Endocrinology and Metabolism

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To test whether chronic enhanced blood flow alters insulin-stimulated glucose uptake, we measured skeletal muscle glucose uptake in chow-fed and high-fat-fed mice injected with adenovirus containing modified angiopoietin-1, COMP-Ang1, via euglycemic-hyperinsulinemic clamp. Blood flow rates and platelet endothelial cell adhesion molecule-1 positive endothelial cells in the hindlimb skeletal muscle were elevated in COMP-Ang1 compared with control LacZ. Whole body glucose uptake and whole body glycogen/lipid synthesis were elevated in COMP-Ang1 compared with LacZ in chow diet. High-fat diet significantly reduced whole body glucose uptake and whole body glycolysis in LacZ mice, whereas high-fat-fed COMP-Ang1 showed a level of whole body glucose uptake that was comparable with chow-fed LacZ and showed increased glucose uptake compared with high-fat-fed LacZ. Glucose uptake and glycolysis in gastrocnemius muscle of chow-fed COMP-Ang1 were increased compared with chow-fed LacZ. High-fat diet-induced whole body insulin resistance in the LacZ was mostly due to ∼40% decrease in insulin-stimulated glucose uptake in skeletal muscle. In contrast, COMP-Ang1 prevented diet-induced skeletal muscle insulin resistance compared with high-fat-fed LacZ. Akt phosphorylation in skeletal muscle was increased in COMP-Ang1 compared with LacZ in both chow-fed and high-fat-fed groups. These results suggest that increased blood flow by COMP-Ang1 increases insulin-stimulated glucose uptake and prevents high-fat diet-induced insulin resistance in skeletal muscle.

  • glucose uptake
  • high-fat diet
  • hyperinsulinemic-euglycemic clamp

insulin resistance, defined as decrease in sensitivity and/or responsiveness to the metabolic action of insulin that promotes glucose disposal to peripheral tissues (1, 13), plays important roles in the pathogenesis of Type 2 diabetes and obesity (16, 30). Since skeletal muscle constitutes 40% of body weight and is one of the major tissues to increase insulin-stimulated glucose uptake, insulin responsiveness in skeletal muscle has an essential role in the development of whole body insulin resistance.

Skeletal muscle blood vessels are fundamental components in the transportation and diffusion of various substances including oxygen, glucose, insulin, and fatty acids from the circulation to skeletal muscle (14, 39). It has been thought that the increase in blood flow in skeletal muscle may facilitate the transfer and diffusion of nutrients and hormones to capillaries of skeletal muscle. Baron and colleagues (17) report showing contribution of vasodilatory effects of insulin on enhanced glucose uptake has boosted up the interest. However, increased blood flow may not necessarily enhance glucose uptake. Increased blood flow by sodium nitroprusside fails to increase insulin-stimulated glucose uptake (21).

Angiopoietin-1 (Ang1) has been identified as a secreted protein ligand of tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) (12, 38). Ang1/Tie2 is thought to be involved in branching and remodeling of the primitive network (5). Ang1- and Tie2-deficient mice display severe vascular remodeling defects, insufficient vessel stabilization, and perturbed vascular maturation (32, 34). Moreover, transgenic overexpression or gene transfer of Ang1 increases vessel formation (3, 33, 35). Furthermore, Ang1 can also counteract vascular endothelial growth factor-induced vascular leakage (36). The amino terminus of Ang1 was replaced with the short coiled coin domain of cartilage oligomeric matrix protein (COMP) and a soluble, stable, and potent Ang1-recombinant chimera, COMP-Ang1, has been recently developed (4). Long-term and sustained treatment with COMP-Ang1 produces long-lasting and stable vascular enlargement and increase in blood flow in trachea (5). The vascular enlargement was also detected in heart, adrenal cortex, and liver (5). Therefore, we hypothesized that treatment of COMP-Ang1 would induce vascular enlargement in skeletal muscle and increase blood flow, leading to enhanced insulin sensitivity and reverse insulin resistance. To our knowledge, no study has assessed insulin sensitivity in chronically modified vessels with angiogenic factors.

In the present study, we increased skeletal muscle blood flow by COMP-Ang1 using an adenoviral vector and tested whether insulin sensitivity was altered in chow-fed or high-fat-fed COMP-Ang1 mice.


