We have previously shown that skeletal muscle capillaries are rapidly recruited by physiological doses of insulin in both humans and animals. This facilitates glucose and insulin delivery to muscle, thus augmenting glucose uptake. In obese rats, both insulin-mediated microvascular recruitment and glucose uptake are diminished; however, this action of insulin has not been studied in obese humans. Here we used contrast ultrasound to measure microvascular blood volume (MBV) (an index of microvascular recruitment) in the forearm flexor muscles of lean and obese adults before and after a 120-min euglycemic-hyperinsulinemic (1 mU ⅐ min ؊1 ⅐ kg ؊1 ) clamp. We also measured brachial artery flow, fasting lipid profile, and anthropomorphic variables. Fasting plasma glucose (5.4 ؎ 0.1 vs. 5.1 ؎ 0.1 mmol/l, P ‫؍‬ 0.05), insulin (79 ؎ 11 vs. 38 ؎ 6 pmol/l, P ‫؍‬ 0.003), and percent body fat (44 ؎ 2 vs. 25 ؎ 2%, P ‫؍‬ 0.001) were higher in the obese than the lean adults. After 2 h of insulin infusion, whole-body glucose infusion rate was significantly lower in the obese versus lean group (19.3 ؎ 3.2 and 37.4 ؎ 2.6 mol ⅐ min ؊1 ⅐ kg ؊1 respectively, P < 0.001). Compared with baseline, insulin increased MBV in the lean (18.7 ؎ 3.3 to 25.0 ؎ 4.1, P ‫؍‬ 0.019) but not in the obese group (20.4 ؎ 3.6 to 18.8 ؎ 3.8, NS). Insulin increased brachial artery diameter and flow in the lean but not in the obese group. We observed a significant, negative correlation between ⌬MBV and BMI (R ‫؍‬ ؊0.482, P ‫؍‬ 0.027) in response to insulin. In conclusion, obesity eliminated the insulin-stimulated muscle microvascular recruitment and increased brachial artery blood flow seen in lean individuals.

Despite intensive study, the relation between insulin's action on blood flow and glucose metabolism remains unclear. Insulin-induced changes in microvascular perfusion, independent from effects on total blood flow, could be an important variable contributing to insulin's metabolic action. We hypothesized that modest, physiologic increments in plasma insulin concentration alter microvascular perfusion in human skeletal muscle and that these changes can be assessed using contrastenhanced ultrasound (CEU), a validated method for quantifying flow by measurement of microvascular blood volume (MBV) and microvascular flow velocity (MFV). In the first protocol, 10 healthy, fasting adults received insulin (0.05 mU ⅐ kg ؊1 ⅐ min ؊1 ) via a brachial artery for 4 h under euglycemic conditions. At baseline and after insulin infusion, MBV and MFV were measured by CEU during continuous intravenous infusion of albumin microbubbles with intermittent harmonic ultrasound imaging of the forearm deep flexor muscles. In the second protocol, 17 healthy, fasting adults received a 4-h infusion of either insulin (0.1 mU ⅐ kg ؊1 ⅐ min ؊1 , n ‫؍‬ 9) or saline (n ‫؍‬ 8) via a brachial artery. Microvascular volume was assessed in these subjects by an alternate CEU technique using an intra-arterial bolus injection of albumin microbubbles at baseline and after the 4-h infusion. With both protocols, muscle glucose uptake, plasma insulin concentration, and total blood flow to the forearm were measured at each stage. In protocol 2 subjects, tissue extraction of 1-methylxanthine (1-MX) was measured as an index of perfused capillary volume. Caffeine, which produces 1-MX as a metabolite, was administered to these subjects before the study to raise plasma 1-MX levels.In protocol 1 subjects, insulin increased muscle glucose uptake (180%, P < 0.05) and MBV (54%, P < 0.01) and decreased MFV (؊42%, P ‫؍‬ 0.07) in the absence of significant changes in total forearm blood flow. In protocol 2 subjects, insulin increased glucose uptake (220%, P < 0.01) and microvascular volume (45%, P < 0.05) with an associated moderate increase in total forearm blood flow (P < 0.05). Using forearm 1-MX extraction, we observed a trend, though not significant, toward increasing capillary volume in the insulin-treated subjects. In conclusion, modest physiologic increments in plasma insulin concentration increased microvascular blood volume, indicating altered microvascular perfusion consistent with a mechanism of capillary recruitment. The increases in microvascular (capillary) volume (despite unchanged total blood flow) indicate that the relation between insulin's vascular and metabolic actions cannot be fully understood using measurements of bulk blood flow alone.

