 associated p85 (IRS-1/p85), immunoprecipitations and subsequent immunoblotting were performed. Muscle pieces were homogenized in 1 ml of ice-cold lysis buffer, and protein concentration was determined. Samples were diluted to 2 mg/ml (IR/pY and IRS-1/pY) or 3 mg/ml (IRS-1/p85). For assessment of IR/pY, 0.5 ml of diluted homogenate was immunoprecipitated with 15 µl of recombinant agarose-conjugated anti-phosphotyrosine antibody (4G10, Upstate Biotechnology). For analysis of IRS-1/pY and IRS-1/p85, 0.5 ml of diluted homogenate was immunoprecipitated with 25 µl of agarose-conjugated anti-IRS-1 antibody (Upstate Biotechnology). After an overnight incubation at 4°C, samples were centrifuged, and the supernatant was removed. The beads were washed three times with ice-cold PBS, mixed with SDS sample buffer, and boiled for 5 min. Proteins were separated by SDS-PAGE on 7.5% polyacrylamide gels (Bio-Rad Laboratories) and transferred to nitrocellulose membranes. Immunoblotting for detection of IR/pY and IRS-1/p85 was completed as described above for detection of protein expression of IR{beta} and p85. For analysis of IRS-1/pY, the nitrocellulose membrane was incubated in anti-phosphotyrosine antibody (PY99, Santa Cruz Biotechnology, Santa Cruz, CA). Thereafter, the membranes were incubated with secondary goat anti-mouse antibody conjugated with HRP (Santa Cruz Biotechnology). Protein bands of interest were exposed, visualized, and quantified as described above.

Statistical analysis. All values are expressed as means ± SE. Differences between two groups were determined using an unpaired Student's t-test. Paired t-tests were used to determine statistical differences between basal and insulin-stimulated values within groups. A level of P < 0.05 was set for statistical significance.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Whole body insulin resistance of TG(mREN2)27 rats. The final average body weight of the hypertensive male heterozygous TG(mREN2)27 rats did not differ significantly from that of normotensive Sprague-Dawley controls (Table 1). Heart weight, expressed in either absolute (g) or relative (g/100 g body wt) terms, was 33–51% greater (P < 0.05) in the TG(mREN2)27 rats than in the normotensive control animals, reflecting the cardiac hypertrophy associated with the hypertensive state of the transgenic animals. Moreover, the amount of total abdominal fat as a percentage of body weight was 18% greater in the TG(mREN2)27 rats (3.71 ± 0.08%) than in the nontransgenic controls (3.15 ± 0.08%, n = 5–6 animals). Whereas fasting plasma glucose was not significantly different between groups (Table 1), fasting plasma insulin was 74% higher (P < 0.05) in the TG(mREN2)27 rats. Fasting plasma FFA concentrations tended to be higher (42%, P < 0.1) in the TG(mREN2)27 rats than in the Sprague-Dawley controls.


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  		Table 1. Body weights, heart weights, and fasting plasma variables in normotensive Sprague-Dawley and hypertensive TG(mREN2)27 rats

 
The glucose value at the 15-min time point was 25% higher (P < 0.05) in the TG(mREN2)27 rats than in the Sprague-Dawley group (Fig. 1A). However, the incremental glucose area under the curve (glucose IAUC) did not differ between groups (Fig. 1B). The insulin concentration at this 15-min time point was more than twice as great (P < 0.05) in the TG(mREN2)27 animals (Fig. 1A), and the insulin IAUC for the insulin response was also twofold greater (P < 0.05) in the TG(mREN2)27 rats compared with the control group (Fig. 1B). The glucose-insulin index is the product of glucose IAUC and insulin IAUC, and an increase in this value reflects a reduction in whole body insulin sensitivity (4). The glucose-insulin index was 88% greater (P < 0.05) in the TG(mREN2)27 rats than in the control animals (Fig. 1B).



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  		Fig. 1. A: glucose and insulin responses to an oral glucose tolerance test in Sprague-Dawley (SD) and TG(mREN2)27 (TG) rats. B: incremental areas under the curves (IAUC) for glucose and insulin and the glucose-insulin index derived from these data are displayed. Values are means ± SE for 4–5 animals per group. *P < 0.05 vs. Sprague-Dawley group.

