Combined use of progesterone inserts, ultrasongraphy, and GnRH to identify and resynchronize nonpregnant cows and heifers 21 days after timed artificial insemination.

The objective was to decrease the reinsemination interval (RI) when dairy cows and heifers are inseminated using all timed artificial insemination (TAI) programs. Holstein cows (n = 211) and heifers (n = 153) were randomly assigned to a control or 21-day Resynch (21dRES) at 13 days after TAI. Animals in 21dRES (n = 109 cows and 77 heifers) had a progesterone device inserted on Day 13 and removed on Day 20 after TAI and ovaries scanned by ultrasonography. Animals found not to have an active CL (<15 mm) or a CL that decreased 10 mm or greater from Days 13 to 20, and to have a follicle of 12 mm or greater received GnRH and TAI on Day 21. Pregnancy diagnosis was performed on Day 32. Nonpregnant control cows (n = 102) were resynchronized immediately using Ovsynch-56, and control heifers (n = 76) were resynchronized using 5-day Cosynch starting on Day 34; therefore, cows and heifers were reinseminated on Day 42. Nonpregnant 21dRES animals that had not been reinseminated on Day 21 were resynchronized concurrently with the control animals. Pregnancy per AI (PAI) for the initial TAI was similar (P = 0.80) for 21dRES and control cows (30.3% vs. 29.4%) and heifers (49.4% vs. 51.3%). Of the nonpregnant 21dRES animals, 33 of 76 cows (43.4%) and 22 of 39 heifers (56.4%) had been reinseminated on Day 21. Therefore, the RI was decreased by 9.9 days (33.3 ± 1.0 vs. 43.2 ± 1.0 days; P < 0.001) in 21dRES cows and by 12.2 days in 21dRES heifers (30.1 ± 1.3 vs. 42.3 ± 1.3 days; P < 0.001) compared with controls. The overall resynchronized PAI was similar for 21dRES cows compared with controls (31.6% vs. 25.0%; P = 0.23). The PAI was 24.2% for 21dRES cows reinseminated on Day 21 and 37.2% for 21dRES cows reinseminated on Day 42. The overall resynchronized PAI was increased for 21dRES heifers compared with controls (57.5% vs. 32.4%; P = 0.03) because 21dRES heifers reinseminated on Day 21 had similar PAI compared with controls (43.5% vs. 32.4%; P = 0.39), but PAI was increased for 21dRES heifers reinseminated on Day 42 compared with controls (76.5% vs. 32.4%; P = 0.003). Consequently, the proportion of animals pregnant from the initial and resynchronized TAI tended to be increased in 21dRES heifers (79.0% vs. 67.1%; P = 0.09). Cost per pregnancy was decreased for the 21dRES in heifers. In conclusion, 21dRES provided a useful method to decrease the RI in cows and heifers, and to increase PAI and decrease cost per pregnancy in heifers.

Relationship between serum resistin concentrations and insulin resistance in nonobese, obese, and obese diabetic subjects.

Early reports suggested that resistin is associated with obesity and insulin resistance in rodents. However, subsequent studies have not supported these findings. To our knowledge, the present study is the first assessment in human subjects of serum resistin and insulin sensitivity by the insulin clamp technique. Thirty-eight nonobese subjects [age, 23 +/- 4 yr; body mass index (BMI), 25.4 +/- 4.3 kg/m(2)], 12 obese subjects (age, 54 +/- 8 yr; BMI, 33.0 +/- 2.5 kg/m(2)), and 22 obese subjects with type 2 diabetes (age, 59 +/- 7 yr; BMI, 34.0 +/- 2.4 kg/m(2)) were studied. Serum resistin concentrations were not different among nonobese (4.1 +/- 1.7 ng/ml), obese (4.2 +/- 1.6 ng/ml), and obese diabetic subjects (3.7 +/- 1.2 ng/ml), and were not significantly correlated to glucose disposal rate during a hyperinsulinemic glucose clamp across groups. Serum resistin was, however, inversely related to insulin sensitivity in nonobese subjects only (r = -0.35; P = 0.05), although this association was lost after adjusting for percent body fat. Serum resistin was not related to percent fat, BMI, or fat cell size. A strong correlation was observed between serum resistin and resistin mRNA expression from abdominal sc adipose tissue in a separate group of obese subjects (r = 0.62; P < 0.01; n = 56). Although the exact function of resistin is unknown, we demonstrated only a weak relationship between resistin and insulin sensitivity in nonobese subjects, indicating that resistin is unlikely to be a major link between obesity and insulin resistance in humans.

Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes.

We examined the hypothesis that an excess accumulation of intramuscular lipid (IMCL) is associated with insulin resistance and that this may be mediated by the oxidative capacity of muscle. Nine sedentary lean (L) and 11 obese (O) subjects, 8 obese subjects with type 2 diabetes mellitus (D), and 9 lean, exercise-trained (T) subjects volunteered for this study. Insulin sensitivity (M) determined during a hyperinsulinemic (40 mU x m(-2)min(-1)) euglycemic clamp was greater (P < 0.01) in L and T, compared with O and D (9.45 +/- 0.59 and 10.26 +/- 0.78 vs. 5.51 +/- 0.61 and 1.15 +/- 0.83 mg x min(-1)kg fat free mass(-1), respectively). IMCL in percutaneous vastus lateralis biopsy specimens by quantitative image analysis of Oil Red O staining was approximately 2-fold higher in D than in L (3.04 +/- 0.39 vs. 1.40 +/- 0.28% area as lipid; P < 0.01). IMCL was also higher in T (2.36 +/- 0.37), compared with L (P < 0.01). The oxidative capacity of muscle determined with succinate dehydrogenase staining of muscle fibers was higher in T, compared with L, O, and D (50.0 +/- 4.4, 36.1 +/- 4.4, 29.7 +/- 3.8, and 33.4 +/- 4.7 optical density units, respectively; P < 0.01). IMCL was negatively associated with M (r = -0.57, P < 0.05) when endurance-trained subjects were excluded from the analysis, and this association was independent of body mass index. However, the relationship between IMCL and M was not significant when trained individuals were included. There was a positive association between the oxidative capacity and M among nondiabetics (r = 0.37, P < 0.05). In summary, skeletal muscle of trained endurance athletes is markedly insulin sensitive and has a high oxidative capacity, despite having an elevated lipid content. In conclusion, the capacity for lipid oxidation may be an important mediator of the association between excess muscle lipid accumulation and insulin resistance.

Fat content in individual muscle fibers of lean and obese subjects.

OBJECTIVE: To examine skeletal muscle intracellular triglyceride concentration in different fiber types in relation to obesity. DESIGN: Skeletal muscle fiber type distribution and intracellular lipid content were measured in vastus lateralis samples obtained by needle biopsy from lean and obese individuals. SUBJECTS: Seven lean controls (body mass index (BMI) 23.0+/-3.3 kg/m(2); mean+/-s.d.) and 14 obese (BMI 33.7+/-2.7 kg/m(2)) individuals; both groups included comparable proportions of men and women. MEASUREMENTS: Samples were histochemically stained for the identification of muscle fiber types (myosin ATPase) and intracellular lipid aggregates (oil red O dye). The number and size of fat aggregates as well as their concentration within type I, IIA and IIB muscle fiber types were measured. The cellular distribution of the lipid aggregates was also examined. RESULTS: The size of fat aggregates was not affected by obesity but the number of lipid droplets within muscle fibers was twice as abundant in obese compared to lean individuals. This was seen in type I (298+/-135 vs 129+/-75; obese vs lean, P<0.05), IIA (132+/-67 vs 79+/-29; P<0.05), and IIB (103+/-63 vs 51+/-13; P<0.05) muscle fibers. A more central distribution of lipid droplets was observed in muscle fibers of obese compared to lean subjects (27.2+/-5.7 vs 19.7+/-6.4%; P<0.05). CONCLUSION: The higher number of lipid aggregates and the disposition to a greater central distribution in all fiber types in obesity indicate important changes in lipid metabolism and/or storage that are fiber type-independent.

Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity.

