Linda A. Jahn

INSULIN AND INSULIN-LIKE GROWTH FACTOR-I ENHANCE HUMAN SKELETAL MUSCLE PROTEIN ANABOLISM DURING HYPERAMINOACIDEMIA BY DIFFERENT MECHANISMS

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 muscles 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 [3H]-phenylalanine kinetics in both forearms of 22 postabsorptive adults during a continuous [3H] phenylalanine 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 hormone-infused 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 than either alone.

Obesity Blunts Insulin-Mediated Microvascular Recruitment in Human Forearm Muscle

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 · min−1 · 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 μmol · min−1 · 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 ΔMBV 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.

Obesity blunts microvascular recruitment in human forearm muscle after a mixed meal.

OBJECTIVE Ingestion 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 METHODS We 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. RESULTS By 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. CONCLUSIONS A 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.

Extreme hyperinsulinemia unmasks insulin's effect to stimulate protein synthesis in the human forearm

Insulin clearly stimulates skeletal muscle protein synthesis in vitro. Surprisingly, this effect has been difficult to reproduce in vivo. As in vitro studies have typically used much higher insulin concentrations than in vivo studies, we examined whether these concentration differences could explain the discrepancy between in vitro and in vivo observations. In 14 healthy volunteers, we raised forearm insulin concentrations 1,000-fold above basal levels while maintaining euglycemia for 4 h. Amino acids (AA) were given to either maintain basal arterial ( n = 4) or venous plasma ( n = 6) AA or increment arterial plasma AA by 100% ( n = 4) in the forearm. We measured forearm muscle glucose, lactate, oxygen, phenylalanine balance, and [3H]phenylalanine kinetics at baseline and at 4 h of insulin infusion. Extreme hyperinsulinemia strongly reversed postabsorptive muscles phenylalanine balance from a net release to an uptake ( P < 0.001). This marked anabolic effect resulted from a dramatic stimulation of protein synthesis ( P < 0.01) and a modest decline in protein degradation. Furthermore, this effect was seen even when basal arterial or venous aminoacidemia was maintained. With marked hyperinsulinemia, protein synthesis increased further when plasma AA concentrations were also increased ( P< 0.05). Forearm blood flow rose at least twofold with the combined insulin and AA infusion ( P< 0.01), and this was consistent in all groups. These results demonstrate an effect of high concentrations of insulin to markedly stimulate muscle protein synthesis in vivo in adults, even when AA concentrations are not increased. This is similar to prior in vitro reports but distinct from physiological hyperinsulinemia in vivo where stimulation of protein synthesis does not occur. Therefore, the current findings suggest that the differences in insulin concentrations used in prior studies may largely explain the previously reported discrepancy between insulin action on protein synthesis in adult muscle in vivo vs. in vitro.Insulin clearly stimulates skeletal muscle protein synthesis in vitro. Surprisingly, this effect has been difficult to reproduce in vivo. As in vitro studies have typically used much higher insulin concentrations than in vivo studies, we examined whether these concentration differences could explain the discrepancy between in vitro and in vivo observations. In 14 healthy volunteers, we raised forearm insulin concentrations 1,000-fold above basal levels while maintaining euglycemia for 4 h. Amino acids (AA) were given to either maintain basal arterial (n = 4) or venous plasma (n = 6) AA or increment arterial plasma AA by 100% (n = 4) in the forearm. We measured forearm muscle glucose, lactate, oxygen, phenylalanine balance, and [3H]phenylalanine kinetics at baseline and at 4 h of insulin infusion. Extreme hyperinsulinemia strongly reversed postabsorptive muscles phenylalanine balance from a net release to an uptake (P < 0.001). This marked anabolic effect resulted from a dramatic stimulation of protein synthesis (P < 0.01) and a modest decline in protein degradation. Furthermore, this effect was seen even when basal arterial or venous aminoacidemia was maintained. With marked hyperinsulinemia, protein synthesis increased further when plasma AA concentrations were also increased (P < 0.05). Forearm blood flow rose at least twofold with the combined insulin and AA infusion (P < 0.01), and this was consistent in all groups. These results demonstrate an effect of high concentrations of insulin to markedly stimulate muscle protein synthesis in vivo in adults, even when AA concentrations are not increased. This is similar to prior in vitro reports but distinct from physiological hyperinsulinemia in vivo where stimulation of protein synthesis does not occur. Therefore, the current findings suggest that the differences in insulin concentrations used in prior studies may largely explain the previously reported discrepancy between insulin action on protein synthesis in adult muscle in vivo vs. in vitro.

