Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Optimized Quantification of Intrahost Viral Diversity in SARS-CoV-2 and Influenza Virus Sequence Data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller, and use of replicate sequencing have the most significant impact on single nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false negative rates. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intrahost viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intrahost variation, viral diversity, and viral evolution. IMPORTANCE: When viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in inclusion of false positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant calling tools. We used simulated and synthetic data to test their performance against a true set of variants, and then used these studies to inform variant identification in data from clinical SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

In vivo evolution of an emerging zoonotic bacterial pathogen in an immunocompromised human host.

Zoonotic transfer of animal pathogens to human hosts can generate novel agents, but the genetic events following such host jumps are not well studied. Here we characterize the mechanisms driving adaptive evolution of the emerging zoonotic pathogen Bordetella hinzii in a patient with interleukin-12 receptor ß1 deficiency. Genomic sequencing of 24 B. hinzii isolates cultured from blood and stool over 45 months revealed a clonal lineage that had undergone extensive within-host genetic and phenotypic diversification. Twenty of 24 isolates shared an E9G substitution in the DNA polymerase III ε-subunit active site, resulting in a proofreading deficiency. Within this proofreading-deficient clade, multiple lineages with mutations in DNA repair genes and altered mutational spectra emerged and dominated clinical cultures for more than 12 months. Multiple enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways were repeatedly mutated, suggesting rapid metabolic adaptation to the human environment. Furthermore, an excess of G:C > T:A transversions suggested that oxidative stress shaped genetic diversification during adaptation. We propose that inactivation of DNA proofreading activity in combination with prolonged, but sub-lethal, oxidative attack resulting from the underlying host immunodeficiency facilitated rapid genomic adaptation. These findings suggest a fundamental role for host immune phenotype in shaping pathogen evolution following zoonotic infection.

In Vivo Efficacy of Olorofim against Systemic Scedosporiosis and Lomentosporiosis.

Clinically relevant members of the Scedosporium/Pseudallescheria species complex and Lomentospora prolificans are generally resistant against currently available systemic antifungal agents in vitro, and infection due to these species is difficult to treat. We studied the in vivo efficacy of a new fungicidal agent, olorofim (formerly F901318), against scedosporiosis and lomentosporiosis in neutropenic animals. Cyclophosphamide-immunosuppressed CD-1 mice infected by Scedosporium apiospermum, Pseudallescheria boydii (Scedosporium boydii), and Lomentospora prolificans were treated by intraperitoneal administration of olorofim (15 mg/kg of body weight every 8 h for 9 days). The efficacy of olorofim treatment was assessed by the survival rate at 10 days postinfection, levels of serum (1-3)-ß-d-glucan (BG), histopathology, and fungal burdens of kidneys 3 days postinfection. Olorofim therapy significantly improved survival compared to that of the untreated controls; 80%, 100%, and 100% of treated mice survived infection by Scedosporium apiospermum, Pseudallescheria boydii, and Lomentospora prolificans, respectively, while less than 20% of the control mice (phosphate-buffered saline [PBS] treated) survived at 10 days postinfection. In the olorofim-treated neutropenic CD-1 mice infected with any of the three species, serum BG levels were significantly suppressed and fungal DNA detected in the target organs was significantly lower than in controls. Furthermore, histopathology of kidneys revealed no or only a few lesions with hyphal elements in the olorofim-treated mice, while numerous fungal hyphae were present in control mice. These results indicate olorofim to be a promising therapeutic agent for systemic scedosporiosis/lomentosporiosis, devastating emerging fungal infections that are difficult to treat with currently available antifungals.

Protracted course of disseminated adenovirus disease with necrotizing granulomas in the liver.

A 52- year-old male with chronic lymphocytic leukemia was hospitalized with disseminated adenovirus disease. More than a month following recovery, hepatic necrotizing granulomas secondary to adenovirus were found. This case illustrates the protracted course that adenovirus disease may take and emphasizes an unusual presentation with hepatic necrotizing granulomas.

Molecular screening for Rickettsia, Anaplasmataceae and Coxiella burnetii in Rhipicephalus sanguineus ticks from Malaysia.