 
Insulin resistance of muscle glucose transport and glycogen synthase in TG(mREN2)27 rats. Basal rates of 2-DG uptake were not different between TG(mREN2)27 rats and Sprague-Dawley controls for either epitrochlearis or soleus muscles (Fig. 2). In contrast, insulin-stimulated 2-DG uptake in the epitrochlearis (30%) and soleus (22%) was significantly less (P < 0.05) in the TG(mREN2)27 rats than in controls. Likewise, the insulin-induced increases in 2-DG uptake above basal (cross-hatched bars in Fig. 2) were 24% smaller in epitrochlearis and 35% smaller in soleus (both P < 0.05) muscles of the TG(mREN2)27 rats than in the nontransgenic controls. These data clearly indicate that skeletal muscle of these transgenic animals is insulin-resistant. GLUT4 protein levels were not different between groups, and activities of total hexokinase and citrate synthase were slightly, though not statistically significantly, greater (11–14%) in soleus muscle of the TG(mREN2)27 rats (data not shown). Interestingly, intramuscular triglycerides were 25% greater in soleus muscle of the TG(mREN2)27 rats compared with the normotensive controls (3.05 ± 0.24 µmol/g muscle vs. 2.44 ± 0.13, n = 8–9 per group). However, the overall activation of PKC in the soleus, as assessed by Ser152/156 phosphorylation of MARCKS, a PKC substrate (30), was not different between groups (data not shown).



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  		Fig. 2. Glucose transport activity in epitrochlearis and soleus muscles of Sprague-Dawley and TG(mREN2)27 rats. 2-Deoxyglucose uptake was assessed in the absence (–) and presence (+) of insulin (2 mU/ml). Increase above basal due to insulin for 2-DG uptake is also presented ({Delta}). Values are means ± SE for 5–7 animals per group. *P < 0.05 vs. Sprague-Dawley group of same incubation condition.

 
Whereas total glycogen synthase activity was not different in the soleus muscle of the TG(mREN2)27 rats compared with the Sprague-Dawley control group (1.99 ± 0.11 vs. 1.91 ± 0.11 nmol·mg muscle–1·min–1, n = 5 per group), it was 39% greater (P < 0.05) in the epitrochlearis of the transgenic animals relative to the Sprague-Dawley rats (1.61 ± 0.04 vs. 1.16 ± 0.06 nmol·mg muscle–1·min–1). Insulin stimulation of glycogen synthase activity in skeletal muscle was compared in TG(mREN2)27 rats and normotensive controls (Fig. 3). The soleus glycogen synthase activity ratio in the absence of insulin was 36% greater (P < 0.05) in the TG(mREN2)27 rats than in controls. More importantly, the increase in glycogen synthase activation due to insulin was reduced in both the epitrochlearis (46%) and the soleus muscles (64; both P < 0.05) of the TG(mREN2)27 rats compared with the Sprague-Dawley rats.



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  		Fig. 3. Glycogen synthase activity in epitrochlearis and soleus muscles of Sprague-Dawley and TG(mREN2)27 rats. Activity ratio of glycogen synthase was assessed in the absence (–) and presence (+) of insulin (2 mU/ml). Increase above basal due to insulin for 2-DG uptake is also presented ({Delta}). Activity ratio is expressed as activity in the absence of glucose 6-phosphate (G-6-P, G6P) divided by activity in the presence of 5 mM G-6-P. Values are means ± SE for 5 animals per group. *P < 0.05 vs. Sprague-Dawley group of same incubation condition.

 
Insulin resistance of muscle insulin signaling in TG(mREN2)27 rats. The ability of insulin to activate elements of the insulin-signaling cascade critical for regulation of glucose transport activity and glycogen synthase activity was assessed. There were no differences in the protein expression of IR{beta}, IRS-1, p85, Akt1/2, and GSK-3{beta} in epitrochlearis and soleus muscles between the transgenic and control groups (data not shown). However, in both epitrochlearis and soleus muscles, insulin stimulation of tyrosine phosphorylation of IR (Fig. 4A) and IRS-1 (Fig. 4B) was significantly reduced by 26–45% (P < 0.05) in the hypertensive TG(mREN2)27 group compared with the normotensive Sprague-Dawley animals. Likewise, the insulin stimulation of IRS-1 associated with the p85 subunit of PI 3-kinase, a surrogate measure of PI 3-kinase activity, was reduced (P < 0.05) in the TG(mREN2)27 rats compared with the normotensive rats in both the epitrochlearis (37%) and the soleus (50%; Fig. 4C).