Generation of Adenovirus Containing COMP-Ang 1

Recombinant adenovirus expressing COMP-Ang1 or bacterial-β-galactosidase (hereafter LacZ) was constructed by using the pAdEasy vector system (Qbiogene, Carlsbad, CA) as described previously (5). Briefly, the COMP-Ang1 gene or LacZ gene was excised from the pcDNA 3.1(+)-FLAG-COMP/CC-Ang/FD plasmid or pcDNA3.1(+)-FLAG LacZ via the HindIII/SalI sites. The resulting fragment was inserted into pShuttle-CMV (Qbiogene) transfer vector and then homologous recombination between the linearized pShuttle-CMV-COMP-Ang-1 or pShuttle-CMV-LacZ and the adenoviral plasmid pAdEasy-1 was mediated by cotransformation in Escherichia coli (BJ5183) (Qbiogene). The selected recombinant plasmid was linearized by PacI digestion and transferred into human embryonic kidney (HEK)-293 cells (American Type Culture Collection, Manassas, VA). Viral particles were precipitated with polyethylene glycol solution followed by further purification by cesium chloride density centrifugation. Purified virus in 10 mM Tris·HCl buffer (pH 8.0) containing 2 mM MgCl2 and 4% sucrose was stored at −80°C. The viral titers were determined by a commercial tissue culture infectious dose 50 method (Qbiogene).

Animals and Treatment

Male C57BL/6J mice weighing 20 g were purchased from Jung-Ang Lab Animals (Seoul, South Korea) and were housed in the animal unit of College of Medicine at Yeungnam University. Mice were housed in a group cage in a room, which was on a 12:12-h light-dark cycle (lights on at 7:00 and off at 19:00). The mice were fed a standard chow diet or high-fat diet for 8 wk and given ad libitum access to water. The standard laboratory chow diet provided 66% energy as carbohydrate, 25% as protein, and 9% as fat (soybean oil). The high-fat diet provided 34% energy as carbohydrate, 15% as protein, and 51% as fat (40% lard and 11% soybean oil). The two diets had the same amount of vitamin and mineral mix. Food intake was monitored by manual weighing, and spillage was taken into account. On the first day of diet treatment, adenovirus containing COMP-Ang1 or LacZ was injected. For adenoviral treatment, Ade-LacZ and Ade-COMP-Ang1 (each 1 × 109 plaque forming units) were diluted in 100 μl of sterile 0.9% NaCl and injected into the tail vein. This study was conducted in accordance with the guidelines for the care and use of laboratory animals provided by Yeungnam University. All experimental protocols were approved by Institutional Review Board of the Yeungnam University Medical Center.

Hyperinsulinemic-Euglycemic Clamp


Four days before experiments, mice were anesthetized by an intraperitoneal injection of a combination of anesthetics (tiletamine and zolezepam, 25 mg/kg body wt; xylazine, 10 mg/kg body wt) and a silicone catheter (Helix Medical, San Mateo, CA) was inserted in the right internal jugular vein. On the day of the experiment, a Y connector was attached to the jugular vein catheter to intravenously deliver glucose or insulin solutions.

Hyperinsulinemic euglycemic clamps.

After an overnight fast, conscious mice were placed in a rat-size restrainer and the tail was restrained with tape to obtain blood samples from the tail vessels. For a 2-h hyperinsulinemic euglycemic clamp, human insulin (Novolin, Novo Nordisk, Bagsvaerd, Denmark) was continuously infused at a rate of 15 pmol·kg−1·min−1 to raise plasma insulin. Blood samples (20 μl) were collected in capillary tubes at 20-min intervals for the immediate measurement of plasma glucose concentration, and 20% glucose was infused at variable rates to maintain glucose at a constant concentration. Insulin-stimulated rates of whole body glucose uptake were estimated with a continuous infusion of [3-3H]glucose (PerkinElmer Life and Analytical Sciences, Boston, MA) throughout the clamps (0.1 μCi/min) (24). To estimate insulin-stimulated glucose uptake in individual tissues, 2-deoxy-d-[1-14C]glucose (2-[14C]DG; PerkinElmer Life and Analytical Sciences) was administered as a bolus (10 μCi) at 75 min after the start of clamps (24). At the end of the clamps, mice were anesthetized and tissues were dissected and stored at −80°C until biochemical and molecular analysis.