Hyperinsulinemia rapidly increases human muscle microvascular perfusion but fails to increase muscle insulin clearance: evidence that a saturable process mediates muscle insulin uptake.

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 · kg−1 · min−1). Microvascular volume was measured using contrast-enhanced ultrasound. RESULTS—Forearm muscle microvascular volume increased within 20 min of insulin infusion (P < 0.01), whereas an effect to increase total forearm flow was not observed until 100 min. Forearm insulin uptake increased with physiological hyperinsulinemia (15 ± 3 and 87 ± 13 fmol · min−1 · 100 ml−1 basal vs. last 40 min of clamp, P < 0.001). However, the extraction fraction and clearance of insulin declined (P = 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.

Salsalate Attenuates Free Fatty Acid–Induced Microvascular and Metabolic Insulin Resistance in Humans

OBJECTIVE Insulin recruits muscle microvasculature, thereby increasing endothelial exchange surface area. Free fatty acids (FFAs) cause insulin resistance by activating inhibitor of κB kinase β. Elevating plasma FFAs impairs insulin’s microvascular and metabolic actions in vivo. Whether salsalate, an anti-inflammatory agent, prevents FFA-induced microvascular and/or metabolic insulin resistance in humans is unknown. RESEARCH DESIGN AND METHODS Eleven healthy, young adults were studied three times in random order. After an overnight fast, on two occasions each subject received a 5-h systemic infusion of Intralipid ± salsalate pretreatment (50 mg/kg/day for 4 days). On the third occasion, saline replaced Intralipid. A 1 mU/kg/min euglycemic insulin clamp was superimposed over the last 2-h of each study. Skeletal and cardiac muscle microvascular blood volume (MBV), microvascular flow velocity (MFV), and microvascular blood flow (MBF) were determined before and after insulin infusion. Whole body glucose disposal rates were calculated from glucose infusion rates. RESULTS Insulin significantly increased skeletal and cardiac muscle MBV and MBF without affecting MFV. Lipid infusion abolished insulin-mediated microvascular recruitment in both skeletal and cardiac muscle and lowered insulin-stimulated whole body glucose disposal (P < 0.001). Salsalate treatment rescued insulin’s actions to recruit muscle microvasculature and improved insulin-stimulated whole body glucose disposal in the presence of high plasma FFAs. CONCLUSIONS High plasma concentrations of FFAs cause both microvascular and metabolic insulin resistance, which can be prevented or attenuated by salsalate treatment. Our data suggest that treatments aimed at inhibition of inflammatory response might help alleviate vascular insulin resistance and improve metabolic control in patients with diabetes.

Physiological hyperinsulinemia stimulates p70(S6k) phosphorylation in human skeletal muscle.

Using tracer methods, insulin stimulates muscle protein synthesis in vitro, an effect not seen in vivo with physiological insulin concentrations in adult animals or humans. To examine the action of physiological hyperinsulinemia on protein synthesis using a tracer-independent method in vivo and identify possible explanations for this discrepancy, we measured the phosphorylation of ribosomal protein S6 kinase (P70(S6k)) and eIF4E-binding protein (eIF4E-BP1), two key proteins that regulate messenger ribonucleic acid translation and protein synthesis. Postabsorptive healthy adults received either a 2-h insulin infusion (1 mU/min.kg; euglycemic insulin clamp; n = 6) or a 2-h saline infusion (n = 5). Vastus lateralis muscle was biopsied at baseline and at the end of the infusion period. Phosphorylation of P70(S6k) and eIF4E-BP1 was quantified on Western blots after SDS-PAGE. Physiological increments in plasma insulin (42 +/- 13 to 366 +/- 36 pmol/L; P: = 0.0002) significantly increased p70(S6k) (P: < 0.01), but did not affect eIF4E-BP1 phosphorylation in muscle. Plasma insulin declined slightly during saline infusion (P: = 0.04), and there was no change in the phosphorylation of either p70(S6k) or eIF4E-BP1. These findings indicate an important role of physiological hyperinsulinemia in the regulation of p70(S6k) in human muscle. This finding is consistent with a potential role for insulin in regulating the synthesis of that subset of proteins involved in ribosomal function. The failure to enhance the phosphorylation of eIF4E-BP1 may in part explain the lack of a stimulatory effect of physiological hyperinsulinemia on bulk protein synthesis in skeletal muscle in vivo.