In obesity and type 2 diabetes, skeletal muscle has been observed to have a reduced oxidative enzyme activity, increased glycolytic activity, and increased lipid content. These metabolic characteristics are related to insulin resistance of skeletal muscle and are factors potentially related to muscle fiber type. The current study was undertaken to examine the interactions of muscle fiber type in relation to oxidative enzyme activity, glycolytic enzyme activity, and muscle lipid content in obese and type 2 diabetic subjects compared with lean healthy volunteers. The method of single-fiber analysis was used on vastus lateralis muscle obtained by percutaneous biopsy from 22 lean, 20 obese, and 20 type 2 diabetic subjects (ages 35+/-1, 42+/-2, and 52+/-2 years, respectively), with values for BMI that were similar in obese and diabetic subjects (23.7+/-0.7, 33.2+/-0.8, and 31.8+/-0.8 kg/m2, respectively). Oxidative enzyme activity followed the order of type I > type IIa > type IIb, but within each fiber type, skeletal muscle from obese and type 2 diabetic subjects had lower oxidative enzyme activity than muscle from lean subjects (P < 0.01). Muscle lipid content followed a similar pattern in relation to fiber type, and within each fiber type, muscle from obese and type 2 diabetic subjects had greater lipid content (P < 0.01). In summary, based on single-fiber analysis, skeletal muscle in obese and type 2 diabetic subjects mani-fests disturbances of oxidative enzyme activity and increased lipid content that are independent of the effect of fiber type.

Skeletal muscle attenuation determined by computed tomography is associated with skeletal muscle lipid content.

The purpose of this investigation was to validate that in vivo measurement of skeletal muscle attenuation (MA) with computed tomography (CT) is associated with muscle lipid content. Single-slice CT scans performed on phantoms of varying lipid concentrations revealed good concordance between attenuation and lipid concentration (r(2) = 0.995); increasing the phantom's lipid concentration by 1 g/100 ml decreased its attenuation by approximately 1 Hounsfield unit (HU). The test-retest coefficient of variation for two CT scans performed in six volunteers was 0.51% for the midthigh and 0.85% for the midcalf, indicating that the methodological variability is low. Lean subjects had significantly higher (P < 0.01) MA values (49.2 +/- 2.8 HU) than did obese nondiabetic (39.3 +/- 7.5 HU) and obese Type 2 diabetic (33.9 +/- 4. 1 HU) subjects, whereas obese Type 2 diabetic subjects had lower MA values that were not different from obese nondiabetic subjects. There was also good concordance between MA in midthigh and midcalf (r = 0.60, P < 0.01), psoas (r = 0.65, P < 0.01), and erector spinae (r = 0.77, P < 0.01) in subsets of volunteers. In 45 men and women who ranged from lean to obese (body mass index = 18.5 to 35.9 kg/m(2)), including 10 patients with Type 2 diabetes mellitus, reduced MA was associated with increased muscle fiber lipid content determined with histological oil red O staining (P = -0.43, P < 0. 01). In a subset of these volunteers (n = 19), triglyceride content in percutaneous biopsy specimens from vastus lateralis was also associated with MA (r = -0.58, P = 0.019). We conclude that the attenuation of skeletal muscle in vivo determined by CT is related to its lipid content and that this noninvasive method may provide additional information regarding the association between muscle composition and muscle function.

Intramuscular lipid content is increased in obesity and decreased by weight loss.

The triglyceride content of skeletal muscle samples determined by lipid extraction correlates with the severity of insulin-resistant glucose metabolism in muscle. To determine whether this reflects increased triglyceride within muscle fibers and to test the hypothesis that the lipid content in muscle fibers is increased in obesity, the present study was undertaken using quantitative histochemistry of Oil Red O staining of vastus lateralis muscle. A percutaneous muscle biopsy was performed in 9 lean subjects, 15 obese subjects without type 2 diabetes mellitus (DM), and 10 obese subjects with type 2 DM (body mass index [BMI], 23.4+/-1.0, 33.6+/-0.6, and 36.0+/-1.1 kg x m(-2) for lean, obese, and DM, respectively). Eight obese and 7 DM subjects had a weight loss and reassessment of muscle lipid content. Transverse muscle cryosections were examined by light microscopy with quantitative image analysis (grayscale images obtained by analog to digital conversion) to determine a lipid accumulation index (LAI) based on the percentage of cross-sectional fiber area occupied by lipid droplets. Muscle fiber lipid content was greater in obese individuals with DM than in lean individuals (3.62%+/-0.65% v 1.42%+/-0.28%, P < .05) but was not different in obese individuals without DM (2.53%+/-0.41%). Weight loss reduced the LAI from 3.43%+/-0.53% to 2.35%+/-0.31%. In summary, lipid accumulation within muscle fibers is significantly increased in obesity and is reduced by weight loss. This provides important information regarding the accumulation and distribution of skeletal muscle triglyceride in type 2 DM and obesity.

Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss.

The current study was undertaken to investigate fatty acid metabolism by skeletal muscle to examine potential mechanisms that could lead to increased muscle triglyceride in obesity. Sixteen lean and 40 obese research volunteers had leg balance measurement of glucose and free fatty acid (FFA) uptake (fractional extraction of [9,10 (3)H]oleate) and indirect calorimetry across the leg to determine substrate oxidation during fasting and insulin-stimulated conditions. Muscle obtained by percutaneous biopsy had lower carnitine palmitoyl transferase (CPT) activity and oxidative enzyme activity in obesity (P < 0.05). During fasting conditions, obese subjects had an elevated leg respiratory quotient (RQ, 0.83 +/- 0.02 vs. 0.90 +/- 0.01; P < 0.01) and reduced fat oxidation but similar FFA uptake across the leg. During insulin infusions, fat oxidation by leg tissues was suppressed in lean but not obese subjects; rates of FFA uptake were similar. Fasting values for leg RQ correlated with insulin sensitivity (r = -0.57, P < 0.001). Thirty-two of the obese subjects were restudied after weight loss (WL, -14.0 +/- 0.9 kg); insulin sensitivity and insulin suppression of fat oxidation improved (P < 0.01), but fasting leg RQ (0.90 +/- 0.02 vs. 0.90 +/- 0.02, pre-WL vs. post-WL) and muscle CPT activity did not change. The findings suggest that triglyceride accumulation in skeletal muscle in obesity derives from reduced capacity for fat oxidation and that inflexibility in regulating fat oxidation, more than fatty acid uptake, is related to insulin resistance.

Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss.

A number of biochemical defects have been identified in glucose metabolism within skeletal muscle in obesity, and positive effects of weight loss on insulin resistance are also well established. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity-related insulin resistance and of the effects of weight loss, though it is evident that muscle contains increased triglyceride. The current study was therefore undertaken to profile markers of human skeletal muscle for fatty acid metabolism in relation to obesity, in relation to the phenotype of insulin-resistant glucose metabolism, and to examine the effects of weight loss. Fifty-five men and women, lean and obese, with normal glucose tolerance underwent percutaneous biopsy of vastus lateralis skeletal muscle for determination of HADH, CPT, heparin-releasable (Hr) and tissue-extractable (Ext) LPL, CS, COX, PFK, and GAPDH enzyme activities, and content of cytosolic and plasma membrane FABP. Insulin sensitivity was measured using the euglycemic clamp method. DEXA was used to measure FM and FFM. In skeletal muscle of obese individuals, CPT, CS, and COX activities were lower while, conversely, they had a higher or similar content of FABP(C) and FABP(PM) than in lean individuals. Hr and Ext LPL activities were similar in both groups. In multivariate and simple regression analyses, there were significant correlations between insulin resistance and several markers of FA metabolism, notably, CPT and FABP(PM). These data suggest that in obesity-related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized toward fat esterification rather than oxidation and that dietary-induced weight loss does not correct this disposition.

CHD1 interacts with SSRP1 and depends on both its chromodomain and its ATPase/helicase-like domain for proper association with chromatin.

CHD1, an Mr approximately 200,000 protein that contains a chromodomain (C), an ATPase/helicase-like domain (H) and a DNA-binding domain (D), was previously shown to be associated with decompacted interphase chromatin in mammalian cells and with transcriptionally active puffs and interbands in Drosophila polytene chromosomes. We now show by transient transfection experiments with genes expressing wild-type and mutant forms of CHD1 that both the C and H domains are essential for its proper association with chromatin. We also present evidence for an in vivo interaction between CHD1 and a novel HMG box-containing protein, SSRP1, which involves an amino-terminal segment of CHD1 that does not include the chromodomain. Immunocytochemical analyses indicated that CHD1 and SSRP1 colocalize in both mammalian nuclei and Drosophila polytene chromosomes.

Overexpression of muscle uncoupling protein 2 content in human obesity associates with reduced skeletal muscle lipid utilization.