Biochemical assays.

Plasma glucose concentration was measured with a Beckman Glucose Analyzer 2 (Beckman, Fullerton, CA) and plasma insulin concentration was measured by enzyme-linked immunosorbent assay (Linco Research, St. Charles, MO). Plasma concentrations of [3-3H]glucose, 2-[14C]DG, and 3H2O were determined after deproteinization of plasma samples (24). The radioactivity of 3H in tissue glycogen was determined by digesting tissue samples in KOH and precipitating glycogen with ethanol. For the determination of tissue 2-[14C]DG 6-phosphate (2-[14C]DG-6-P) content, tissue samples were homogenized, and the supernatants were subjected to an ion exchange column to separate 2-DG-6-P from 2-DG (24).


Rates of basal hepatic glucose production (HGP) and insulin-stimulated whole body glucose uptake were determined the ratio of the [3H]glucose infusion rate [disintegrations per minute (dpm)] to the specific activity of plasma glucose (dpm/μmol) at the end of basal period and during the final 30 min of clamps, respectively. HGP during the clamps was determined by subtracting the glucose infusion rate from the whole body glucose uptake rate. Whole body glycolysis was calculated from the rate of increase in plasma 3H2O concentration, determined by linear regression of the measurements at 80, 90, 100, 110, and 120 min of clamps. Whole body glycogen plus lipid synthesis was estimated by subtracting whole body glycolysis from whole body glucose uptake (25), assuming that glycolysis and glycogen plus lipid synthesis account for the majority of insulin-stimulated glucose uptake. Because 2-DG is a glucose analog that is phosphorylated but not metabolized, insulin-stimulated glucose uptake in individual tissues can be estimated by determining the tissue (e.g., skeletal muscle, heart) content of 2-[14C]DG-6-P. On this basis, glucose uptake in individual tissues was calculated from the plasma 2-[14C]DG profile, which was fitted with a double exponential or linear curve by using MLAB (Civilized Software, Bethesda, MD) and tissue 2-[14C]DG-6-P content. Skeletal muscle glycogen synthesis was calculated from 3H incorporation into muscle glycogen, and skeletal muscle glycolysis was estimated as the difference between muscle glucose uptake and muscle glycogen synthesis.

Histological and Morphometric Analysis

Mouse muscles tissues were prefixed by perfusion of 1% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in cryofreezing medium for cryosection after postfixation by 4% paraformaldehyde. Cryosectioned muscle tissues (20-μm thickness) were washed three times with PBS and were incubated for 1 h at room temperature with a blocking solution containing 5% normal goat serum (Jackson ImmunoResearch, West Grove, PA) and 0.3% Triton X-100 in PBS. Then tissue sections were incubated for 2 h at room temperature with a 1:200 dilution of hamster clone 2H8 anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody (Chemicon International, Temecula, CA). For nuclear counterstaining, a 1:2,000 dilution of TOTO-3 (Invitrogen, Carlsbad, CA) was used. After several washes in PBS, sections were incubated with a 1:500 dilution of Cy3-conjugated anti-hamster IgG antibody (Jackson ImmunoResearch) for 2 h at room temperature. For control experiments, the primary antibody was omitted or replaced by preimmune serum. Signals were visualized and digital images were obtained by using a Zeiss LSM 510 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss, Oberkochen, Germany). PECAM-1 positive vascular area densities (percentage of immunopositive area per total tissue area) were measured at ×100 magnification by analysis of pixel-based fluorescence intensities using ImageJ software. Color images were converted to eight-bit grayscale image and specific signal was detected from nonspecific background by a threshold value between 55 and 60. Area densities of PECAM-1 positive were calculated as the proportion of pixels having higher fluorescent intensities than threshold value.