A total of 44 Rhipicephalus sanguineus ticks collected from 23 dogs from Malaysia were screened for Rickettsia, Anaplasmataceae and Coxiella burnetii. Coxiella burnetii was detected in 59% (26/44) of ticks however Rickettsia and Anaplasmataceae were not detected in any of the ticks. In order to genotype the strains of C. burnetii, multispacer sequence typing (MST) was carried out using three different spacers. One of the spacers; Cox2 successfully amplified a fragment for which the full length sequence of 397 bp was obtained. The sequenced product revealed only a single nucleotide difference with the Cox2.3 type sequence.

In vitro antimicrobial effect and in vivo preventive and therapeutic effects of partially purified lantibiotic lacticin NK34 against infection by Staphylococcus species isolated from bovine mastitis.

Lantibiotics are small (<5 kDa), polycyclic peptides produced by gram-positive bacteria; they are also known as gram-positive bacteriocins. The high antimicrobial activity of lacticins and the continuing appearance of antibiotic-resistant bacteria in recent years have resulted in a renewed interest in lantibiotics. A partially purified form of lacticin NK34 (a Lactococcus lactis product isolated from the Korean fermented fish jeotgal) was tested to determine its antimicrobial effects against Staphylococcus aureus (n=20) and coagulase-negative Staphylococcus (CNS, n=20) strains isolated from the raw milk of cows with subclinical bovine mastitis in the present study. The spot-on-lawn assay was used to identify the 2 strains from each group with the greatest lacticin NK34 susceptibility, and the minimal lethal dose (MLD) was measured in ICR (imprinting control region) mice. The preventive and therapeutic effects of lacticin NK34 on the mouse infection model were determined for the first time. Lacticin NK34 demonstrated antimicrobial effects in 14 of 20 (70%) S. aureus indicator strains and in 18 of 20 (90%) CNS strains. Staphylococcus aureus 69 and S. simulans 55 demonstrated the greatest susceptibility to lacticin NK34 in the spot-on-lawn assay. The S. aureus 69 MLD was measured at 1.53 x 10(9) cfu/mouse, whereas the S. simulans 55 MLD was 3.59 x 10(9) cfu/mouse. Mice infected experimentally with S. aureus 69 MLD or S. simulans 55 MLD were treated with lacticin NK34. Treated mice demonstrated an 80% survival rate (48 of 60 mice) compared with a survival rate of 7.5% (3 of 40 mice) in control mice treated with distilled water. These data suggest that lacticin NK34 might be useful in the control of bovine mastitis and systemic bacterial infection.

CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells.

Cancer stem cells (CSCs) have been identified in solid tumors and cancer cell lines. In this study, we isolated a series of cancer cell clones, which were heterogeneous in growth rate, cell cycle distribution and expression profile of genes and proteins, from ovarian tumor specimens of a patient and identified a sub-population enriched for ovarian CSCs defined by CD24 phenotype. Experiments in vitro demonstrated CD24(+) sub-population possessed stem cell-like characteristics of remaining quiescence and more chemoresistant compared with CD24(-) fraction, as well as a specific capacity for self-renewal and differentiation. In addition, injection of 5 x 10(3) CD24(+) cells was able to form tumor xenografts in nude mice, whereas equal number of CD24(-) cells remained nontumorigenic. We also found that CD24(+) cells expressed higher mRNA levels of some 'stemness' genes, including Nestin, beta-catenin, Bmi-1, Oct4, Oct3/4, Notch1 and Notch4 which were involved in modulating many functions of stem cells, and lower E-cadherin mRNA level than CD24(-) cells. Altogether, these observations suggest human ovarian tumor cells are organized as a hierarchy and CD24 demarcates an ovarian cancer-initiating cell population. These findings will have important clinical applications for developing effective therapeutic strategies to treat ovarian cancer.

Electrophilic cyclizations of vinylcyclopropanols to tethered aldehydes.

[reaction: see text]. The intramolecular, stereoselective addition of 1-vinylcyclopropanols to tethered aldehydes has been achieved under mild conditions. Thus, sequential application of the titanium-mediated cyclopropanation of alpha,beta-unsaturated esters and the electrophilic cyclization of the aldehyde-tethered cyclopropanol products provides the facile formation of carbocyclic rings.