Intense exercise and insulin each increases total limb blood flow and recruits muscle capillaries, presumably to facilitate nutrient exchange. Whether mixed meals or light exercise likewise recruits capillaries is unknown. We fed 18 (9 M, 9 F) healthy volunteers a 480-kcal liquid mixed meal. Plasma glucose, insulin, brachial artery flow, and forearm muscle microvascular blood volume were measured before and after the meal. Brachial artery flow and microvascular volume were also examined with light (25% max), moderate (50%), and heavy (80%) forearm contraction every 20 s in 5 (4 M, 1 F) healthy adults. After the meal, glucose and insulin rose modestly (to ϳ7 mM and ϳ270 pM) and peaked by 30 min, whereas brachial artery blood flow (P Ͻ 0.05) and the microvascular volume (P Ͻ 0.01) each increased significantly by 60 min, and microvascular flow velocity did not change. For exercise, both 50 and 80%, but not 25% maximal handgrip, increased average forearm and brachial artery blood flow (P Ͻ 0.01). Flow increased immediately after each contraction and declined toward basal over 15 s. Exercise at 25% max increased microvascular volume threefold (P Ͻ 0.01) without affecting microvascular flow velocity or total forearm blood flow. Forearm exercise at 80% maximal grip increased both microvascular volume and microvascular flow velocity (P Ͻ 0.05 each). We conclude that light exercise and simple meals each markedly increases muscle microvascular volume, thereby expanding the endothelial surface for nutrient exchange, and that capillary recruitment is an important physiological response to facilitate nutrient/hormone delivery in healthy humans. INSULIN (1, 7, 18) AND EXERCISE (8, 13) each increase total limb blood flow, and each recruits flow to muscle capillaries (13,18). These vascular responses facilitate the delivery of oxygen, insulin, and glucose to muscle cells. Flow to individual capillary networks is controlled by small, ϳ20 m diameter terminal arterioles (21). We previously showed that during insulin infusion, relaxation of larger-resistance arterioles (which contribute to the regulation of total blood flow) and terminal arterioles can occur independently in rat (25, 29) and human (7) muscle.Muscle microvascular responses to physiological stimuli have been difficult to assess in humans due to the lack of noninvasive techniques suitable for assessing microvascular blood volume. We have developed two independent techniques for determining muscle microvascular volume noninvasively.The first technique is based on the metabolism of exogenously infused 1-methylxanthine (1-MX) to 1-methylurate by capillary endothelial xanthine oxidase (18,29). This has worked well in rodent models but has not been adapted to human studies. The second technique, contrast-enhanced ultrasound (CEU), relies on the contrast introduced by intravenously infused microbubbles (27). To measure microvascular volume, CEU takes advantage of the fact that during their continuous infusion it is possible to disrupt microbubbles by a single pulse of high...

OBJECTIVEIngestion of a mixed meal recruits flow to muscle capillaries and increases total forearm blood flow in healthy young lean people. We examined whether these vascular responses are blunted by obesity.RESEARCH DESIGN AND METHODSWe fed eight middle-aged lean and eight obese overnight-fasted volunteers a liquid mixed meal (480 kcal). Plasma glucose and insulin were measured every 30 min, and brachial artery flow and muscle microvascular recruitment (contrast ultrasound) were assessed every 60 min over 2 h after the meal.RESULTSBy 30 min, plasma glucose rose in both the lean (5.1 ± 0.1 vs. 6.7 ± 0.4 mmol/l, P < 0.05) and the obese groups (5.4 ± 0.2 vs. 6.7 ± 0.4 mmol/l, P < 0.05). Plasma insulin rose (28 ± 4 vs. 241 ± 30 pmol/l, P < 0.05) by 30 min in the lean group and remained elevated for 2 h. The obese group had higher fasting plasma insulin levels (65 ± 8 pmol/l, P < 0.001) and a greater postmeal area under the insulin-time curve (P < 0.05). Brachial artery flow was increased at 120 min after the meal in the lean group (38 ± 6 vs. 83 ± 16 ml/min, P < 0.05) but not in the obese group. Muscle microvascular blood volume rose by 120 min in the lean group (14.4 ± 2.2 vs. 24.4 ± 4.2 units, P < 0.05) but not in the obese group.CONCLUSIONSA mixed meal recruits muscle microvasculature in lean subjects, and this effect is blunted by obesity. This impaired vascular recruitment lessens the endothelial surface available and may thereby impair postprandial glucose disposal.