Measurement of Hindlimb Blood Flow

Blood flow in hindlimb muscle was measured using laser Doppler flowmeter, as described previously (5, 6). Briefly, mice were anesthetized and placed in a supine position on a heated table, and the skin of a hindlimb was incised ∼1 cm to expose the adductor muscle and branches of the femoral artery. An N series flowprobe (Transonic Systems, Ithaca, NY) was placed perpendicular to the second and third branches of the femoral artery. The flow probe was kept in place on the position of the highest sensitivity by a micromanipulator and was connected to a laser Doppler flowmeter (model BLF21; Transonic Systems), which can measure microcirculation in 1 mm3 of tissue assuming that this volume weighs ∼0.001 g. Two or three measurements were done in the same area and the average value of each measurement was used for data analysis. These analog signals were digitized at 100 Hz (Digidata 1200; Axon Instruments, Foster City, CA) and were continuously displayed by a data acquisition program. The average value of tissue perfusion rate (ml/min per 100 g of tissue) was analyzed by using AXOSCOPE 9.0 software (Axon Instruments).

Real-Time PCR

About 40 mg of liver was homogenized in TRI Reagent (Sigma) by use of an Ultra-Turrax T25 (Janke & Kunkel, IKA-Labortechnik, Staufel, Germany). RNA was reverse transcribed to cDNA from 1 μg of total RNA by using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using the Real-Time PCR 7500 Software system and Power SYBR Green PCR master mix (Applied Biosystems), according to the manufacturer's instructions. The reactions were incubated at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s, 55°C for 20 s, and 72°C for 35 s. Expression levels of β-actin were used for sample normalization. Primers for mouse β-actin, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) were based on NCBI's Nucleotide database and designed using the Primer Express program (Applied Biosystems): β-actin (121 bp: forward, 5′-TGG ACA GTG AGG CAA GGA TAG-3′; reverse, 5′-TAC TGC CCT GGC TCC TAG CA-3′), G6Pase (71 bp: forward, 5′-CTC TTG CTA TCT TTC GAG GAA A-3′; reverse, 5′-CCA ACC ACA AGA TGA CGT TC), PEPCK (71 bp: forward, 5′-AGA GAG CTG TGG ACG AGA GAT T-3′; reverse, 5′-CCA TGC TGA ATG GAA GCA-3′).

Western Blotting

Skeletal muscle samples obtained at the end of clamp were used for measurements of Akt phosphorylation. Approximately 40 mg of each muscle sample was homogenized in a lysis buffer (Invitrogen) containing 1% NP40, 150 mM NaCl, 5 mM MgCl, 10 mM HEPES, leupeptin, and pepstatin A. A 60-μg sample of total protein per lane was separated by 12% SDS-PAGE. Separated proteins were then transferred to a 0.45-μm polyvinylidene fluoride membrane (Gelman Sciences, East Hill, NY). After blocking with 5% skim milk-10 mM Tris·HCl, pH 7.4–150 mM NaCl-0.1% Tween 20, the membrane was incubated overnight at 4°C with primary antibody diluted 1:1,000. Specific antibody binding was detected by using a 1:2,000 dilution of sheep anti-rabbit IgG horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and visualized by using an enhanced chemiluminescence detection regent (Amersham, Buckinghamshire, UK).


The results are expressed as means ± SE. Differences of tissue blood flow, area density of endothelial cells, and Akt phosphorylation between LacZ and COMP-Ang1 were analyzed by the Student's t-test. Otherwise, differences among the groups were assessed via one-way analysis of variance followed by least significant difference test. All statistical analyses were conducted with the SPSS ver. 10 (SPSS, Chicageo. IL).


Vascular Density and Blood Flow Rate

The skin of mice treated with adenovirus containing COMP-Ang1 appeared markedly redder than skin of mice treated with LacZ containing virus, especially hair-sparse portions such as foot, beginning 2 wk after treatment (Fig. 1A). Skin redness was maintained as long as 8 wk when hyperinsulinemic-euglycemic clamp was conducted. Consistent with the present study, persistence of skin redness for up to 16 wk has been noted (5). Blood flow rate in hindlimb skeletal muscle at 8 wk was significantly elevated in COMP-Ang1-treated mice compared with LacZ-treated mice (Fig. 1B). PECAM-1 positive blood vascular densities were greater in skeletal muscle of mice treated with COMP-Ang1 compared with LacZ-treated mice (Fig. 1C). Previously, these changes in vessel were noticeable 1 wk after treatment and further increased until 6 wk. The plateau at 6 wk lasted as long as 16 wk, although circulating COMP-Ang1 level returned to the control level at 6 wk (5, 24).

Fig. 1.