Free fatty acids induce peripheral insulin resistance without increasing muscle hexosamine pathway product levels in rats.

To evaluate the role of the hexosamine biosynthesis pathway (HBP) in fat-induced insulin resistance, we examined whether fat-induced insulin resistance is additive to that induced by increased HBP flux via glucosamine infusion and, if so, whether such additive effects correlate with muscle HBP product levels. Prolonged hyperinsulinemic (approximately 550 pmol/l) euglycemic clamps were conducted in conscious overnight-fasted rats. After the initial 150 min to attain steady-state insulin action, rats received an additional infusion of saline, Intralipid, glucosamine, or Intralipid and glucosamine (n = 8 or 9 for each) for 330 min. At the conclusion of clamps, skeletal muscles (soleus, extensor digitorum longus, and tibialis anterior) were taken for the measurement of HBP product levels. Intralipid and glucosamine infusions decreased insulin-stimulated glucose uptake (Rd) by 38 and 28%, respectively. When the infusions were combined, insulin-stimulated Rd decreased 47%, significantly more than with Intralipid or glucosamine alone (P < 0.05). The glucosamine-induced insulin resistance was associated with four- to fivefold increases in muscle HBP product levels. In contrast, the Intralipid-induced insulin resistance was accompanied by absolutely no increase in HBP product levels in all of the muscles examined. Also, when infused with glucosamine, Intralipid decreased insulin action below that with glucosamine alone without changing HBP product levels. In a separate study, short-term (50 and 180 min) Intralipid infusion also failed to increase muscle HBP product levels. In conclusion, increased availability of plasma free fatty acids induces peripheral insulin resistance without increasing HBP product levels in skeletal muscle.

Short-term K(+) deprivation provokes insulin resistance of cellular K(+) uptake revealed with the K(+) clamp.

We aimed to test the feasibility of quantifying insulin action on cellular K(+) uptake in vivo in the conscious rat by measuring the exogenous K(+) infusion rate needed to maintain constant plasma K(+) concentration ([K(+)]) during insulin infusion. In this "K(+) clamp" the K(+) infusion rate required to clamp plasma [K(+)] is a measure of insulin action to increase net plasma K(+) disappearance. K(+) infusion rate required to clamp plasma [K(+)] was insulin dose dependent. Renal K(+) excretion was not significantly affected by insulin at a physiological concentration ( approximately 90 microU/ml, P > 0.05), indicating that most of insulin-mediated plasma K(+) disappearance was due to K(+) uptake by extrarenal tissues. In rats deprived of K(+) for 2 days, plasma [K(+)] fell from 4.2 to 3.8 mM, insulin-mediated plasma glucose clearance was normal, but insulin-mediated plasma K(+) disappearance decreased to 20% of control, even though there was no change in muscle Na-K-ATPase activity or expression, which is believed to be the main K(+) uptake route. After 10 days K(+) deprivation, plasma [K(+)] fell to 2.9 mM, insulin-mediated K(+) disappearance decreased to 6% of control (glucose clearance normal), and there were 50% decreases in Na-K-ATPase activity and alpha2-subunit levels. In conclusion, the present study proves the feasibility of the K(+) clamp technique and demonstrates that short-term K(+) deprivation leads to a near complete insulin resistance of cellular K(+) uptake that precedes changes in muscle sodium pump expression.

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats.

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).

Acute effect of growth hormone to induce peripheral insulin resistance is independent of FFA and insulin levels in rats.