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  		Fig. 4. Tyrosine phosphorylation of insulin receptor (IR; A), tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1; B), and IRS-1 associated with the p85 subunit of phosphatidylinositol (PI) 3-kinase (C) in epitrochlearis and soleus muscles of Sprague-Dawley and TG(mREN2)27 rats. Muscles were incubated in the absence (–) and presence (+) of insulin (2 mU/ml). Representative bands from the autoradiograph are displayed at the top of each panel. Values are means ± SE for 4–8 animals per group. *P < 0.05 vs. Sprague-Dawley group of same incubation condition.

 
Downstream elements of the insulin-signaling pathway were also assessed. Insulin-stimulated Ser473 phosphorylation of Akt1/2 was reduced (P < 0.05) in both the epitrochlearis (31%) and soleus (54%) muscles of the transgenic vs. control rats (Fig. 5A). The insulin stimulation of Ser9 phosphorylation of GSK-3{beta}, a target of Akt phosphorylation and a regulator of glycogen synthase activity (5, 34), was also diminished by ~30% in the epitrochlearis and soleus (both P < 0.05) muscles of the TG(mREN2)27 rats (Fig. 5B).



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  		Fig. 5. Akt serine phosphorylation (A) and glycogen synthase kinase (GSK)-3{beta} serine phosphorylation (B) in epitrochlearis and soleus muscles of Sprague-Dawley and TG(mREN2)27 rats. Ser473 phosphorylation of Akt1/2 and Ser9 phosphorylation of GSK-3{beta} were assessed in the absence (–) and presence (+) of insulin (2 mU/ml). Representative bands from the autoradiograph are displayed at the top of each panel. Values are means ± SE for 4–5 animals per group. *P < 0.05 vs. Sprague-Dawley group of same incubation condition.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have demonstrated that the hypertensive male heterozygous TG(mREN2)27 rat displays characteristics of insulin resistance at the whole body and skeletal muscle levels. These animals exhibited impaired glucose tolerance (Fig. 1), decreased whole body insulin sensitivity (Fig. 1), fasting hyperinsulinemia and dyslipidemia (Table 1), and reduced insulin action of skeletal muscle glucose transport (Fig. 2), confirming previous findings of our research group (19) and others (18). Moreover, we have presented the new finding that insulin resistance in skeletal muscle of the TG(mREN2)27 rat is observed not only for the glucose transport process but is also manifested for the activation of glycogen synthase (Fig. 3), the rate-limiting enzyme in the pathway for glycogenesis in muscle.

We have presented further novel information indicating that the skeletal muscle of the TG(mREN2)27 rat is also characterized by defects in insulin-signaling elements, including reduced insulin action on IR and IRS-1 tyrosine phosphorylation (Fig. 4), IRS-1 associated with the p85 regulatory subunit of PI 3-kinase (Fig. 4), and serine phosphorylation of Akt and GSK-3 (Fig. 5). Our results are consistent with the interpretation that a defect in an upstream component of the insulin-signaling cascade exists, which manifests itself in reduced functionality of downstream signaling elements. The defects in the insulin stimulation of the association of IRS-1 with p85 and of the serine phosphorylation of Akt appear to be of particular importance as a possible mechanism for the insulin resistance of glucose transport activity observed in skeletal muscle of the TG(mREN2)27 rats (Fig. 2 and Ref. 19), as the functionality of these factors is critical for the normal translocation of GLUT4 and an increase in facilitated glucose transport (reviewed in Ref. 40; also see Refs. 14, 20, and 36).

Moreover, our new data support a potential mechanistic connection between the reductions in serine phosphorylation of Akt and GSK-3{beta} (Fig. 5) and the lesser insulin stimulation of glycogen synthase activity in the skeletal muscle of the TG(mREN2)27 rat (Fig. 3). A reduced insulin-stimulated Akt activity (as reflected by the diminished serine phosphorylation state) would allow GSK-3 to remain in a more active state, thereby increasing the capacity of GSK-3 to inactivate glycogen synthase, a direct target of GSK-3 action (5). Our findings therefore provide further evidence supporting an important physiological role of GSK-3 in the regulation of glycogen synthase activity and of glycogen metabolism in mammalian skeletal muscle.