Effect of adenoviral cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1) on skin color, hindlimb blood flow, and endothelial cell numbers of skeletal muscle at 8 wk after treatment. Adenoviral LacZ-injected mice were used as control. Skin color of hind foot was red at the second week of treatment (A). Hindlimb blood flow measured in femoral artery was significantly increased in COMP-Ang1 mice (B). Platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive vessel densities of gastrocnemius muscle were also significantly increased in COMP-Ang1 (C). Values are means ± SE for 7 or 8 experimental cases. *P < 0.05 vs. LacZ.

Body Composition and Plasma Profiles

Food intake was not different between LacZ and COMP-Ang1 in both chow-fed and high-fat-fed mice (data not shown). On the standard chow diet, body weight, fat mass, and fasting plasma insulin and glucose levels did not differ between the LacZ and COMP-Ang1 mice (Table 1 and Fig. 2). Plasma free fatty acids and triglycerides also did not differ between the two groups. High-fat feeding caused a comparable increase in body weight, epididymal fat mass, and plasma levels of insulin, free fatty acids and triglycerides in the LacZ and COMP-Ang1 mice compared with chow-fed mice and did not affect plasma glucose level in either group. However, body weight and plasma profiles did not differ between LacZ and COMP-Ang1 in the high-fat-fed group (Table 1 and Fig. 2).

Fig. 2.

Effect of adenoviral COMP-Ang1 on body weight (A), epididymal fat mass (B), plasma levels of free fatty acids (C) and triglycerides (D), and glucose infusion rate (E) in chow-fed or high-fat-fed mice. Adenoviral LacZ injected mice were used as control. Values are means ± SE for 7 or 8 experimental cases. □, LacZ; ▪, COMP-Ang1. *P < 0.05 vs. chow diet LacZ and #P < 0.05 vs. high-fat diet LacZ.

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Table 1.

Metabolic parameters of mice injected with adenovirus containing LacZ and COMP-Ang1 at basal and during a 2-h hyperinuslinemic-euglycemic clamp experiments

Insulin-Stimulated Glucose Metabolism

Whole body glucose metabolism.

The effect of insulin action on glucose metabolism was examined in whole body and peripheral tissues during a 2-h hyperinsulinemic euglycemic clamp in LacZ and COMP-Ang1 mice fed chow or high-fat diet. During the clamps, plasma glucose level was maintained at euglycemia and plasma insulin levels were similarly elevated in all groups of mice (Table 1). Steady-state rates of glucose infusion required to maintain euglycemia were elevated in COMP-Ang1 mice fed chow diet (288 ± 17 vs. 347 ± 27 μmol·kg−1·min−1 in LacZ vs. COMP-Ang1, respectively) (Fig. 2C). Similarly, insulin-stimulated whole body glucose uptake was elevated in COMP-Ang1 mice fed the chow diet (355 ± 11 vs. 399 ± 6 μmol·kg−1·min−1 in LacZ vs. COMP-Ang1, respectively). Furthermore, whole body glycogen/lipid synthesis showed a similar pattern of change to whole body glucose uptake, indicating that whole body insulin sensitivity was increased in COMP-Ang1 mice (Fig. 3). High-fat diet caused a reduction in the steady-state rate of glucose infusion (Fig. 2C), whole body glucose uptake, and whole body glycolysis in LacZ mice, indicating that the high-fat diet induced whole body insulin resistance in mice (Fig. 3). In contrast, COMP-Ang1 mice showed a level of glucose uptake that was comparable to chow-fed LacZ mice and significantly elevated whole body glucose uptake compared with high-fat-fed LacZ mice (252 ± 27 vs. 328 ± 23 μmol·kg−1·min−1 in LacZ vs. COMP-Ang1, respectively). However, glucose uptake in high-fat-fed mice was lower than that of chow-fed mice in the COMP-Ang1group (Fig. 3). Similar to glucose uptake, the steady-state rates of glucose infusion rate and glycogen/lipid synthesis were elevated in COMP-Ang1 mice compared with LacZ mice in the high-fat diet group (Figs. 2C and 3C). These results indicate that COMP-Ang1 mice reduce high-fat diet-induced insulin resistance.

Fig. 3.