Insulin inhibits proteolysis in human muscle thereby increasing protein anabolism. In contrast, IGF-I promotes muscle protein anabolism principally by stimulating protein synthesis. As increases or decreases of plasma amino acids may affect protein turnover in muscle and also alter the muscle's response to insulin and/or IGF-I, this study was designed to examine the effects of insulin and IGF-I on human muscle protein turnover during hyperaminoacidemia. We measured phenylalanine balance and [3HI -phenylalanine kinetics in both forearms of 22 postabsorptive adults during a continuous [3HIphenylalanine infusion.Measurements were made basally and at 3 and 6 h after beginning a systemic infusion of a balanced amino acid mixture that raised arterial phenylalanine concentration about twofold. Throughout the 6 h, 10 subjects received insulin locally (0.035 mU/min per kg) into one brachial artery while 12 other subjects were given intraaterial IGF-I (100 ng/min per kg) to raise insulin or IGF-I concentrations, respectively, in the infused arm. The contralateral arm in each study served as a simultaneous control for the effects of amino acids (aa) alone.Glucose uptake and lactate release increased in the insulin-and IGF-I-infused forearms (P < 0.01) but did not change in the contralateral (aa alone) forearm in either study. In the aa alone arm in both studies, hyperaminoacidemia reversed the postabsorptive net phenylalanine release by muscle to a net uptake (P < 0.025, for each) due to a stimulation of muscle protein synthesis. In the hormoneinfused arms, the addition of either insulin or IGF-I promoted greater positive shifts in phenylalanine balance than the aa alone arm (P < 0.01). With insulin, the enhanced anabolism was due to inhibition of protein degradation (P < 0.02), whereas IGF-I augmented anabolism by a further stimulation of protein synthesis above aa alone (P < 0.02). We conclude that: (a) hyperaminoacidemia specifically stimulates muscle protein synthesis; (b) insulin, even with hyperaminoacidemia, improves muscle protein balance solely by inhibiting proteolysis; and (c) hyperaminoacidemia combined with IGF-I enhances protein synthesis more

Compared to saline, lipid infusion raises plasma FFA concentrations and blocks the ability of insulin or meal to recruit muscle microvasculature. High plasma FFA concentrations may contribute to muscle insulin resistance and the microvascular complications of diabetes.

OBJECTIVE-Transport of insulin from the central circulation into muscle is rate limiting for the stimulation of glucose metabolism. By recruiting muscle microvasculature, insulin may promote its own movement into muscle interstitium. We tested whether in humans, as in the rat, insulin exerts an early action to recruit microvasculature within skeletal muscle. We further hypothesized that expansion of the microvascular volume of muscle would enhance muscle insulin clearance.RESEARCH DESIGN AND METHODS-Microvascular volume, total blood flow, and muscle insulin and glucose uptake (forearm balance method) were measured in 14 lean, healthy volunteers before and during a 2-h hyperinsulinemic-euglycemic clamp (1 mU ⅐ kg Ϫ1 ⅐ min Ϫ1 ). Microvascular volume was measured using contrast-enhanced ultrasound.RESULTS-Forearm muscle microvascular volume increased within 20 min of insulin infusion (P Ͻ 0.01), whereas an effect to increase total forearm flow was not observed until 100 min. Forearm insulin uptake increased with physiological hyperinsulinemia (15 Ϯ 3 and 87 Ϯ 13 fmol ⅐ min Ϫ1 ⅐ 100 ml Ϫ1 basal vs. last 40 min of clamp, P Ͻ 0.001). However, the extraction fraction and clearance of insulin declined (P ϭ 0.02, for each), indicating saturability of muscle insulin uptake at physiological hyperinsulinemia.CONCLUSIONS-Skeletal muscle contributes to peripheral insulin clearance both in the basal state and with physiological hyperinsulinemia. Insulin promptly expands human muscle microvascular volume but only slowly increases blood flow. Despite increased microvascular volume available for insulin uptake, muscle insulin clearance decreases significantly. These findings are consistent with the presence of a saturable transport mechanism facilitating the transendothelial transport of insulin into human muscle.

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn 2؉ -dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as ⌬ffhda1 ⌬ffhda2 mutants resemble the uninfected control plant. Microarray analysis with a ⌬ffhda1 mutant that has lost the major HDAC revealed differential expression of secondarymetabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the ⌬ffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.