There are a number of possible scenarios that can help explain the etiology of the defective insulin signaling in skeletal muscle of the TG(mREN2)27 rat. Angiotensin II, which is locally elevated in tissues of these hypertensive transgenic rats (24), has been shown to induce an insulin-resistant state when infused into both rats (26) and dogs (29). Direct negative effects of angiotensin II on insulin signaling in myocytes have been demonstrated. Acute exposure of heart tissue (11, 38) and cultured aortic smooth muscle cells (10, 11) to angiotensin II caused increased serine phosphorylation of IRS-1 by Janus kinase-2 (associated with the AT1 receptor), leading to a reduction in insulin-stimulated docking of IRS with the p85 regulatory subunit of PI 3-kinase. This is consistent with our finding of reduced insulin-stimulated IRS-1 associated with p85 in skeletal muscle of the TG(mREN2)27 rat (Fig. 4). Collectively, these findings suggest that the angiotensin II-induced reduction in PI 3-kinase activity occurs via cross talk between the angiotensin II and insulin-signaling pathways.

In contrast, Ogihara et al. (26) have presented evidence that the negative effects of angiotensin II on insulin action are manifested distal to the insulin-signaling pathway, possibly by affecting GLUT4 translocation in a oxidative stress-dependent mechanism. These investigators demonstrated that chronic angiotensin II infusion into normal rats induced the accumulation of plasma cholesteryl ester hydroperoxides, indicative of increased oxidative stress, and that this alteration was normalized by treatment with tempol, the membrane-permeable superoxide dismutase mimetic. Moreover, the antioxidant treatment ameliorated the insulin resistance of insulin-stimulated glucose transport and PI 3-kinase activity in the angiotensin II-infused rats (26). These findings suggest that the mechanism underlying angiotensin II-induced insulin resistance involves oxidative stress, which may impair insulin signaling at a point distal to PI 3-kinase activation.

Finally, the roles of augmented plasma FFAs and muscle lipids as potential contributors to the defective insulin signaling in skeletal muscle of the TG(mREN2)27 rat merit discussion. A greater degree of central adiposity was detected in the TG(mREN2)27 rats compared with the nontransgenic controls (see RESULTS). Importantly, plasma FFAs are increased in the male heterozygous TG(mEN2)27 rat (Ref. 19 and Table 1), and the connection between elevated plasma FFAs and insulin resistance has long been recognized (1). Increasing evidence supports the effect of increased long-chain fatty acyl-CoAs in the induction of defective IR and IRS-1 functionality (3, 32, 39), possibly via an upregulation of PKC (3, 32, 39) and serine phosphorylation of these signaling elements (39). However, in the present study we were unable to detect an upregulation of overall PKC activity, as assessed by phosphorylation of the PKC substrate MARCKS. We did observe a significant 25% elevation of intramuscular triglycerides (and presumably fatty acid derivatives in the soleus muscle of the transgenic animals), and this may have been sufficient to induce at least part of the defective IR and IRS-1 functionality and insulin resistance of glucose transport observed in these muscles. In addition, the hyperinsulinemia present in the TG(mREN2)27 animals (Table 1) may have also contributed to the development of this skeletal muscle insulin resistance (27). It is clear that further study is needed to clarify the molecular mechanisms responsible for the defective insulin signaling seen in skeletal muscle of this hypertensive rodent model.