Effect of adenoviral COMP-Ang1 on insulin-stimulated whole body and hepatic glucose metabolism in chow-fed and high-fat-fed mice. Adenoviral LacZ-injected mice were used as control. A: insulin-stimulated whole body glucose uptake in vivo. B: insulin-stimulated whole body glycolysis. C: insulin-stimulated whole body glycogen plus and lipid synthesis. D: hepatic glucose production (HGP) reflected as the percent suppression of basal HGP during insulin clamp. Gene expression of glucose-6-phosphatase (G6Pase) (E) and phosphoenolpyruvate carboxykinase (PEPCK) (F) in liver are also shown. Values are means ± SE for 7 or 8 experimental cases. □, LacZ; ▪, COMP-Ang1. *P < 0.05 vs. chow diet LacZ and #P < 0.05 vs. high-fat diet LacZ.

Hepatic insulin action.

The rate of basal HGP did not differ between LacZ and COMP-Ang1 in chow-fed and high-fat-fed mice (data not shown). Insulin decreased basal HGP by ∼60% in the both groups of chow-fed mice. High-fat feeding reduced the effect of insulin on HGP in LacZ and COMP-Ang1 mice. Insulin action on HGP was not different between the two groups in both chow-fed and high-fat fed mice (Fig. 3D). Consistent with these data, there was no difference of gene expression of gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK), between two groups in chow diet. High-fat feeding reduced the gene expression of both enzymes that may be caused by increased plasma insulin levels (2, 26). Again there was no difference between two groups in high-fat-fed mice (Fig. 3, E and F).

Skeletal muscle glucose metabolism.

On the chow diet, insulin-stimulated glucose uptake (266 ± 31 vs. 342 ± 39 nmol·g−1·min−1 in LacZ vs. COMP-Ang1, respectively) and glycolysis (217 ± 23 vs. 271 ± 20 nmol·g−1·min−1 in LacZ vs. COMP-Ang1, respectively) in gastrocnemius muscle were increased in COMP-Ang1 mice. High-fat-diet-induced whole body insulin resistance in the LacZ mice was mostly due to an ∼40% decrease in insulin-stimulated glucose uptake in skeletal muscle. In contrast COMP-Ang1 significantly reduced diet-induced insulin resistance compared with high-fat-fed LacZ mice (178 ± 22 vs. 234 ± 26 nmol·g−1·min−1in LacZ vs. COMP-Ang1, respectively). However, glucose uptake in high-fat-fed mice was lower than that of chow-fed mice in the COMP-Ang1group. Insulin-stimulated glycolysis in the skeletal muscle of COMP-Ang1 mice showed the same pattern with glucose uptake (Fig. 4).

Fig. 4.

Effect of adenoviral COMP-Ang1 on skeletal muscle (gastrocnemius) glucose metabolism in vivo and Akt phosphorylation in chow-fed and high-fat-fed mice. Adenoviral LacZ-injected mice were used as control. Glucose uptake (A), glycolysis (B), and glycogen synthesis (C) were measured during hyperinsulinemic-euglycemic clamp study. D: Akt phosphorylation. Values are means ± SE for 7 or 8 experimental cases. □, LacZ; ▪, COMP-Ang1. *P < 0.05 vs. chow diet LacZ and #P < 0.05 vs. high-fat diet LacZ. *P < 0.05 vs. LacZ in Akt phosphorylation.

Akt Phosphorylation

To determine whether the increase in insulin-stimulated glucose uptake in COMP-Ang1 was accompanied with alteration in insulin signaling activity, we assessed insulin-stimulated phosphorylation of Akt, which is one of the key intracellular mediators in the insulin signaling pathway, in tissues obtained at the end of insulin clamp experiments. Increase in insulin stimulated-glucose uptake was associated with increase in Akt phosphorylation in skeletal muscle (Fig. 4D).


A previous study (5) reported that treatment of modified Ang1, COMP-Ang1, by adenoviral injection results in the enlargement of vessels in the trachea, which leads to enhanced blood flow. The present study shows the treatment of COMP-Ang1 by adenoviral injection induces enhanced blood flow in skeletal muscle. Importantly, this increased blood flow in turn increases insulin sensitivity and prevents high-fat-diet-induced insulin resistance in skeletal muscle.