Uncoupling proteins (UCP) may influence thermogenesis. Since skeletal muscle plays an important role in energy homeostasis and substrate oxidation, this study was undertaken to test the hypotheses that skeletal muscle UCP2 content is altered in obesity and could be linked to basal energy expenditure, insulin sensitivity, or substrate oxidation within skeletal muscle under postabsorptive (fasting) conditions. To examine these possibilities, limb basal energy expenditure and respiratory quotient (bRQ) were measured in 18 obese nondiabetic (Ob) and lean individuals (L). Total body fat (%) ranged from 11% to 46%. In addition, insulin-stimulated rates of glucose disposal (Rd) were measured under euglycemic hyperinsulinemic conditions. Biopsy of vastus lateralis muscle was used to measure cytochrome c oxidase (COX) enzyme activity and UCP2 content. Whereas low muscle COX activity was found in the Ob compared to L (6.9+/-1.6 vs. 9.6+/-1.2 U/g; P<0.001), skeletal muscle UCP2 content in Ob was significantly higher than in L (48+/-9 vs. 33+/-12 arbitrary units/g; P<0.05). Moreover, UCP2 content was positively correlated with percent of total body fat (r=0.57; P<0. 05) and bRQ (r=0.59; P<0.01), but not with visceral fat (r=0.17; P=0. 49), basal energy expenditure (r=0.07; P=0.79) or Rd (r=-0.23; P=0. 34). In summary, these results indicate that if development of obesity in humans is mediated by defective expression of UCP2 within skeletal muscle, then this effect is not observed in people with established obesity. The present study also suggests that skeletal muscle UCP2 content is not related to basal energy expenditure or insulin sensitivity in humans. However, the increased content of UCP2 within skeletal muscle in obesity appears to coincide with a reduced postabsorptive lipid utilization by muscle.

Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM.

The insulin resistance of skeletal muscle in glucose-tolerant obese individuals is associated with reduced activity of oxidative enzymes and a disproportionate increase in activity of glycolytic enzymes. Because non-insulin-dependent diabetes mellitus (NIDDM) is a disorder characterized by even more severe insulin resistance of skeletal muscle and because many individuals with NIDDM are obese, the present study was undertaken to examine whether decreased oxidative and increased glycolytic enzyme activities are also present in NIDDM. Percutaneous biopsy of vatus lateralis muscle was obtained in eight lean (L) and eight obese (O) nondiabetic subjects and in eight obese NIDDM subjects and was assayed for marker enzymes of the glycolytic [phosphofructokinase, glyceraldehyde phosphate dehydrogenase, hexokinase (HK)] and oxidative pathways [citrate synthase (CS), cytochrome-c oxidase], as well as for a glycogenolytic enzyme (glycogen phosphorylase) and a marker of anaerobic ATP resynthesis (creatine kinase). Insulin sensitivity was measured by using the euglycemic clamp technique. Activity for glycolytic enzymes (phosphofructokinase, glyceraldehye phosphate dehydrogenase, HK) was highest in subjects with subjects with NIDDM, following the order of NIDDM > O > L, whereas maximum velocity for oxidative enzymes (CS, cytochrome-c oxidase) was lowest in subjects with NIDDM. The ratio between glycolytic and oxidative enzyme activities within skeletal muscle correlated negatively with insulin sensitivity. The HK/CS ratio had the strongest correlation (r = -0.60, P < 0.01) with insulin sensitivity. In summary, an imbalance between glycolytic and oxidative enzyme capacities is present in NIDDM subjects and is more severe than in obese or lean glucose-tolerant subjects. The altered ratio between glycolytic and oxidative enzyme activities found in skeletal muscle of individuals with NIDDM suggests that a dysregulation between mitochondrial oxidative capacity and capacity for glycolysis is an important component of the expression of insulin resistance.

Insulin-stimulated Glut 4 translocation in human skeletal muscle: a quantitative confocal microscopical assessment.