In summary, the present investigation has demonstrated that the hypertensive male heterozygous TG(mREN2)27 rat displays glucose intolerance, reduced whole body insulin sensitivity, and a marked reduction in insulin-stimulated glucose transport activity and glycogen synthase activity. Moreover, the defects in these physiological variables are associated with an increase in central adiposity and muscular lipid and a reduced insulin stimulation of insulin-signaling elements in skeletal muscle, including tyrosine phosphorylation of IR and IRS-1, IRS associated with PI 3-kinase, and serine phosphorylation of Akt1/2 and GSK-3{beta}. These results further support the close association between hypertension and insulin resistance, possibly due to local elevations in angiotensin II.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported by Grant-in-Aid 0256016Z from the Pacific Mountain Affiliate of the American Heart Association to E. J. Henriksen.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. J. Henriksen, Dept. of Physiology, Ina E. Gittings Bldg. #93, Univ. of Arizona, Tucson, AZ 85721–0093 (E-mail: ejhenrik{at}u.arizona.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Boden G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46: 3–10, 1997.[Abstract]
  2. Campbell DJ, Rong P, Kladis A, Rees B, and Skinner SL. Angiotensin and bradykinin peptides in the TGR(mREN2)27 rat. Hypertension 25: 1014–1020, 1995.[Abstract/Free Full Text]
  3. Cooney GJ, Thompson AL, Furler SM, Ye J, and Kraegen EW. Muscle long-chain acyl CoA esters and insulin resistance. Ann NY Acad Sci 967: 196–207, 2002.[ISI][Medline]
  4. Cortez MY, Torgan CE, Brozinick JT, and Ivy JL. Insulin resistance of obese Zucker rats exercise trained at two different intensities. Am J Physiol Endocrinol Metab 261: E613–E619, 1991.[Abstract/Free Full Text]
  5. Cross DA, Alessi DR, Cohen P, Andjelkovich M, and Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789, 1995.[CrossRef][Medline]
  6. DeFronzo RA and Ferrannini E. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14: 173–194, 1991.[Abstract]
  7. Denton RM and Randle PJ. Concentration of glycerides and phospholipids in rat heart and gastrocnemius muscles: effects of alloxan-diabetes and perfusion. Biochem J 104: 416–422, 1967.[ISI][Medline]
  8. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico MA, Oleggini M, Graziadei L, Pedrinelli R, Brandi L, and Bevilacqua S. Insulin resistance in essential hypertension. N Engl J Med 317: 350–357, 1987.[Abstract]
  9. Folch J, Lees M, and Sloane Stanley JH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497–509, 1957.[Free Full Text]
  10. Folli F, Kahn CR, Hansen H, Bouchie JL, and Feener EP. Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels. A potential role for serine phosphorylation in insulin/angiotensin II crosstalk. J Clin Invest 100: 2158–2169, 1997.[ISI][Medline]
  11. Folli F, Saad MJ, Velloso LA, Hansen H, Feener EP, and Kahn CR. Crosstalk between insulin and angiotensin II signaling systems. Exp Clin Endocrinol Diabetes 107: 133–139, 1999.[ISI][Medline]
  12. Frayn KN and Maycock PF. Skeletal muscle triacylglycerol in the rat: methods for sampling and measurement, and studies of biological variability. J Lipid Res 21: 139–144, 1980.[Abstract]
  13. Ginsburg HN. Insulin resistance and cardiovascular disease. J Clin Invest 106: 453–458, 2000.[ISI][Medline]
  14. Hajduch E, Alessi DR, Hemmings BA, and Hundal HS. Constitutive activation of protein kinase B{alpha} by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes 47: 1006–1013, 1998.[Abstract]
  15. Hansen PA, Gulve EA, and Holloszy JO. Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J Appl Physiol 76: 979–985, 1994.[Abstract/Free Full Text]
  16. Henriksen EJ and Halseth AE. Early alterations in soleus GLUT-4, glucose transport, and glycogen in voluntary running rats. J Appl Physiol 76: 1862–1867, 1994.[Abstract/Free Full Text]
  17. Henriksen EJ, Tischler ME, and Johnson DG. Increased response to insulin of glucose metabolism in the 6-day unloaded rat soleus muscle. J Biol Chem 261: 10707–10712, 1986.[Abstract/Free Full Text]
  18. Holness MJ and Sugden MC. The impact of genetic hypertension on insulin secretion and glucoregulatory control in vivo: studies with the TGR(mREN2)27 transgenic rat. J Hypertens 16: 369–376, 1998.[CrossRef][ISI][Medline]