To examine whether growth hormone (GH) induces peripheral insulin resistance by altering plasma free fatty acid (FFA) or insulin levels, the effects of GH infusion on insulin-stimulated glucose fluxes were studied in conscious rats under two protocols. In study 1, either saline (n = 7) or human recombinant GH (21 microg. kg(-1). h(-1); n = 8) was infused for 300 min, and insulin-stimulated glucose fluxes were estimated during the final 150-min period of hyperinsulinemic euglycemic clamps. In study 2, hyperinsulinemic euglycemic clamps were first conducted for 150 min (to raise plasma insulin and suppress FFA levels), and saline or GH (n = 7 for each) was subsequently infused for the following 300-min clamp period. In study 1, GH infusion in the basal state did not significantly alter plasma FFA or insulin levels. In contrast, GH infusion decreased insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis by 32, 27, and 40%, respectively (P < 0.05). In study 2, GH infusion during hyperinsulinemic euglycemic clamps did not alter plasma FFA or insulin levels (P > 0.05). GH infusion had no effect on insulin-stimulated glucose uptake during the initial 150 min but eventually decreased insulin-stimulated glucose uptake by 37% (P < 0. 05), similar to the results in study 1. These data indicate that GH induces peripheral insulin resistance independent of plasma FFA and insulin levels. The induction of insulin resistance was preceded by suppression of glycogen synthesis, consistent with the hypothesis that metabolic impairment precedes and causes development of peripheral insulin resistance.

Temporal responses of oxidative vs. glycolytic skeletal muscles to K+ deprivation: Na+ pumps and cell cations.

When K+ output exceeds input, skeletal muscle releases intracellular fluid K+ to buffer the fall in extracellular fluid (ECF) K+. To investigate the mechanisms and muscle specificity of the K+ shift, rats were fed K+-deficient chow for 2-10 days, and two muscles at phenotypic extremes were studied: slow-twitch oxidative soleus and fast-twitch glycolytic white gastrocnemius (WG). After 2 days of low-K+ chow, plasma K+ concentration ([K+]) fell from 4.6 to 3.7 mM, and Na+-K+-ATPase alpha2 (not alpha1) protein levels in both muscles, measured by immunoblotting, decreased 36%. Cell [K+] decreased from 116 to 106 mM in soleus and insignificantly in WG, indicating that alpha2 can decrease before cell [K+]. After 5 days, there were further decreases in alpha2 (70%) and beta2 (22%) in WG, not in soleus, whereas cell [K+] decreased and cell [Na+] increased by 10 mM in both muscles. By 10 days, plasma [K+] fell to 2.9 mM, with further decreases in WG alpha2 (94%) and beta2 (70%); cell [K+] fell 19 mM in soleus and 24 mM in WG compared with the control, and cell [Na+] increased 9 mM in soleus and 15 mM in WG; total homogenate Na+-K+-ATPase activity decreased 19% in WG and insignificantly in soleus. Levels of alpha2, beta1, and beta2 mRNA were unchanged over 10 days. The ratios of alpha2 to alpha1 protein levels in both control muscles were found to be nearly 1 by using the relative changes in alpha-isoforms vs. beta1- (soleus) or beta2-isoforms (WG). We conclude that the patterns of regulation of Na+ pump isoforms in oxidative and glycolytic muscles during K+ deprivation mediated by posttranscriptional regulation of alpha2beta1 and alpha2beta2 are distinct and that decreases in alpha2-isoform pools can occur early enough in both muscles to account for the shift of K+ to the ECF.

Reduced glucose clearance as the major determinant of postabsorptive hyperglycemia in diabetic rats.

The relationships between postabsorptive glucose concentration and hepatic glucose output (HGO) and glucose clearance were studied in rats one day after treatment with various doses of streptozotocin (STZ; 0, 15, 30, 40, 50, or 75 mg/kg; n = 6 per dose; study 1). Glucose fluxes were estimated using a prolonged (6-h) infusion of [3-3H]glucose to ensure complete tracer equilibration at hyperglycemia. Postabsorptive glucose was significantly increased at the high doses of STZ (50 and 75 mg/kg; P < 0.01) and was strongly correlated with glucose clearance across all doses (r = -0.85, P < 0.001) but less strongly with HGO (r = 0.46, P < 0.01). In the group treated with 50 mg/kg STZ, postabsorptive glucose was increased twofold compared with the control (i.e., zero dose) group, with no change in HGO and a 45% decrease in glucose clearance, indicating that the hyperglycemia was due to a decrease in glucose clearance. To understand the cellular mechanisms of decreased glucose clearance in STZ diabetic rats, skeletal muscle glucose clearance and intracellular glucose and glucose 6-phosphate (G-6-P) concentrations were determined in normal and STZ (50 mg/kg) diabetic rats at their postabsorptive glucose levels as well as at matched hyperglycemia (12 mM; study 2). Glucose clearance was significantly decreased in soleus (P < 0.05) muscles of the diabetic rats, and this was associated with significantly decreased intracellular glucose and G-6-P levels at matched hyperglycemia (P < 0.05), suggestive of decreased glucose transport. In conclusion, postabsorptive hyperglycemia in STZ diabetic rats was largely due to decreased glucose clearance, although increased HGO may also have been a contributing factor at the highest STZ dose. The decrease in postabsorptive glucose clearance in STZ diabetic rats appeared to be associated with an impairment of glucose transport in soleus (type I) muscles.