Insulin stimulation of glucose transport in skeletal muscle is considered to involve translocation of the skeletal muscle/adipose tissue glucose transporter isoform, Glut 4, from cytosolic vesicles to the cell surface. The current study was undertaken to investigate Glut 4 translocation in skeletal muscle of healthy volunteers during euglycaemic insulin infusion. Previous quantitative studies of glucose transport have depended on differential centrifugation methods, which demand large biopsy samples. In this study we have developed and applied a quantitative method using confocal laser microscopy, well suited to the small needle biopsies that are typically available clinically. Percutaneous biopsy of vastus lateralis skeletal muscle was performed during basal and euglycaemic insulin-stimulated conditions, and Glut 4 translocation was assessed using immunohistochemical labelling and confocal laser microscopy imaging in 14 healthy lean subjects. At physiological hyperinsulinaemia (536 +/- 16 pM), mean systemic glucose utilization was 9.27 +/- 0.78 mg/kg-min, indicative of normal insulin sensitivity. The presence of Glut 4 at the sarcolemma increased significantly (p < 0.01), with a ratio of insulin-stimulated to basal sarcolemmal Glut 4 of 1.85 +/- 0.33, indicative of insulin-stimulated Glut 4 translocation. The area of Glut 4-labelled sites also increased significantly (p < 0.01) in response to insulin infusion; this ratio was 1.56 +/- 0.13. Thus, at physiological hyperinsulinaemia, the amount of Glut 4 at the cell surface of skeletal muscle in healthy, lean individuals increases approximately twofold over basal conditions, and this process can be measured using immunohistochemical labelling imaged by confocal laser scanning microscopy.

The effect of non-insulin-dependent diabetes mellitus and obesity on glucose transport and phosphorylation in skeletal muscle.

Defects of glucose transport and phosphorylation may underlie insulin resistance in obesity and non-insulin-dependent diabetes mellitus (NIDDM). To test this hypothesis, dynamic imaging of 18F-2-deoxy-glucose uptake into midthigh muscle was performed using positron emission tomography during basal and insulin-stimulated conditions (40 mU/m2 per min), in eight lean nondiabetic, eight obese nondiabetic, and eight obese subjects with NIDDM. In additional studies, vastus lateralis muscle was obtained by percutaneous biopsy during basal and insulin-stimulated conditions for assay of hexokinase and citrate synthase, and for immunohistochemical labeling of Glut 4. Quantitative confocal laser scanning microscopy was used to ascertain Glut 4 at the sarcolemma as an index of insulin-regulated translocation. In lean individuals, insulin stimulated a 10-fold increase of 2-deoxy-2[18F]fluoro-D-glucose (FDG) clearance into muscle and significant increases in the rate constants for inward transport and phosphorylation of FDG. In obese individuals, the rate constant for inward transport of glucose was not increased by insulin infusion and did not differ from values in NIDDM. Insulin stimulation of the rate constant for glucose phosphorylation was similar in obese and lean subjects but reduced in NIDDM. Insulin increased by nearly twofold the number and area of sites labeling for Glut 4 at the sarcolemma in lean volunteers, but in obese and NIDDM subjects translocation of Glut 4 was attenuated. Activities of skeletal muscle HK I and II were similar in lean, obese and NIDDM subjects. These in vivo and ex vivo assessments indicate that impaired glucose transport plays a key role in insulin resistance of NIDDM and obesity and that an additional impairment of glucose phosphorylation is evident in the insulin resistance of NIDDM.

Skeletal muscle utilization of free fatty acids in women with visceral obesity.

Visceral obesity is strongly associated with insulin resistance. One potential cause is increased availability of FFA. Alternatively, it has been proposed that there is impaired oxidation of lipid in individuals at risk for obesity. The extent to which either concept involves skeletal muscle is uncertain. To examine these opposing hypotheses, 17 healthy lean and obese premenopausal women, among whom cross-sectional area of visceral fat ranged from 18 to 180 cm2, participated in leg balance studies for measurement of FFA and glucose utilization during basal and insulin-stimulated conditions. A metabolic profile of skeletal muscle, based on enzyme activity, was determined in vastus lateralis muscle obtained by percutaneous biopsy. Visceral fat content was negatively correlated with insulin sensitivity (rates of leg glucose uptake and storage), but insulin resistance was not caused by glucose-FFA competition. During hyperinsulinemia, neither leg FFA uptake nor oxidation was increased in women with visceral obesity. During fasting conditions, however, rates of FFA uptake across the leg were negatively correlated with visceral adiposity as were activities of muscle carnitine palmitoyl transferase and citrate synthase. In summary, visceral adiposity is clearly associated with skeletal muscle insulin resistance but this is not due to glucose-FFA substrate competition. Instead, women with visceral obesity have reduced postabsorptive FFA utilization by muscle.

Skeletal muscle glycolytic and oxidative enzyme capacities are determinants of insulin sensitivity and muscle composition in obese women.