  19. Kinnick TR, Youngblood EB, O'Keefe MP, Saengsirisuwan V, Teachey MK, and Henriksen EJ. Modulation of insulin resistance and hypertension by voluntary exercise training in the TG(mREN2)27 rat. J Appl Physiol 93: 805–812, 2002.[Abstract/Free Full Text]
  20. Kohn AD, Summers SA, Birnbaum MJ, and Roth RA. Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulated glucose uptake and glucose transporter 4 translocation. J Biol Chem 271: 31372–31378, 1996.[Abstract/Free Full Text]
  21. Landsberg L. Insulin resistance and hypertension. Clin Exp Hypertens 21: 885–894, 1999.[ISI][Medline]
  22. Langheinrich M, Lee MA, Bohm M, Pinto YM, Ganten D, and Paul M. The hypertensive Ren-2 transgenic rat TGR(mREN2)27 in hypertension research: characteristics and functional aspects. Am J Hypertens 9: 506–512, 1996.[CrossRef][ISI][Medline]
  23. Lee MA, Bohm M, Kim S, Bachmann S, Bachmann J, Bader M, and Ganten D. Differential gene expression of rennin and angiotensinogen in the TGR(mREN2)27 transgenic rat. Hypertension 25: 570–580, 1995.[Abstract/Free Full Text]
  24. Lee MA, Bohm M, Paul M, Bader M, Ganten U, and Ganten D. Physiological characterization of the hypertensive transgenic rat TGR(mREN2)27. Am J Physiol Endocrinol Metab 270: E919–E929, 1996.[Abstract/Free Full Text]
  25. Mullins JJ, Peters J, and Ganten D. Fulminant hypertension in transgenic rats harboring the Ren-2 gene. Nature 344: 541–544, 1990.[CrossRef][Medline]
  26. Ogihara T, Asano T, Katsuyuki A, Chiba Y, Sakoda H, Anai M, Shojima N, Ono J, Onishi Y, Fujishiro M, Katagiri H, Fukushima Y, Kikuchi M, Noguchi N, Aburatani H, Komuro I, and Fujita T. Angiotensin II-induced insulin resistance is associated with enhanced insulin signaling. Hypertension 40: 872–879, 2002.[Abstract/Free Full Text]
  27. Pirola L, Bonnafous S, Johnston AM, Chaussade C, Portis F, and Van Obberghen E. Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells. J Biol Chem 278: 15641–15651, 2003.[Abstract/Free Full Text]
  28. Reaven GM. Are insulin resistance and/or compensatory hyperinsulinemia involved in the etiology and clinical course of patients with hypertension? Int J Obes 19: S2–S5, 1995.
  29. Richey JM, Ader M, Moore D, and Bergman RN. Angiotensin II induces insulin resistance independent of changes in interstitial insulin. Am J Physiol Endocrinol Metab 277: E920–E926, 1999.[Abstract/Free Full Text]
  30. Rodriguez-Pena A and Rozengurt E. Phosphorylation of an acidic mol. wt 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity. EMBO J 5: 77–83, 1986.[ISI][Medline]
  31. Sander M, Bader M, Djavidani B, Maser-Gluth C, Vecsei P, and Mullins J. The role of the adrenal gland in hypertensive transgenic rat TGR(mREN2)27. Endocrinology 131: 807–814, 1992.[Abstract]
  32. Schmitz-Peiffer C. Protein kinase C and lipid-induced insulin resistance in skeletal muscle. Ann NY Acad Sci 967: 146–157, 2002.[ISI][Medline]
  33. Srere PA. Citrate synthase. Methods Enzymol 13: 3–10, 1969.[CrossRef]
  34. Summers SA, Kao AW, Kohn AD, Backus GS, Roth RA, Pessin JE, and Birnbaum MJ. The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. J Biol Chem 274: 17934–17940, 1999.[Abstract/Free Full Text]
  35. Thomas JA, Schlender KK, and Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem 25: 486–499, 1968.[CrossRef][ISI][Medline]
  36. Ueki K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BMT, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, and Kadowaki T. Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273: 5315–5322, 1998.[Abstract/Free Full Text]
  37. Uyeda K and Racker E. Regulatory mechanisms in carbohydrate metabolism. VII. Hexokinase and phosphofructokinase. J Biol Chem 240: 4682–4688, 1965.[Free Full Text]
  38. Velloso LA, Folli F, Sun XJ, White MF, Saad MJ, and Kahn CR. Cross-talk between the insulin and angiotensin signaling systems. Proc Natl Acad Sci USA 93: 12490–12495, 1996.[Abstract/Free Full Text]
  39. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, Bergeron R, Kim JK, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, and Shulman GI. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 277: 50230–50236, 2002.[Abstract/Free Full Text]
  40. Zierath JR, Krook A, and Wallberg-Henriksson H. Insulin action and insulin resistance in human skeletal muscle. Diabetologia 43: 821–835, 2000.[CrossRef][ISI][Medline]



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