Copper vapour laser treatment of port-wine stains in brown skin.

Forty-seven Korean patients with port-wine stains were treated with a copper vapour laser and clinical responses were assessed at three months after the last treatment by comparing photographs taken before each treatment. The immediate histologic changes within 15 min after laser treatment were also observed by routine H&E and nitroblue tetrazolium chloride staining. When we treated port-wine stains with minimal whitening doses of 6-8 J/cm2, no or slight colour changes were obtained. Thus, all port-wine stain lesions in this study were treated with non-specific energy densities ranging from 10-20 J/cm2. Good to excellent results were obtained in 18 (38.2%) of 47 Korean patients with port-wine stains. Repeated treatment can continue to reduce colour. Darker lesions (purple or red) are more likely to result in a marked colour change. At above threshold dose, there was wedge-shaped diffuse coagulation necrosis and loss of viability of the epidermis and underlying dermis. Even though copper vapour laser treatment of port-wine stains in brown skin is not as selective as in white skin because of epidermal melanin, our clinical data demonstrate the usefulness of the copper vapour laser for the treatment of port-wine stains in brown skin.

Prolonged suppression of glucose metabolism causes insulin resistance in rat skeletal muscle.

To determine whether an impairment of intracellular glucose metabolism causes insulin resistance, we examined the effects of suppression of glycolysis or glycogen synthesis on whole body and skeletal muscle insulin-stimulated glucose uptake during 450-min hyperinsulinemic euglycemic clamps in conscious rats. After the initial 150 min to attain steady-state insulin action, animals received an additional infusion of saline, Intralipid and heparin (to suppress glycolysis), or amylin (to suppress glycogen synthesis) for up to 300 min. Insulin-stimulated whole body glucose fluxes were constant with saline infusion (n = 7). In contrast, Intralipid infusion (n = 7) suppressed glycolysis by approximately 32%, and amylin infusion (n = 7) suppressed glycogen synthesis by approximately 45% within 30 min after the start of the infusions (P < 0.05). The suppression of metabolic fluxes increased muscle glucose 6-phosphate levels (P < 0.05), but this did not immediately affect insulin-stimulated glucose uptake due to compensatory increases in other metabolic fluxes. Insulin-stimulated whole body glucose uptake started to decrease at approximately 60 min and was significantly decreased by approximately 30% at the end of clamps (P < 0.05). Similar patterns of changes in insulin-stimulated glucose fluxes were observed in individual skeletal muscles. Thus the suppression of intracellular glucose metabolism caused decreases in insulin-stimulated glucose uptake through a cellular adaptive mechanism in response to a prolonged elevation of glucose 6-phosphate rather than the classic mechanism involving glucose 6-phosphate inhibition of hexokinase.

The development of insulin resistance with high fat feeding in rats does not involve either decreased insulin receptor tyrosine kinase activity or membrane glycoprotein PC-1.