Regional fat distribution is an important determinant of insulin resistance in obesity. In the current study, the relationship between skeletal muscle insulin sensitivity, mid-thigh muscle composition, and the metabolic profile of muscle was investigated. Muscle composition was assessed by computed tomography of the mid-thigh, and by activities of marker enzymes of aerobic-oxidative and glycolytic pathways and muscle fiber typing using biopsies of the vastus lateralis muscle. Muscle with reduced Hounsfield attenuation on computed tomography scans was increased in proportion to obesity, and was strongly related to insulin resistance, reduced muscle oxidative capacity, and increased anaerobic and glycolytic capacities by muscle. These findings suggest that as part of its expression of insulin resistance, skeletal muscle of obese individuals is also poorly equipped for substrate oxidation and manifests increased storage of fat.

Impaired free fatty acid utilization by skeletal muscle in non-insulin-dependent diabetes mellitus.

This study was undertaken to assess utilization of FFA by skeletal muscle in patients with non-insulin-dependent diabetes mellitus (NIDDM). 11 NIDDM and 9 nondiabetic subjects were studied using leg balance methods to measure the fractional extraction of [3H]oleate. Limb indirect calorimetry was used to estimate RQ. Percutaneous muscle biopsy samples of vastus lateralis were analyzed for muscle fiber type distribution, capillary density, and metabolic potential as reflected by measurements of the activity of seven muscle enzyme markers of glycolytic and aerobic-oxidative pathways. During postabsorptive conditions, fractional extraction of oleate across the leg was lower in NIDDM subjects (0.31 +/- 0.08 vs. 0.43 +/- 0.10, P < 0.01), and there was reduced oleate uptake across the leg (66 +/- 8 vs. 82 +/- 13 nmol/min, P < 0.01). Postabsorptive leg RQ was increased in NIDDM (0.85 +/- 0.03 vs. 0.77 +/- 0.02, P < 0.01), and rates of lipid oxidation by skeletal muscle were lower while glucose oxidation was increased (P < 0.05). In subjects with NIDDM, proportions of type I, IIa, and IIb fibers were 37 +/- 2, 37 +/- 6, and 26 +/- 5%, respectively, which did not differ from nondiabetics; and capillary density, glycolytic, and aerobic-oxidative potentials were similar. During 6 h after ingestion of a mixed meal, arterial FFA remained greater in NIDDM subjects. Therefore, despite persistent reduced fractional extraction of oleate across the leg in NIDDM (0.34 +/- 0.04 vs. 0.38 +/- 0.03, P < 0.05), rates of oleate uptake across the leg were greater in NIDDM (54 +/- 7 vs. 45 +/- 8 nmol/min, P < 0.01). In summary, during postabsorptive conditions there is reduced utilization of FFA by muscle, while during postprandial conditions there is impaired suppression of FFA uptake across the leg in NIDDM. During both fasting and postprandial conditions, NIDDM subjects have reduced rates of lipid oxidation by muscle.

Interaction between glucose and free fatty acid metabolism in human skeletal muscle.

The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.

Comparative utilization of transcription factor GABP by the promoters of ribosomal protein genes rpL30 and rpL32.

The promoters of two mouse ribosomal protein genes, rpL30 and rpL32, contain a similarly located element, called beta, which was previously shown to interact with the same nuclear protein. This protein has now been identified as the GA-binding protein (GABP) on the basis of studies with recombinant GABP subunits and GABP-specific antibodies. The rpL30 element consists of two contiguous GABP binding sites that can form a tetrameric complex with two alpha and two beta 1 subunits of GABP, as well as dimeric complexes with alpha and either the beta 1 or beta 2 subunit. The rpL32 element consists of a solitary GABP binding site that can form only dimeric complexes with alpha and beta 1 or beta 2. Footprint analysis and a comparison of the effects of mutations in each of the tandem rpL30 binding sites demonstrated that the site nearest to the transcriptional start point is strongly favored for dimeric complex formation and is correspondingly more important for rpL30 promoter function. The contributions to overall promoter activity of the proximal rpL30 site and the solitary rpL32 site are virtually the same. Paradoxically, the potential for tetramer formation afforded by the tandem sites in rpL30 has a relatively minor effect on overall promoter strength. These findings illustrate the subtlety of mechanisms by which fine-tuning of rp promoters is achieved.