Physiological Hyperinsulinemia Stimulates p70S6k Phosphorylation in Human Skeletal Muscle

Using tracer methods, insulin stimulates muscle protein synthesis in vitro, an effect not seen in vivo with physiological insulin concentrations in adult animals or humans. To examine the action of physiological hyperinsulinemia on protein synthesis using a tracer-independent method in vivo and identify possible explanations for this discrepancy, we measured the phosphorylation of ribosomal protein S6 kinase (P70(S6k)) and eIF4E-binding protein (eIF4E-BP1), two key proteins that regulate messenger ribonucleic acid translation and protein synthesis. Postabsorptive healthy adults received either a 2-h insulin infusion (1 mU/min.kg; euglycemic insulin clamp; n = 6) or a 2-h saline infusion (n = 5). Vastus lateralis muscle was biopsied at baseline and at the end of the infusion period. Phosphorylation of P70(S6k) and eIF4E-BP1 was quantified on Western blots after SDS-PAGE. Physiological increments in plasma insulin (42 +/- 13 to 366 +/- 36 pmol/L; P: = 0.0002) significantly increased p70(S6k) (P: < 0.01), but did not affect eIF4E-BP1 phosphorylation in muscle. Plasma insulin declined slightly during saline infusion (P: = 0.04), and there was no change in the phosphorylation of either p70(S6k) or eIF4E-BP1. These findings indicate an important role of physiological hyperinsulinemia in the regulation of p70(S6k) in human muscle. This finding is consistent with a potential role for insulin in regulating the synthesis of that subset of proteins involved in ribosomal function. The failure to enhance the phosphorylation of eIF4E-BP1 may in part explain the lack of a stimulatory effect of physiological hyperinsulinemia on bulk protein synthesis in skeletal muscle in vivo.

Insulin Enhances Endothelial Function Throughout the Arterial Tree in Healthy But Not Metabolic Syndrome Subjects.

CONTEXT Insulin reportedly impairs endothelial function in conduit arteries but improves it in resistance and microvascular arterioles in healthy humans. No studies have assessed endothelial function at three arterial levels in healthy or metabolic syndrome (METSYN) subjects. OBJECTIVE The objective of the study was to compare endothelial responsiveness of conduit arteries, resistance, and microvascular arterioles to insulin in healthy and METSYN subjects. DESIGN We assessed conduit, resistance, and microvascular arterial function in the postabsorptive and postprandial states and during euglycemic hyperinsulinemia (insulin clamp). SETTING The study was conducted at a clinical research unit. PARTICIPANTS Age-matched healthy and METSYN subjects participated in the study. INTERVENTIONS We used brachial flow-mediated dilation, forearm postischemic flow velocity, and contrast-enhanced ultrasound to assess the conduit artery, resistance arteriole, and microvascular arteriolar endothelial function, respectively. We also assessed the conduit artery stiffness (pulse wave velocity and augmentation index) and measured the plasma concentrations of 92 cardiovascular disease biomarkers at baseline and after the clamp. RESULTS Postabsorptive and postprandial endothelial function was similar in controls and METSYN in all tested vessels. METSYN subjects were metabolically insulin resistant (P < .005). In controls, but not METSYN subjects, during euglycemic hyperinsulinemia, endothelial function improved at each level of arterial vasculature (P < .05 or less for each). Conduit vessel stiffness (pulse wave velocity) was increased in the METSYN group. Twelve of 92 biomarkers differed at baseline (P < .001) and remained different at the end of the insulin clamp. CONCLUSIONS We conclude that insulin enhances arterial endothelial function in health but not in METSYN, and this vascular insulin resistance may underlie its increased cardiovascular disease risk.

Androgenic Modulation of the Growth Hormone—IGF Axis and Its Impact on Metabolic Outcomes

The maintenance of lean body mass, particularly skeletal muscle mass, has much relevance to human health because skeletal muscle serves two important functions. First, it is a major protein reservoir and provides amino acids for the synthesis of protein in more critical tissues during periods of caloric depletion and catabolic illness (1,2). Second, the loss of skeletal muscle that occurs with age or chronic illness likely contributes to the risk of falls, fractures, and subsequent deterioration of functional capacity and health. Comprehension of the physiological factors that regulate skeletal muscle (and whole-body) protein mass are therefore relevant to human health.