Recent studies have suggested that the insulin receptor tyrosine kinase inhibitor, membrane glycoprotein PC-1, may play a role in certain insulin resistant states. In the present study, we examined whether either insulin receptor function or PC-1 activity was altered during the development of insulin resistance that occurs with high fat feeding in normal rats. Over the course of 14 days of high fat feeding, both maximal and submaximal (physiological) insulin-stimulated skeletal muscle glucose uptake decreased gradually; after 14 days of high fat feeding, submaximal and maximal insulin-stimulated glucose uptake decreased by approximately 40 and approximately 50%, respectively. In contrast, in the same muscles (tibialis anterior) of these animals, neither insulin receptor content nor insulin-stimulated insulin receptor autophosphorylation was altered after 14 days of high fat feeding. PC-1 has both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) enzyme activities. These enzyme activities showed no changes during the course of 14 days of high fat feeding. Individual data revealed that there was no significant correlation between insulin-stimulated glucose uptake and alkaline phosphodiesterase or nucleotide pyrophosphatase activity (P > 0.05). Together, these data indicate that neither defects in insulin receptor function nor elevated PC-1 activities are involved in the development of insulin resistance in rats with high fat feeding, and the insulin resistance induced with high fat feeding is likely due to postreceptor defects in skeletal muscle.

Extracellular glucose distribution is not altered by insulin: analysis of plasma and interstitial L-glucose kinetics.

We examined the effects of insulin on leg blood flow, whole body extracellular glucose distribution, and glucose diffusion into the interstitial fluid (ISF) surrounding skeletal muscle cells in anesthetized dogs. Extracellular glucose distribution and glucose diffusion into the muscle ISF were assessed by studying the kinetics of L-[1-14C]glucose in plasma and hindlimb lymph. Femoral artery blood flow was not increased with insulin (7.9 +/- 0.7 vs. 7.1 +/- 1.4 ml.min-1.kg-1; P = 0.54). Plasma and lymph dynamics of L-glucose after intravenous administration were superimposable between saline and insulin infusion experiments, indicating that insulin did not affect L-glucose disappearance from plasma or appearance in muscle ISF. Plasma L-glucose kinetics were best described by a four-compartment model, and one of the remote pools (intermediate) predicted the lymph L-glucose dynamics well. Estimation of maximum glucose diffusion capacity indicated that this pool, rather than the slowest pool, represents insulin-sensitive tissues. In conclusion, our data indicate that insulin does not increase transcapillary glucose diffusion to insulin-sensitive cells. In addition, hindlimb lymph represents primarily skeletal muscle ISF, which is represented by an intermediate, rather than the slowest, remote pool from whole body compartmental analysis.

Metabolic impairment precedes insulin resistance in skeletal muscle during high-fat feeding in rats.

To examine whether impairment of intracellular glucose metabolism precedes insulin resistance, we determined the time courses of changes in insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis during high-fat feeding in rats. Animals were fed with a high-fat (66.5%) diet ad libitum for 0, 2, 4, 7, or 14 days (n = 10-11 in each group) after 5 days of a low-fat (12.5%) diet. Submaximal and maximal insulin-stimulated glucose fluxes were estimated in whole body and individual skeletal muscles using the glucose clamp technique combined with D-[3-3H]glucose infusion and 2-[1-14C]deoxyglucose injection. Both submaximal and maximal insulin-stimulated glucose uptake in whole body decreased gradually with high-fat feeding. However, the decreases were minimal and not statistically significant during the initial few days (i.e., 2 and 4 days) of high-fat feeding (P > 0.05). In contrast, insulin-stimulated whole-body glycolysis (both maximal and submaximal) significantly decreased by approximately 30% with 2 days of high-fat feeding and remained suppressed thereafter (P < 0.05). Similar patterns of changes in insulin-stimulated glucose uptake and glycolysis were also observed in skeletal muscle. Insulin-stimulated glycogen synthesis and glucose-6-phosphate (G-6-P) concentrations in skeletal muscle increased significantly during the initial few days of high-fat feeding and gradually returned to control levels by day 14, suggesting that increased G-6-P concentrations were responsible for increased glycogen synthesis. Thus, suppression of insulin-stimulated glycolysis and a compensatory increase in glycogen synthesis (presumably arising from the glucose-fatty acid cycle) preceded decreases in insulin-stimulated glucose uptake in skeletal muscle during high-fat feeding. These findings suggest that the insulin resistance may develop as a secondary response to impaired intracellular glucose metabolism.