There has been a discrepancy between blood flow and glucose uptake in skeletal muscle. The acute increase of blood flow with vasodilators such as l-arginine (23) and bradykinin (15) elevates whole body glucose uptake and skeletal muscle glucose uptake, respectively. On the contrary, the infusion of sodium nitroprusside into the forearm increases blood flow but fails to increase insulin-stimulated glucose uptake in hypertensive patients (21). Here we show that chronic increase of blood flow with COM-Ang1 increases insulin-stimulated glucose uptake.

Recently, it has been suggested that the distribution of blood flow may impact more intensely in glucose uptake especially in the presence of insulin (7). The treatment of COMP-Ang1 induces significant enlargement of specific sites of vessel such as terminal arterioles, venous end of capillaries, postcapillary venules, and collecting venules (5). Terminal arteries, the last part of the arterioles that feed into capillaries, controls capillary blood flow distribution and recruitment (11). Since the capillary is indeed a primary site of exchange, enlargement of terminal arterioles may increase capillary blood flow and/or recruitment of the capillary bed to enhance diffusion of glucose and insulin through the endothelium. Insulin increases capillary recruitment and glucose uptake prior to an increase in total blood flow (8, 28), and exercise also increases capillary recruitment to increase glucose uptake (29). Inhibition of insulin-mediated recruitment of capillary flow by α-methyserotonin decreases insulin-stimulated glucose uptake (27). Currently, we do not know which blood vessels are affected in skeletal muscle by COMP-Ang1. On the basis of previous studies and our results, we suggest that COMP-Ang1 may increase capillary blood flow through enlargement of terminal arterioles and capillaries resulting in insulin-stimulated glucose uptake in skeletal muscle. Further studies for the characterization of affected blood vessel in skeletal muscle by COMP-Ang1 should clear our suggestion and may provide the mechanism of increased insulin sensitivity by COMP-Ang1.

Importantly, COMP-Ang1 also inhibits insulin resistance in high-fat-fed mice. High-fat feeding induces insulin resistance in rodents and humans with an increase in fat mass (19, 24, 25). Although increased accumulation of fat metabolites attenuates the insulin signaling pathway to induce insulin resistance (25), a high-fat diet is also associated with arteriolar dysfunction (31), which may lead to deterioration of tissue perfusion. Furthermore, lipid infusion impairs physiological insulin-mediated capillary recruitment and muscle uptake in vivo (9), and obesity blunts insulin-mediated microvascular recruitment in human forearm muscle and increased brachial artery blood flow in lean individuals (10). Therefore, it is possible that deteriorated capillary blood flow induced by a high-fat diet may be compensated by COMP-Ang1, which inhibits the insulin resistance of skeletal muscle. It thus appears that a COMP-Ang1-induced increase in microvascular blood flow is one of effective ways to attenuate insulin resistance. This view is supported by the previous demonstration that COMP-Ang1 by adenoviral injection to db/db mice decreases the glucose level in an intraperitoneal glucose tolerance test (18).

COMP-Ang1-induced increase in glucose uptake is mediated by increased insulin sensitivity, which is supported by activated insulin signaling molecule. Binding of insulin to insulin receptor leads to the activation of a variety of signaling pathways involving specific protein kinases including protein kinase Bα/Akt kinase (37). This signaling cascade translocates intracellular GLUT4 to the plasma membrane and transports glucose from the extracellular space to intracellular cytoplasm (37). The present study also shows that COMP-Ang1 increases phosphorylation of Akt in both chow-fed and high-fat-fed mice.

However, HGP was not altered by COMP-Ang1 in chow-fed and high-fat-fed mice. Reduction in liver blood flow in septic rats precedes increased glucose production (20), and decreased blood flow of liver by intense exercise reduces lactate extraction in humans (22), suggesting an association between blood flow and liver metabolism. Few studies, however, have evaluated the effect of increased blood flow on liver glucose metabolism. The present data indicate that COMP-Ang1-mediated increased blood flow does not alter glucose production in the liver.

In conclusion, increased skeletal muscle blood flow by COMP-Ang1 enhances insulin sensitivity and prevents high-fat diet-induced insulin resistance in skeletal muscle. These results will provide a new insight for the prevention and treatment of insulin resistance.


This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Aging-Associated Vascular Disease Research Center at Yeungnam University (R13-2005-005-01003-0) and also supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-531-E00004).


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